Issue 28
Spring 2012
the market source for life science
I
Page 3
Precise cell counting using the Moxi ZTM mini automated cell counter I Page 16 Expansion and
differentiation of human mesenchymal stromal cells I Page 30 DNA quantification in microlitre volumes
the market source for life science
editorial
This is the first edition of the VWRbioMarke Magazine for 2012,
where we welcome back our VWRbioMarke partners bringing
the high quality of innovation and application support that
makes them eligible to join this unique programme craft for life
scientists.
We are very excited this year to include some new names:
Orflo - Orflo Technologies is a Seattle-based, privately-held
leading edge biotechnology company with a strong intellectual
property portfolio focused on developing high-precision,
inexpensive portable instruments for cell analysis. Learn more
about the Moxi Z TM mini, the revolutionary automated cell
counting system.
Amresco - Amresco Inc. has been acquired by VWR early 2011
and is our new biochemical brand and product range for life
science research. Amresco offers high quality biochemicals across
all life science applications and features innovative products that
save time, simplify protocols and increase safety as the EZ-Vision®
DNA Dye, a non-toxic, non-mutagenic DNA visualisation dye that
eliminates hazardous ethidium bromide use in DNA gels
Biotix - Biotix, Inc. is a manufacturer of high-quality life science
consumables and engineered pipette tips. See how you can
optimise high sensitivity protein assays with Biotix X-RESINTM
Included with this issue of the VWRbioMarke magazine is the
VWRbioMarke Shop, the tabloid filled with special offers on
key products – often linked to the magazine articles. This year
everyone is feeling the pinch of rising prices and spending
restrictions so make sure that you have a look through this flyer to
help you get the most of your budget!
Very best regards
The VWRBioMarke team
CONTENTS
Cell biology
Orflo - Precise cell counting for life science applications using the Moxi™ Z mini automated cell counter . . . 3
BD - Scaling up cells in BD Falcon Cell Culture Multi-Flasks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Polyplus - Delivery of siRNA using INTERFERin® transfection reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
BTX - Preparing clinical grade myeloid dendritic cells by electroporation mediated transfection of in vitro
amplified tumour derived mRNA and safety testing in stage IV malignant melanoma . . . . . . . . . . . . . . . . . . . . 12
Biochrom - Serum-free media, supplements and enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Applichem - Detection and elimination of Mycoplasma in cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Corning - Cell migration, chemotaxis and invasion assay protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Thermo Scientific - Expansion and differentiation of human mesenchymal stromal cells . . . . . . . . . . . . . . . . . . 20
Wheaton - Simple solutions for biospecimen management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Proteomics
VWR International Europe bvba
Researchpark Haasrode 2020
Geldenaaksebaan 464
3001 Leuven
Belgium
Copywriting
VWR International Europe bvba
Layout and typesetting
Spectrum - The use of Spectra/Por Regenerated Cellulose membrane tubing . . . . . . . . . . . . . . . . . . . . . . . . . . 26
®
5Prime - The Rapid Translation System (RTS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
GE Healthcare - High throughput screening for antibodies and histidine tagged proteins using
Mag Sepharosetm magnetic beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Thermo Scientific - Human SRM ATLAS Peptide Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Pall Life Sciences - New PRC and LRC chromatography columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
genomics
Thermo Scientific - DNA quantification in microlitre volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Amresco - EZ-Vision® DNA Dye featured in AMRESCO’s line of People-Planet-Safe products . . . . . . . . . . . . . . 38
Supporting products
Thermo Scientific - ART® Essentials kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Biotix - Optimising high sensitivity protein assays with Biotix X-RESIN™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Brand - Detection of light absorbing leachable chemicals from disposable filter tips . . . . . . . . . . . . . . . . . . . . . 42
2
Editor
I VWR International I VWRbioMarke Issue 28 I April 2012
Marketing Services VWR
Printing
Stork, Bruchsal, Germany
No part of this publication may be reproduced
or copied without prior permission by writing of
VWR International Europe.
Run
83 300 copies
Publication date: April 2012
Due to the high sales volume of promoted articles
some items may be temporarily out of stock VWR Terms and Conditions of Sale apply.
Cell biology
®
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
Precise cell counting for life science
applications using the Moxi ZTM
mini automated cell counter
Cell counts are routinely performed in life science, clinical, and industrial
laboratories to ensure proper cell growth rates, to measure passage/seeding
densities, as well as to establish initial counts for experimental protocols.
Traditionally, these counts are performed manually by researchers using a
hemocytometer, mechanical counter, and a microscope. In addition to being
extremely laborious, this approach suffers from large errors and variability in
the resulting count information due to subjective interpretation of cells and
debris, loading errors, improper counting technique, difficulty in tracking high
concentration counts, challenges in counting of (3-D) clustered cells, and poor
statistical robustness of low cell concentration counts.
Alternatives to this unreliable and painstaking
approach of counting include high end flow
cytometers, Coulter counting systems, and
most recently imaging-based systems. The
former systems are prohibitively expensive and
can require significant training for proper use.
Imaging‑based systems present researchers
with the trade-off of realising lower cost,
enhanced convenience, and ease of use at the
expense of the count precision and accuracy
provided by their higher end counterparts.
Recently, the Orflo Moxi Z™ cell counter has
been introduced as a new alternative that
bridges this gap in performance vs. cost and
usability. Specifically, the Moxi Z™ delivers
cell count and sizing information that mirrors
the performance of the higher end systems
while simultaneously offering the significantly
improved ease of use, functionality, speed,
lower cost, and maintenance-free operation that
has characterised the newer imaging systems.
This application note examines and compares
the features and performance of the Moxi Z™
cell counter as compared to a high end Coulter
system and a leading imaging system*.
Precision and accuracy
Figure 1. Photograph of the three systems
evaluated in this application note.
Juxtaposition of the systems highlights the
relative size of each and the small footprint
of the Moxi Z TM. From back to front: Beckman
Coulter Z2, BioRad TC10, Orflo Moxi Z TM.
The foremost criteria in evaluating a counting
system’s performance is the quality of the count
information that is generated. This information
often serves as the foundation for experimental
protocols such as in the determination of the
quantities of (costly) reagents and the cell
seeding densities necessary for downstream
processing. Count information also is often
applied to the normalisation of results in data
analysis and presentation, thereby imposing
a strict requirement for both consistency and
accuracy.
April 2012 I VWRbioMarke Issue 28 I VWR International I
3
the market source for life science
Figure 2. Comparison of histogram displays on the a) Moxi Z TM versus
the b) Imaging system for a mixed sample of five types of precision
calibrated beads (mean diameters of 4,1 µm, 6 µm, 7,9 µm, 10,1 µm,
and 15,6 µm). Moxi Z TM dynamic gating enables precise counting and
sizing of particle sub-populations (10,1 µm bead gated count and size
shown here). The imaging system presents only a total count (on a
different screen).
To achieve accuracy and precision, the Moxi Z™
cell counter has implemented the same Coulter
Technique of cell counting that is used in the
higher end Coulter systems. At the core of this
technique is a precise, volumetric (3-D) electrical
measurement of cells as they pass through an
aperture. In contrast, imaging systems take
an image (2-D) of a cell sample and apply
software algorithms to extract cell profiles
and corresponding counts. This interpretive
approach is subject to errors in focusing, debris
contamination and overall processing limitations,
all of which are reflected in the quality of
the corresponding count results. As would
be expected, the image processing approach
provides extremely coarse information regarding
sample size profiles, particularly as compared
to the exact volumetric information reported
by the Moxi Z™. An example highlighting the
discrepancy in size quality is shown in Figure
2 for a mixed sample of five types of precision
calibrated beads (mean diameters of 4,1 µm, 6
µm, 7,9 µm, 10,1 µm, and 15,6 µm) displayed
on the Moxi Z™ and imaging system (note: The
Coulter system was not displayed as there was
not a sufficient amount of beads to generate the
5 ml of sample required for a test).
Figure 3.
a) Serial Dilution Counts were performed
with the Moxi Z TM, imaging system and
Coulter system. As the Coulter Z2 is
the established standard in counting
technologies, both the Moxi Z TM counts
(blue circles) and imaging system counts
(green triangles) were plot with respect
to this system (black line ideal count).
Error bars are representative of ± one
standard deviation of the mean.
b) Bar graph representation of the
count errors (% with respect to Coulter
Z2 system, blue bars) and coefficients
of variation (CV’s %, red bars) for the
different systems.
4
I VWR International I VWRbioMarke Issue 28 I April 2012
Differences in the performance of an imaging
system vs. the Moxi Z™ were evaluated
through serial dilution experiments of CHO-K1
and Jurkat E6-1 cells and comparisons to
corresponding counts from the gold standard
Coulter system. As the resulting data shows
(Figure 3), the Moxi Z™ yields improved linearity
of the counts (Moxi Z™ r2=.9958 vs. imaging
system r2=.9317 with CHO-K1 cells and Moxi Z™
r2=.9972 vs. imaging system r2=.9939 for Jurkat
E6-1 cells) vs the imaging system. More notably,
the imaging system routinely and significantly
underestimates the true counts of the sample
and also exhibits substantial count-to-count
variations (large error bars) for identical samples.
Figure 3b quantifies this error (as a percentage
of the Coulter system results) and presents the
overall variability (coefficient of variation, CV%)
of the counts for each system. As the underlying
implementation of the imaging system is similar
to (and therefore subject to many of the same
limitations of) the hemocytometer technology,
it expectedly exhibits a similar error value (31%)
and CV (~21%) range. In stark contrast, the
Moxi Z™ achieved similar performance to the
reference standard with an error of just 5% and
a CV of only 4%.
Cell biology
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
®
Cassette architecture
Figure 4. Loading procedure of
the Moxi Z TM. The cassette-based
architecture and exterior sample
loading eliminate the potential for
instrument contamination.
Automated cell counters have brought
dramatic improvements in eliminating system
maintenance. A key breakthrough in this regard
is the use of disposables for test processing. This
format, used by both imaging systems and the
Moxi Z™, minimises the potential for sample
contact with the system and contrasts with
the high end Coulter systems in which samples
are in direct contact with the system aperture
and internal fluidics. This sample exposure
adds a substantial degree of complexity to the
operation and maintenance of the system. At
a minimum, considerable care has to be taken
to prevent frequent blockage of the fluidic
path. Beyond this, significant cost and effort is
required in the routine disinfecting of system
components. No such maintenance is required
by the Moxi Z™. In addition, the Moxi Z™
sample loading method ( Figure 4) offers an
added degree of protection from instrument
contamination (compared to imaging systems) by
pulling the sample into the detection chamber
from the exterior (as opposed to insertion of the
sample loaded end of the disposable into the
system).
Finally, while the imaging systems use simple
clear plastic slides, the Moxi Z™ cassettes are
sophisticated microfluidic sensors that include
pre-filters for dissociation of cell aggregates and
clog prevention as well as electrical circuitry for
cell detection. This circuitry is also used to make
the electronic volumetric measurement of each
particle for precise particle sizing. Furthermore,
because of the cassette-based circuitry, new
capabilities of the system can be implemented
via new cassette types. This allows all existing
Moxi Z™ instruments to take advantage of
evolving technology improvements.
Culture health assessment
Through the Moxi Population Index (MPI), the
Moxi Z™ provides a rapid, general assessment
of the ongoing health of cell cultures without
the need for reagents. The underlying principle
of MPI is that there are morphological changes
that occur with time as cells die including a
shrinking of the cells and, ultimately, a breaking
apart of the cells. This, in addition to potential
microbial contamination, contribute to increased
sample debris/particulate counts. All these
changes generate cell/particle populations that
can be size differentiated from the healthy
cell populations by Moxi Z™. In this regard,
the Moxi Z™ MPI provides a valuable, new,
and different perspective from traditional live/
dead viability staining regarding the health of
a culture. Furthermore, because the MPI and
detailed particle size histogram are automatically
calculated with each curve-fit test, users can
automatically track the ongoing health of
a culture. Alternatively, the imaging system
requires mixing of the Trypan Blue stain with the
cell sample for each health assessment test. The
instrument then attempts to isolate the dead
cells from the live cells colorimetrically, based
upon dye exclusion from the cell membranes.
However, because of the automated processing
of the image lacks the subjective (user)
interpretation of the traditional hemocytometer
counts, the imaging system analysis is subject to
unpredictable interference from non specifically
stained debris. Finally, the Coulter system does
not provide any health assessment information.
Operation - ease of use and
functionality
In addition to the overall maintenance of the
systems, the Moxi Z™ delivers considerably
improved ease of use and overall functionality
with respect the higher end counting systems
and even the newer imaging systems. To
begin with, the Moxi Z™ implements a fully
automated paradigm that requires no pretest configuration or focusing of the sample.
Furthermore, the Moxi Z™ also generates a high
resolution histogram of the data that, coupled
with the color touchscreen display, enables
interactive analysis of the results (Figure 5a). This
analysis includes both user-adjustable gating
(Figure 5a-right) for regional size analysis (i.e.
multi-cell populations) as well as curve-fitting
(Figure 5a-left) for more precise counts, more
accurate cell size information, and reagent-less
assessment of cell culture health (MPI). Regional
analysis with gating enables the identification
of counts and mean diameters within a mixed
population of cells/particles (i.e. 10,1 µm bead
count and size gating example in Figure 2a).
Unlike the Coulter system, this analysis can be
performed after the test has been run as well
as with saved tests. Furthermore, the Coulter
system provides no information on the health of
the sample. The imaging system lacks the post
processing capabilities and dynamic size analysis
all together.
In addition to count and histogram data,
the Moxi Z™ automatically displays average
diameter and volume information for each
test, and provides functionality to quickly
rescale both the vertical (counts) and horizontal
(diameter) axes. This compares to the Coulter
April 2012 I VWRbioMarke Issue 28 I VWR International I
5
the market source for life science
®
the imaging system and no data storage for
the Coulter unit. Furthermore, in stark contrast
to the simple count-only information stored
by the imaging system, the Moxi Z™ stores
the full histogram information and enables
on-unit analysis by switching from curve-fit
mode to gating mode, adjustable gating, and
vertical scaling. ORFLO also provides a free PC
and Macintosh compatible software package,
MoxiChart (Figure 6a), that enables data
transfer from the Moxi units via Bluetooth®
or USB, firmware updates for functionality
improvements, data management, and data
analysis capabilities. Additionally, the complete
histogram information is transferred and stored
in a comma separated value (.csv) file format for
facilitated loading and subsequent processing
by external data analysis programmes such as
Microsoft Excel or Wavemetrics IGOR Pro (Figure
6b). This is a significant enhancement over the
functionality provided by the imaging system
that merely provides a text file output (Figure 6c)
with count totals only and is transferrable only
by using a USB flash drive.
Figure 5. Images of an
identical HEK-293 cell sample
processed on the a) Moxi Z TM,
b) Coulter Z2 and c) TC10
cell counters. The Moxi Z TM
interface has advantages
over the other technologies
with its color touch screen
display, high resolution
histogram, post processing
analysis and the quantity of
information presented.
system which requires the scaling (gating range)
to be specified a priority and creates lower
resolution histograms (Figure 5b) only after
pressing the menu option after each test. The
imaging system provides only simple count
information initially (Figure 5c - left). A low
resolution, coarse size histogram (Figure 5c right) can be accessed immediately after a test
after navigating through the menu. However,
it lacks critical mean cell size information and is
not saved with the count results.
Data transfer and management
Up to 500 tests can be stored at a time on the
Moxi Z™ unit. This compares to 100 tests for
A)
6
B)
I VWR International I VWRbioMarke Issue 28 I April 2012
C)
Summary
The Moxi Z™ cell counter is a revolutionary
cell counting system that combines the count
precision and accuracy of higher end cell
counters and flow cytometers with the ease
of use, maintenance-free operation and lower
cost of imaging systems. In addition, Moxi Z™
adds to existing technologies with dramatically
improved overall functionality including dynamic
gating following a test, curve-fitting for more
accurate counts, reagent-less cell health
assessment and improved data management
capabilities. As a result, the Moxi Z™ provides
a level of performance and usability that is
unparalleled in the industry.
Figure 6.
a) Orflo MoxiChart application
enables data transfer from the
Moxi Z TM unit, data management,
analysis and image generation/
printing.
b) The .csv file output format has
complete histogram information for
each test enabling further analysis
in data analysis programmes.
c) Imaging system output consists
of a text file with count total
information.
Cell biology
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
Scaling up cells
in BD Falcon
Cell Culture
Multi-Flasks
The BD Falcon Multi-Flask advantages:
•
•
•
•
•
•
Stackable 3- or 5-layer formats (525 cm2 or 875 cm2 growth
surface respectively)
Pipette access for aspiration/addition of cells and reagents
directly into the flask
Efficient recovery of materials with reduced risk of
contamination
Minimal residual liquid retention reducing wastage of
valuable cells and reagents
Mixing/equilibration port allowing in-vessel mixing, uniform
distribution of cells and reagents to all layers
Vessel design facilitates use of a wide range of liquid
volumes (5 to 50 ml per layer)
A growing number of cell-based applications
require large numbers of cells. Usage of
single layer T-flasks become cumbersome,
laborious and time-consuming when large
numbers of cells are required. For efficient
scale-up of adherent mammalian cell cultures,
it is important to use vessels that generate
homogeneous cells on a consistent basis while
retaining original cellular characteristics such
as morphology, growth and function, and are
designed for optimal cell or product recovery.
To address these needs, BD Biosciences have
engineered two new multi-layered cell culture
vessels with intuitive design to facilitate easy
scaling up of cells from single layered T-flasks.
BD FalconTM Multi-Flask vessel design is
compatible with reagent usage and cell seeding
densities that are typically suited for T-175
flasks making scaling up a familiar and simple
process. The current study demonstrates that
BD Falcon Multi-Flasks features translate into
homogeneous cell culture within and between
layers of each vessel and consistently produce
cells that can be cultured in an environment
that is congruent to T-175 flasks and are
equivalent in characteristic and performance
of cells grown in T-175 flasks. Diverse cell
lines and primary cultures can be scaled up
efficiently without the need for re- optimizing
existing culture conditions or compromising
quality, homogeneity or performance of cells.
April 2012 I VWRbioMarke Issue 28 I VWR International I
7
the market source for life science
Even media distribution and
homogeneous cell growth on all layers
A key objective in developing cell-based
assays is to reduce variability (% CV) and
maximize signal-to-noise ratio for a given
cellular response. Cell-based assay outcome
can be highly influenced by the cell culture
environment, cell health and uniformity of cell
populations. Using an optimized ratio of culture
medium volume to cell density per unit area
of the culture vessel is critical for controlling
nutrient to metabolite ratio, gas exchange rate
between air and media interface, and overall
cell growth. Lower than optimal volumes of
medium result in rapid depletion of nutrients
and build up of deleterious metabolites which in
turn can cause early cell death and detachment.
Conversely, higher than optimal volumes of
medium can result in rapid cell growth, overconfluence and for some cell types, lead to
contact inhibition. The BD Falcon Multi-Flasks
have been designed with a mixing/equilibration
port to evenly distribute cell suspensions and
maintain a consistent volume of medium per
layer to provide the same environment for all
cells and thereby enable growth of homogenous
cells on a consistent basis. Moreover, culture
medium to cell ratio per unit area on all layers
of the BD Falcon Multi-Flasks are identical to
those used in T-175 flasks thus enabling efficient
expansion of cells without the need for protocol
re-optimization.
To further evaluate homogeneity of cultures, cell
confluence was quantified in the BD Falcon MultiFlasks and control T-175 flasks. Measurements at
various regions of interest across the bottom layer
of 3- and 5-layer BD Falcon Multi-Flasks revealed
uniform cell confluence with low intra-layer CV
(< 10%) comparable to T-175 flasks (figures next
page, top left).
A
B
C
Cell patterning, media distribution and cell yield on individual layers of BD Multi-Flasks.
A) EcoPack 2-293 cells grown to >80% confluence in 3-layer BD Multi-Flasks and T-175 were fixed and stained with crystal
violet for observing cell attachment and growth patterns. The vessel was cut and each stained layer was scanned.
B) Each bar shows medium distribution per layer as a percentage of total volume added per flask. Cell culture medium
(150 ml; maximum recommended volume) was added to 3-layer BD Multi-Flasks and equilibrated. Medium was pumped
out of individual layers and fluid weights were recorded from each layer. The data shown is mean + SD of 7 flasks.
For competitor vessels, a parallel experiment was conducted using vendor recommended media volume (100 mL) and
equilibration steps.
C) CHO-M1 WT3 cells were seeded at a density of 6 x 10 6 cells in 105 ml of total medium per vessel in 3-layer BD Falcon
Multi- Flasks and competitor vessels. Following 72 hours of culture, cells were harvested from each individual layer of the
vessel and cell yield per layer was determined using a Vi-CELL XR automated cell counter. Each bar on the graph illustrates
the normalized mean cell yield per layer (% mean + SD) relative to the bottom layer for three flasks
8
I VWR International I VWRbioMarke Issue 28 I April 2012
Cell biology
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
Cell confluence in BD Falcon MultiFlasks. Shown is mean intra-layer
confluence (top panel) calculated by
imaging 134 regions across the bottom
layer of 3- and 5-layer Multi-Flasks
and control T-175 flasks using IncuCyte
(ESSEN). BHK-21 cells grown to >80%
confluence in 3-layer BD Multi-Flasks
and T-175 control vessels were fixed
and stained with crystal violet prior to
cell confluence measurements. Intralayer CV (%) values were calculated
for mean confluence levels across each
growth layer (bottom panel).
Easy Scale Up from T-175 Flasks
A
Cell yield from BD Falcon MultiFlaskscompared to T-175 flasks.
Three and five times the number of
BHK-21 cells were grown and recovered
from 3- and 5- layer BD Falcon™ MultiFlasks compared to T-175 flasks (left
panel; n=4 flasks). Expected yield was
calculated by multiplying the mean cell
yield of T-175 flasks by three and five
times for the 3- and 5-layer BD Falcon
Multi-Flasks respectively. Cell yield per
unit area (cm2) was equivalent in 3- and
5-layer BD Multi- Flasks and T-175
flasks for BHK-21, LnCap, Hep-G2 and
EcoPack 2-293 cells (right panel). Each
bar represents mean of 3 to 6 flasks.
BD Falcon Cell Culture Multi-Flasks have
three and five times the growth surface area
compared to T-175 flasks. Both formats have
a familiar T-175 footprint and are compatible
with the same reagent volume and cell density
requirements as T-flasks. Even media and cell
distribution in all layers of the vessels, and
convenient pipette access to recover cells and
reagents, allows easy 3X and 5X scale up from
T-175 flasks. This is illustrated in Figure A where
three and five times the number of BHK‑21
cells were grown and recovered from 3- and
5-layer BD Falcon Multi-Flasks as compared to
T-175 flasks. Equivalent cell yield per unit area
compared to control T-175 flasks was observed
with various cell types for both 3- and 5-layer
Multi-Flasks (Figure B). Furthermore, scale up of
primary cultures such as MSCs in the MultiFlasks using serum- containing and serum-free
media yielded equivalent cell number per unit
area compared to control T-175 flasks (see Table
below)
Conclusions
•
B
•
•
•
Cell type*
BD T-175
(cells/cm2)
2,47 x 105
2,49 x 104
9,95 x 104
BHK-21
HUVEC-2
LnCap
Hep-G2
EcoPack 2-293 (HEK 293 derived)
CHO-M1
MSC (with Serum)
MSC (Serum Free
6,04 x 104
4,04 x 105
2,40 x 105
1,32 x 104
4,41 x 104
BD Falcon Multi-flask (3-layer)
(cells/cm2)
% of T-175
2,48 x 105
100 %
3,18 x 104
128 %
9,92 x 104
106 %
6,29 x 104
4,31 x 105
2,31 x 105
1,37 x 104
4,36 x 104
104 %
107 %
97 %
104 %
99 %
* Cell lines: LnCAP, HepG2, CHO-M1 cell lines were purchased from ATCC. BHK-21 cells
were obtained from Sigma Aldrich, EcoPackTM 2-293 cells from Clontech, hMSCs from
Lonza and BDTM HUVEC-2 cells from BD Biosciences. Vendor’s protocols were followed
for culturing each of these cell lines.
BD Falcon Multi-Flasks offer scientists the
option to enhance productivity by increasing
overall cell yield during expansion without
compromising cell behavior.
Homogeneous cell growth is observed within
and between layers of BD Falcon Multi-Flasks.
Three and five times the number of cells can
be grown and recovered from 3- and 5-layer
BD Falcon Multi-Flasks respectively, compared
to T-175 flasks.
Cell growth (cells/cm2) for diverse cell lines
and primary cells (cultured with and without
serum) is equivalent for BD Falcon Multi-Flasks
compared to T-175 flasks.
Table. Equivalent cell yield per cm2 from T-175 and
BD Falcon Multi-Flasks for diverse cell types and
primary cultures
BD Falcon Cell Culture Multi-Flask
Description
Qty/Pack Qty/Case Cat.No.
2
12
734-2456
3-Layer, Tissue CultureTreated, 525 cm2
1
8
734-2457
5-Layer, Tissue CultureTreated, 875 cm2
April 2012 I VWRbioMarke Issue 28 I VWR International I
9
the market source for life science
Delivery of siRNA using
INTERFERin® transfection reagent
Transfection of chemically synthesised siRNA is the simplest way of transiently suppressing the expression
of a gene in cell culture. Gene silencing is selective and allows accurate conclusions to be derived, providing
the delivery process itself is not “interfering”. Among other factors, off target effects are concentration
dependent. The efficiency of siRNA delivery into the cytoplasm is therefore of prime importance in order to
keep concentrations as low as possible.
Efficient and uniform gene silencing
As a sensitive readout, antiGL3 luciferase siRNA
was delivered with INTERFERin® into A549
Luc cells stably expressing the GL3 luciferase
gene. A mismatched siRNA (anti-GL2 luciferase)
was used as a control and the GL3 luciferase
gene expression was measured 48 hours after
transfection. As shown in Figure 1, over 90%
inhibition of gene expression was observed with
1 nM siRNA. Yet even at 10 picomolar siRNA,
50% inhibition of the luciferase gene expression
was still observed.
INTERFERin is not just based on electrostatic
carrier/cargo attraction like cationic reagents
used for plasmid DNA or RNA transfection.
Its active ingredient was developed from first
principles to bind into the deep and shallow
major groove of double stranded RNA. The
carrier/cargo interaction is as a result tight
enough to transfect even the short siRNA
into the cytoplasm. Finally, the INTERFERin®
formulation and protocol were optimised and
further developed into a robust and easy-to-use
siRNA transfection reagent.
®
INTERFERin® is a stable aqueous solution
shipped ready-to-use. The transfection protocol
is as fast and simple as plasmid transfection
(Figure 3).
One day before transfection, adherent cells are
plated in order to reach 30 to 50% confluency
the day of transfection. INTERFERin® (1 - 2 µl) is
added to siRNA in 100 µl cell culture medium.
After incubation, the siRNA/INTERFERin®
complexes are added to the cell culture well.
Unlike manufacturer’s recommendation of
most other siRNA delivery reagents, the
80
60
40
20
0
1 nM
100 pM
siRNA concentration
10
The transfection protocol remains
standard
24-well plates
100
% inhibition
Figure 1. INTERFERin®-mediated
siRNA transfection inhibits luciferase
expression in A549-GL3Luc cells.
Cells were transfected in 24 well
plates in the presence of serum
with decreasing concentrations of
Luciferase siRNA (GL3Luc) duplexes
using INTERFERin®. Luciferase
expression was measured after
48 hours. No inhibition was
observed with control siRNA
duplexes (mismatch GL2Luc, data
not shown).
Efficient delivery at picomolar levels ensures
uniform delivery at nanomolar levels. Poor siRNA
delivery leads to uneven RNA interference within
the cell population that can generate different
or partial phenotypes. Using INTERFERin®,
transfection of 1 nM lamin A/C siRNA shows
that lamin A/C expression is reduced to barely
detectable levels in >95% CaSki cells (Figure 2).
I VWR International I VWRbioMarke Issue 28 I April 2012
10 pM
Figure 2. Efficient Lamin A/C silencing using
INTERFERin®. CaSki cells were transfected with
1 nM of 21-mer siRNA duplexes matching the
lamin A/C sequence using INTERFERin®. After
48 hours, lamin A/C silencing efficiency was
determined by immunofluorescence microscopy.
Cell biology
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
Figure 4. Comparison of cell morphology 48 hours
after siRNA delivery using INTERFERin® or competitor
reagent. A549-GL3Luc cells were transfected in
the presence of serum with 1 nM of GL3Luc siRNA
duplexes using INTERFERin® or competitor S according
to the manufacturer’s protocol.
INTERFERin® protocol calls for the cells to be
kept in their original growth medium with
serum and antibiotics. This is not only time
and money saver, but it also helps keep cells
healthy during transfection (Figure 4 shows
A549 cells transfected with INTERFERin® and
with a serumless transfection reagent). Gene
expression analysis can be performed between
24 and 96 hours post transfection. Protocols
for multiplicates and larger or smaller well sizes
are simply derived from this basic protocol. If
required, specific protocols for suspension cells
such as THP-1 and K562 are also available.
For High Throughput Screening experiments,
INTERFERin®-HTS reagent should be
recommended. This siRNA transfection reagent
was specially developed for HTS applications
and comes with a reverse protocol for 96 and
384 well plates.
A versatile and robust reagent
Transfection efficiency and gene silencing
vary with cell type and target gene. Thanks
to the powerful delivery and low toxicity
of INTERFERin® (vide supra), only the siRNA
concentration needs to be varied, generally
in the 1 - 10 nM range, in order to reach
satisfactory silencing results (Table 1)*.
*Suspension cells are cultured and hence
transfected in different conditions.
A table with nearly 150 entries each providing
cell type and silencing efficiency/publication
reference, when available, can be found on the
Polyplus website (www.polyplus-transfection.
com, Cell Transfection Database with keyword
“INTERFERin®”).
Figure 3. INTERFERin® standard
protocol in 24 well plates
Table 1. Selected successfully transfected cell lines and primary cells and silencing efficiencies
obtained with INTERFERin®.
Adherent cell lines
A549
CaSki
HCT-116
HeLa
HuH-7
MCF7
MRC-5
NIH-3T3
PC-3
RAW 264.7
SiHa
SW480
SW620
CaCo2
HepG2
Luciferase
GAPDH/Lamin A/C
GAPDH
GAPDH/Lamin A/C
GAPDH
GAPDH/Lamin A/C
GAPDH
Vimentin
GAPDH
Eg5
GAPDH/Lamin A/C
GAPDH
GAPDH
GAPDH
GAPDH
1 nM
1 nM
10 nM
1 nM
25 nM
1 nM
10 nM
1 nM
25 nM
1 nM
1 nM
20 nM
20 nM
10 nM
1 nM
>90%
Primary cells
Murine embryonic fibroblasts
Primary human fibroblasts
Primary human hepatocytes
Primary human keratinocytes
Primary human melanocytes
Suspension cell lines
K562
THP-1
GAPDH
GAPDH/Lamin A/C
GAPDH
GAPDH
GAPDH
GAPDH
GAPDH
Description
INTERFERin® siRNA transfection reagent
85%
60 - 70%
1 nM
1 nM
1 nM
10 nM
10 nM
5 nM
5 nM
Size (ml)
0,1
1
5x1
>90%
>80%
>80%
Cat. No.
PPLU409-01
PPLU 409-10
PPLU 409-10
April 2012 I VWRbioMarke Issue 28 I VWR International I
11
the market source for life science
Preparing clinical grade myeloid dendritic cells by electroporation
mediated transfection of in vitro amplified tumour derived mRNA
and safety testing in stage IV malignant melanoma
Dendritic cells (DCs) are
increasingly used in vaccine
research as immunotherapy for
cancer and other diseases. DCs
can be transfected with DNA
or RNA to produce anti-tumour
antigens that break tolerance
to tumours and induce tumourspecific therapeutic immunity.
While there are several ways to
transfect DCs, electroporation
significantly enhances transfection
efficiency. This paper describes
the optimisation of parameters
for electroporation-mediated
transfection (electrotransfection)
of myeloid dendritic cells (DCs)
with in vitro expanded RNA
isolated from tumour tissue to
produce clinical grade DC vaccines.
12
RESULTS
CONCLUSIONS
1.Operating parameters were optimised for
electroporation-mediated transfection
of eGFP mRNA into normal immature
dendritic cells (IDCs) (Figure 2). The
optimal settings were 1,0-kV/cm pulses
of 150-ms duration for 10 mg RNA /106
cells.
Standardised preparation of viable clinical
grade DCs transfected with tumour
derived and in vitro amplified mRNA is
feasible and their administration is safe.
Electroporation-mediated transfection was
optimised for maximal efficiency and cell
viability.
2.Electrotransfection of patient DCs with
mRNA isolated from tumour tissue
shows strong expression of CD83 and
CD86 surface markers.
METHODS
I VWR International I VWRbioMarke Issue 28 I April 2012
Overall scheme of dendritic cell
vaccine preparation
The overall scheme of DC vaccine
preparation was to separate immature
dendritic cells (IDCs) from autologous
CD14-positive cells and isolate the total
RNA from autologous tumour tissue. RNA
was reversely transcribed to obtain cDNA
and amplified using cDNA as template
incorporating a T7 RNA promoter.
Amplified cDNA was in vitro transcribed
and loaded into IDCs by electroporation.
The DCs were subsequently matured in
the presence of inflammatory cytokines
and cryopreserved as single aliquots prior
to use.
Cell biology
Dendritic cell electrotransfection
with RNA
For experiments aimed at optimising
electroporation conditions, a cDNA
encoding the enhanced green fluorescent
protein (eGFP) gene and containing a
T7 promoter and polyadenylation signal
suitable for DC transfection was prepared
using standard methods. For transfection
into DCs, mRNA was dissolved in
water at 1,0 mg/ml. Electrotransfection
parameters were optimized by monitoring
transfection efficiency and DC viability as
a function of electrode separation, pulse
amplitude and length and amplified mRNA
concentration in the medium (Figure 2).
In all experiments we employed a PA4000 PulseAgile square-wave generator
and the proprietary cGMP grade low
conductivity (80 mS/cm) Cytoporation
Medium Formula R medium (both Cyto
Pulse Sciences, Glen Burnie, MD). We
transfected IDCs with mRNA encoding the
eGFP gene, matured the cells for 48 hours
and measured transfection efficiency
(by eGFP fluorescence) and viability (by
exclusion of 7-amino-actinomycin D,
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
7-AAD; Pharmingen, San Diego, CA)
following transfection in electroporation
cuvettes with a 4 mm electrode separation
(400 ml). The mRNA concentration was
varied between 4,0 mg/ml and 25 mg/
ml, pulse amplitude between 0,5 kV/
cm and 2,5 kV/cm and pulse width
between 0,05 ms and 0,45 ms. From the
dependence of transfection efficiency
and viability on mRNA concentration
(Figure 2A), we optimized the effect of
pulse amplitude for mRNA concentration
in the 20 - 25 mg/ml range (Figure 2B).
Because the pulse of 1,0 kV/cm resulted
in acceptable transfection efficiency and
reasonable viability, we further studied
the effect of pulse width at 20 - 25 mg/ml
RNA and 1,0 kV/cm (Figure 2C).
Electrotransfection of patient
DCs with mRNA isolated from
tumour tissue
Immature DCs manufactured from
the blood of melanoma patients were
electrotransfected with mRNA isolated
from autologous tumour tissue and in
vitro amplified. The cells were prepared as
above except that the IDCs were washed,
suspended in the Cytoporation Formula
R Medium (Cyto Pulse Sciences, Inc.,
Glen Burnie, MD) at a density of 1µ 107/
ml in the presence of 20 - 50 mg/ml of
autologous mRNA. The cell suspension
was transferred to sterile, disposable
electroporation cuvettes with a 4-mm
electrode gap (Molecular BioProducts, San
Diego, CA). The cells were subjected to
two square 400-V pulses of 50 ms each
from the PA-4000 PulseAgile generator.
Following electroporation the cells were
rested in X-VIVO 15 medium containing
HABS, GM-CSF and IL-4 as above at 37°C
in humidified 5 percent CO2 for one hour.
Subsequently the cells were washed once
and suspended in the maturation medium
containing 1100 IU/ml TNF- and 1,0 mg/
ml PGE2 for two more days. MDCs were
collected, assayed for compliance with
release criteria.
Reference:
Markovic, SN, Dietz, AB, Greiner, CW, Maas, Ml,
Butler, GW, Padley, DJ, Bulur, PA, Allred, JB, Creagan,
ET, Ingle, JN, Gastineau, and Vuk-Pavlovic, S. (2006)
Preparing clinical-grade myeloid dendritic cells by
electroporation-mediated transfection of in vitro
amplified tumour-derived mRNA and safety testing
in stage IV malignant melanoma. J Transl. Med 2006,
4:35-48. doi:10.1186/1479-5876-4-35
Figure 2. Identifying conditions for electrotransfection
of immature dendritic cells. Normal IDCs were
electro-transfected with in vitro transcribed eGFPmRNA. Following electrotransfection, the cells
were matured for 48 hours when viability (open
symbols) and transfection efficiency (closed symbols)
were quantified (by 7-AAD exclusion and eGFP
fluorescence, respectively). Shown are the data
from the final iteration in the analysis where mRNA
concentration varied from 4,0 mg/ml to 25 mg/ml (A),
pulse amplitude from 0,5 kV/cm to 2,5 kV/cm (B) and
pulse width from 0,05 ms to 0.45 ms (C). Symbols
denote mean values of measurements in cells from
three or more individuals ± standard deviation (except
in panel C that is an example of an entire experiment
conducted with cells from one individual).
Figure 5. Expression of CD83 (left) and CD86 (right)
by patients’ RNA-transfected DCs (red) used for
vaccination. Isotype controls are shown in green.
April 2012 I VWRbioMarke Issue 28 I VWR International I
13
the market source for life science
Serum-free media, supplements and enzymes
Serum-free media can be used to establish monitorable and reproducible
cultivation conditions. Biochrom AG offers serum-free media for the cultivation
of, inter alia, hybridoma, CHO, ceratinocytes, or insect cells.
In order to ensure serum-free work, supplements and growth factors need to
be added to the media. Animal-free enzymes are needed for the detachment of
adherent cells. All serum-free cell culture products can be found in this overview.
What cell types do you intend to cultivate?
We recommend some suitable serum-free media
for your cells. (Table 1.)
Supplements for serum-free media
Representing a defined medium for the clonal
growth of human ceratinocytes, MCDB 153
basal medium needs to be added by 5 ng/ml
EGF, 5 mg/l insulin, 1,4 mM hydrocortisone,
0,1 mM ethanolamine, and 0,1 mM
phosphoethanolamine.1
HAT medium is used for the selection
of hybridoma cells. HAT is composed of
hypoxanthine, thymidine and aminopterin.
Following a successful selection, the cells are
cultivated in HT medium for several passages,
before being transferred into normal hybridoma
medium.
In order to allow cells to grow ideally under
serum-free conditions, ITS (insulin, transferrin
and selenium) may be added to the media.
Insulin has different growth promoting effects
on animal cells, such as, for example, promoting
the absorption of glucose and amino acids2.
Transferrin serves as carrier protein for iron ions.
Selenium has an antioxidant effect when inside
the medium.
Table 1. Serum-free media for
your cells.
Enzyme for serum-free cell culture
The source material of Biotase is obtained from
invertebrates. Biotase can be used to detach
cells carefully. Significant surface structures of
the cells remain intact.
When using serum-free media, trypsinisation
necessitates a trypsin inhibitor.
Our tip:
Serum-free cell freezing with BIOFREEZE
Biofreeze is a serum and DMSO-free freezing
medium for the cryopreservation of cell cultures
in liquid nitrogen. It is suitable for freezing
a wide range of cell lines. Biofreeze has no
cytotoxic effect and may be used within the
framework of all traditional freezing methods.
Serum-free transport and cold storage of
cells with ChillProtec®
Adherent cells, cell suspensions or small tissue
pieces are able to remain intact after cold
storage when kept in the new Chillprotec®
medium. The protective medium reduces
cell damage caused by cold. Primary human
hepatocytes, for example, remained intact at
2 - 8 °C for several days.
References:
1. Boyce, S.T. et al. (1983): Calcium-regulated differentiation
of normal human epidermal keratinocytes in chemically
defined clonal culture and serum-free serial culture,
J of Invest. Dermatol., 81, 33-40.
2. Freshney R.I. Culture of animal cells. 5. Edition.
Wiley-Liss. 2005
Cell type (recommended)
CHO
FRTL 5
Serum-free media (Biochrom AG)
Octomed, ISF-1
Coon’s F-12 serum-free with additives
Hybridoma
Insect cells
Ceratinocytes
HybridoMed DIF 1000, ISF-1
Insectomed SF express, TC-100
Grace’s insect cell medium
MCDB 153 serum-free with additives
Lymphocytes
Neuroblastoma, glioma hybrid cells, neuronal primary cells
Neuronal primary rat cells
Sebocytes
Vero, 3T6
Iscove’s (IMDM) serum-free with additives
TNB 100 serum-free with additives
Start V
Sebomed™
PFEK-1
14
I VWR International I VWRbioMarke Issue 28 I April 2012
Additives
Insulin, hydrocortisone, transferrin, glycyl-L-histidylL-lysine-acetate, somatostatin,thyrotropin
EGF, insulin, hydrocortisone, ethanolamine,
phosphoethanol-amine
Recombinant BSA, soybean, lipides, transferrin
Lipide-protein complex
Cell biology
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
See the BioMarke Shop
for Free samples of
Biofreeze, ChillProtec®
and ChillProtec® plus!
General recommendations:
Adaptation of cells to serum-free media
Adapting cells to serum-free
media can be performed
either directly or gradually in
accordance with the following
protocols.
The source material should be
in the logarithmic growth phase
featuring the maximum number
of living cells (>90%). In principle,
a successful adaptation also
depends on the nature of the cell
line used. It is as a result highly
recommended that the retained
cultures are kept in the original
medium until the transfer into
serum-free medium has been
completed successfully.
Direct transfer of the cells:
1.Transfer the cells from serum containing medium into serum-free
medium that has been warmed to reach +37 °C. The seeding
density should correspond to that in the original culture. Incubate
the cells at +37 °C and 5 - 10% CO2 depending on the medium.
2.Passage the cells for a minimum of 4 to 8 passages, while closely
monitoring growth and viability.
3.If there is a significant decrease of growth and viability during
these passages, the user should switch to the gradual adaptation
method.
Gradual adaptation of the cells:
1.Seed the cells with a density twice as high as in the normal
inoculum in a 3:1 mixture of serum containing to serum-free
medium.
2.Having reached a density of 106 viable cells/ml, transfer the cells
into a 1:1 mixture of serum containing to serum-free medium.
3.Once the cell density is 1x 106 viable cells/ml, transfer the culture
into a 1:3 mixture of serum containing to serum-free medium.
4.Having reached a cell density of 1x 106 viable cells/ml, transfer the
cells into 100% serum-free medium.
April 2012 I VWRbioMarke Issue 28 I VWR International I
15
the market source for life science
Detection and elimination of
Mycoplasma in cell culture
Here we review
AppliChem’s proven
solutions and latest
additions in the
field of Mycoplasma
contamination and
treatment.
Surveys of cultures from labs all over the world
reveal a strong prevalence of contamination by
Mycoplasma and other mollicutes. Depending on
the method of detection 10 - 40% of continuous
cell lines have been tested positively. The
species most frequently found are Mycoplasma
orale, M. fermentans (human), M. arginini,
Acholeplasma laidlawii (bovine), and M. hominis
(swine).
Effects. Mycoplasmas are parasitic bacteria
that lack a cell wall. Depending on the species
Mycoplasma penetrate the surface of mammalian
host cells or live on the cell surface. The complete
loss of a cell culture due to Mycoplasma growth
is rarely observed. But in most cases the parasites
grow unnoted while dramatically affect the
growth and behaviour of the mammalian
cells. There is an almost infinite variety of
possible effects. To name a few, inhibition
of cell proliferation, chromosome breakage,
translocation events, degradation of DNA/RNA,
induction of inflammatory cytokines and other
factors such as Interleukins (IL-1, IL-6, IL-10),
or TNF.
Sources. There are various possible sources
for contamination by Mycoplasma. During
recent years, a rising awareness of the problem
may have changed the contribution of the
individual sources. Culture reagents such as
bovine serum have been a considerable source
of contamination in the past. Today, most labs
prefer Mycoplasma-free tested sera. Laboratory
personnel may introduce Mycoplasma into
cultures, are now trained to avoid contamination
during the handling of cultures. However,
other sources are even more difficult to avoid.
Mycoplasma detection by microscopy
Synonym
Specification
UV spectrum:
Directions
Detection procedure (outline)
DAPI BioChemica
4’,6-Diamidino-2-phenylindole dihydrochloride
Assay (TLC): Min. 98%
Solubility (1%; H2O): Clear
λ max 223 nm, 261 nm, 342 nm
λ min 246 nm, 282 nm
To prepare a stock solution, dissolve DAPI in double distilled water to a final concentration of 1 - 5 mg/ml
Working solution: Dilute the stock solution with methanol to a final concentration of 1 μg/ml
Examine cells grown on cover slips and fixed by methanol under fluorescence microscope
(excitation: 365 nm; emission maximum at 450 nm)
Mycoplasma detection by standard PCR
Possible band patterns
Kit components
Taq DNA polymerase
Form of delivery
Shipping
Cat. No.
16
PCR Mycoplasma Test Kit
M
1
2
3
4
5
M DNA marker
1. Positive control
2. Positive control
3. Negative control (water)
4. Negative control (buffer only)
5. Positive sample
• Reaction mix (dNTPs, PCR primers, Taq DNA polymerase)
• Buffer solution
• Positive template control
Included
Ready-to-use master mix, liquid
Cooled
A3744.0010
10 tests
A3744.0025
25 tests
I VWR International I VWRbioMarke Issue 28 I April 2012
PCR Mycoplasma Test Kit II
M
1
2
3
4
5
6
M DNA marker
1. Negative control
2. Positive control
3. Inhibited sample
4. Negative sample
5. Contaminated positive sample
6. Heavily contaminated positive sample
• Reaction mix (PCR primers, dNTPs)
• PCR grade water
• Buffer solution
• Positive template control
• Internal control DNA
This kit meets criteria of section 2.6.7 of Ph. Eur.
Not included
Single components, lyophilised
Ambient temperature
A8994.0025
25 tests
A8994.0050
50 tests
A8994.0100
100 tests
Cell biology
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
Mycoplasma detection by Real Time/qPCR
qPCR Mycoplasma Test Kit
Application
This qPCR Mycoplasma test kit is suited for use in combination
with different instruments: LightCycler® 1,2, 1,5, 2,0, 480,
Rotor-GeneTM 3000, 6000, ABI Prism® 7000, iCycler iQ®,
iQ™ 5, Opticon 2, Chromo 4, MX300P®, MX4000®.
Kit components
• Reaction mix (including primers, probes, nucleotides)
• Positive template control
• Buffer solution
• PCR grade water
• Internal control DNA
This kit meets criteria of section 2.6.7 of Ph. Eur.
Taq DNA polymerase
Not included
Form of delivery
Single components, lyophilised
Shipping
Ambient temperature
Cat. No.
A9019.0025
25 tests
A9019.0100
100 tests
Any addition to the culture is relevant, such
as virus suspensions, antibody solutions, or
media ingredients. Mycoplasma from original
tissue isolates contribute to less than 1% to the
reported cases. The most common source by far
is cross contamination from infected cultures.
Labs exchange infected cultures and thereby
inadvertently distribute Mycoplasma.
PCR Mycoplasma Test Kit
(A3744)
AppliChem provides the tools for detection and
treatment of Mycoplasma for every cell culture
laboratory. For the detection by microscopy we
are offering the proven fluorescent dye DAPI.
Detection by PCR. In recent years the
sensitive polymerase chain reaction (PCR)
Treatment
Application
Components
Form of delivery
Cat. No.
Treatment
Application
Components
Form of delivery
Cat. No.
Treatment
Application
Components
Form of delivery
Cat. No.
became a standard method for the detection
of Mycoplasma contamination in biological
samples such as mammalian cell cultures. PCR is
established in almost all life science labs either
as standard PCR or Real-Time/quantitative
PCR. For your preferred set-up, we offer three
different kits to choose from. rRNA gene
sequences of prokaryotes including Mycoplasma
are well conserved, whereas the lengths and
sequences of the spacer region in the rRNA
differ from species to species. The detection
procedure utilises the PCR for amplification of a
conserved and Mycoplasma specific 16S rRNA
gene region. This system does not allow the
amplification of DNA originating from other
sources, such as cultured cells or bacteria, which
affect the detection result. Amplification of the
gene sequence with PCR using this primer set
enhances not only the sensitivity, but also the
specificity of detection. Amplified products are
detected by agarose gel electrophoresis or by
Real-Time/quantitative PCR.
Treatment of Mycoplasma infections
in cell cultures
AppliChem offers well proven treatments to
achieve reliable elimination of Mycoplasma
infections from mammalian cell cultures. Precious
cell cultures that are infected cannot always be
simply discarded and replaced by new ones.
For both biological and economic reasons, it
is important to eliminate Mycoplasma from
cell cultures being used for basic research,
diagnostics and biotechnological production.
Myco-1 &-2
For the treatment of all mammalian cell lines including embryonic stem cells (ES cells).
Both agents are used in combination, one after another.
Myco-1 (A5222), based on the antibiotic Tiamulin (from Pleurotus mutilus)
Myco-2 (A5233), based on the antibiotic Minocyclin
Sterile 100X concentrated antibiotic solutions
A8360.0010
1 set (2x 10 ml)
A8360.0020
1 set (2x 20 ml)
A8360.0100
1 set (2x 100 ml)
Myco-3
Eliminates the most common Mycoplasma species including M. orale, M. hyorhinis, M. fermentans, M. arginini, as well as
A. laidlawii. At the concentrations recommended for use (1 μg/ml), no cytotoxic effects have been found.
Myco-3 is based on the antibiotic Ciprofloxacin
100X concentrated antibiotic solution
A5240.0010
10 ml
A5240.0020
20 ml
A5240.0100
100 ml
Myco-4
Novel combination of antibiotic and biophysical agents. For maximum efficiency and a broad spectrum.
Almost 100% of permanent eradication of Mycoplasma is achieved.
One kit is needed for a treatment.
Each kit contains
• 1 vial of Starter Treatment solution
• 3 vials of Main Treatment solution
Sterile, ready-to-use solutions
A8366.0002
2 kits
A8366.0005
5 kits
A8366.0010
10 kits
April 2012 I VWRbioMarke Issue 28 I VWR International I
17
the market source for life science
Cell migration, chemotaxis and
Cell migration, the movement of cells from one area to another generally in
response to a chemical signal, is central to achieving functions such as wound
repair, cell differentiation, embryonic development and the metastasis of tumours.
Cell invasion is similar to cell migration; however, it requires a cell to migrate
through an extracellular matrix (ECM) or basement membrane extract (BME)
barrier by first enzymatically degrading the barrier in order to become established
in a new location. Cell invasion is exhibited by both normal cells in responses
such as inflammation and by tumour cells in the process of metastasis, therefore
understanding the underlying mechanisms of this process are important for a wide
array of biological systems.
The advent of disposable permeable supports, such as Transwell® inserts from
Corning Life Sciences, provides a relatively simple in vitro approach to performing
chemotaxis and cell invasion assays. Common barriers employed for invasion
assays include collagen, fibronectin and laminin coatings as well as more complex
extracellular or basement membrane extracts. More elaborate invasion assays
establish a monolayer of endothelial cells on the permeable support in place of, or
in addition to, the protein coatings or BME listed above. Similarly, cells that secrete
a paracrine growth factor can be cultured in the receiver wells of the permeable
support system to act as the source of chemoattractant in either simple chemotaxis
assays or for more elaborate invasion assays.
This protocol outlines the steps for conducting a cell invasion assay through a
BME barrier with special notes for conducting a chemotaxis assay (similar to an
invasion assay, however, no BME or ECM is present). It is a generalised protocol
and should be adapted to suit your needs. This protocol utilises Corning’s 96 well
HTS Transwell® permeable supports, however, tables are provided with the proper
volumes and amounts of pertinent materials and reagents to scale the assay for use
with large permeable supports.
18
I VWR International I VWRbioMarke Issue 28 I April 2012
Cell biology
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
invasion assay protocol
Hilary Sherman, Pilar Pardo and Todd Upton, Corning Incorporated, Life Sciences
Materials
Procedure
Cell lines:
Grow enough cells in advance to
accommodate the different cell
concentrations required to set-up an assay
(Table 2). A separate flask should be set-up
at a lower concentration in order to run
standard curve on day of assay (day 3 of
the protocol).
•
Non invasive MCF-7 cells (human breast adenocarcinoma, ATCC® no. HTB-22™)
• Invasive HT1080 cells (human fibrosarcoma ATCC No. CCL-121™)
Assay plates:
•
96 well HTS Transwell® permeable supports with 8 μm pores (Corning Cat. No. 3374)
96 well black receiver plate (Corning Cat. No. 3583)
• 96 well solid black microplates (Corning Cat. No. 3916)
• 6 well Transwell® permeable supports with 8 μm pores (Corning Cat. No. 3428)
• 12 well Transwell® permeable supports with 12 μm pores (Corning Cat. No. 3403)
• 24 well Transwell® permeable supports with 8 μm pores (Corning Cat. No. 3422)
•
Reagents:
5x Basement Membrane Extract (BME) coating solution (Trevigen Cat. No. 3455-096-02)
10x Coating Buffer (Trevigen Cat. No. 3455-096-03)
10x Cell Dissociation Solution for preparing the assay dissociating solution (Trevigen Cat.
No. 3455-096-05).
Protocol overview:
Day 1 – Starve cells and coat Transwell®
inserts with Basement Membrane Extract
(BME).
Day 2 – Plate cells in Transwell® inserts and
stimulate with FBS attractant; optional setup of standard curve.
Day 3 – Detect cells that pass through the
membrane and prepare standard curve.
This solution can not be used to harvest cells.
HyQ®Tase™ Dissociation Solution for harvesting cells (Hyclone® Cat. No. JQH2447)
• Calcein AM (Molecular Probes Cat. No. C3100MP) dissolved to 1,67 μg/μl in DMSO
• IMDM medium with 10% FBS (Invitrogen Cat. No. 12440046 and 14037036,
respectively)
• Serum-free medium (SFM), IMDM medium without serum containing 1x ITS (Invitrogen
Cat. No. 41400045)
• Wash buffer – Dulbecco’s Phosphate Buffered Saline (DPBS) with calcium and
magnesium Sterile deionised water
•
Instruments:
•
37 ºC CO2 incubator
Laminar flow hood
• Fluorescent plate reader with a 485 nm excitation and 520 nm emission filter package
•
To read and receive the full protocol email cceurnl@corning.com
using ‘Protocol Transwell’ in the subject line.
April 2012 I VWRbioMarke Issue 28 I VWR International I
19
the market source for life science
Expansion and differentiation of human
mesenchymal stromal cells
Louise Gjelstrup and Thomas Stelzer, Thermo Fisher Scientific
Human mesenchymal stromal cells (hMSC) are
candidates for clinical use because they are
readily expanded in culture, have immunomodulatory potential and can differentiate into
the osteogenic, chondrogenic and adipogenic
lineages. Their therapeutic potential is currently
studied as part of clinical trials to treat
diseases such as graft-versus-host disease1 and
osteoarthritis2, as well as in the regeneration of
cardiac muscle following myocardial infarcts3.
Whether the requirements are for clinical or
research use, obtaining a substantial number
of cells can constitute a bottleneck for the
investigator. hMSC display some plasticity in
their culture conditions, but several investigators
report a higher growth index and increased
differentiation potential at lower seeding
densities4,5. Here we present a protocol enabling
the clinician or researcher to rapidly expand
a population of hMSCs on Thermo Scientific
NunclonTM Delta cell culture treated surface
utilising the potential of Thermo Scientific
HyClone AdvanceSTEMTM Mesenchymal Stem
Cell Basal Medium, developed specifically
for the optimal expansion and maintenance
of undifferentiated hMSCs. A definitive
120
A
test of multi-potency is a functional test. In
consequence, we subjected the expanded hMSC
to differentiation. The cells were differentated
into osteoblast or adipocytes in Thermo
Scientific NuncTM 48 well MultiDishes®.
Methods
Cultivation of hMSC. Human mesenchymal
stromal cells (Lonza, USA) were maintained
in α-MEM medium containing 10% FBS, 1%
Penicillin/Streptomycin and 2 mm UltraGlutamine,
or AdvanceSTEMTM Mesenchymal Stem Cell
Basal Medium supplemented with 10%
AdvanceSTEMTM Growth Supplement.
In order to test the effect of cell disassociation
on the growth and differentiation of the hMSCs.
Cell culture: hMSC was incubated at 37 °C in a
humidified atmosphere of 5% CO2 in air using
a Thermo Scientific Revco® Ultima II Series CO2
incubator.
Cell counting: Cells were counted using an
integrated automated fluorescence microscope
(Nucleocounter, Chemometec, Denmark).
B
100
Advanc eS TEM - 100 c ells /c m 2
Advanc eS TEM - 350 c ells /c m 2
80
% Confluence
Advanc eS TEM - 1000 c ells /c m 2
Advanc eS TEM - 4000 c ells /c m 2
60
α-MEM - 100 c ells /c m 2
C
α-MEM - 350 c ells /c m 2
40
α-MEM - 1000 c ells /c m 2
20
0
α-MEM - 4000 c ells /c m 2
0
50
100
150
200
250
300
350
Incubation time (hours)
Figure 1.
A: Growth of hMSC in T25 flasks with NunclonTM Delta surface in α-MEM medium or AdvanceSTEMTM Mesenchymal Stem Cell Basal
Medium. Cells were seeded at four different densities: 100 – 350 – 1000 - 4000 cells/cm2. Cultures were incubated for 12 days under
standard culture conditions (5% CO2 with media change every fourth day. Culture confluence was measured using automated microscopy
in the incubator (IncuCyteTM Imager) every three hours. Each data point represents the mean of 50 measurements in one flask.
B: hMSC morphology in AdvanceSTEMTM Mesenchymal Stem Cell Basal Medium after 7 days of incubation. Seeding density: 350 cells/cm2.
C: hMSC morpholgy in α-MEM medium after 7 days of incubation. Seeding density: 350 cells/cm2.
20
I VWR International I VWRbioMarke Issue 28 I April 2012
Cell biology
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
hMSCs maintain their multi-potency after large scale expansion in NunclonTM
Delta treated TripleFlasks. In order to verify that the cells had maintained
their ability to differentiate, the cells expanded in TripleFlasks, in both types of
growth media, using our in-house protocol, was differentiated into osteoblasts and
adipocytes in 48 well multidish at a density of 5000 cells/cm2. The differentiation
was induced using either osteogenic or adipogenic differentiation media. The
differentiation was monitored at days 3, 7, and 18 using commercial kits.
18000
16000
Figure 2. OsteoImage data from
hMSCs osteogenically differentiated
in 48 well multidish and assayed
at day 3, 7, and 18 post change to
differentiation media. Data displayed
as relative fluorescence units.
AdvanceSTEM, HyQtase
14000
AdvanceSTEM, Trypsin
Fluorescence units
12000
α-MEM, Trypsin
10000
AdvanceSTEM,
undifferentiated control
8000
6000
4000
2000
0
Day 3
Day7
Day 18
Impact of AdvanceSTEMTM Mesenchymal Stem Cell Basal Medium on
differentiation. Marked differences between hMSCs expanded in AdvanceSTEMTM
Mesenchymal Stem Cell Basal Medium and hMSCs expanded in α-MEM medium was
observed during differentiation. hMSCs expanded in AdvanceSTEMTM Mesenchymal
Stem Cell Basal Medium displayed a 59% higher signal using the OsteoImage assay
compared to cells expanded in α-MEM medium (Figure 2). Regarding adipogenic
differentiation, hMSCs expanded in α-MEM medium were unable to differentiate into
adipocytes displaying baseline signals in the AdipoRed® assay. In contrast, hMSCs
expanded in AdvanceSTEMTM Mesenchymal Stem Cell Basal Medium differentiated
successfully into adipocytes (Figure 3).
4000
3500
Advanc eS TEM , HyQtas e
Advanc eS TEM , Tryps in
Fluorescence units
3000
2500
2000
α-MEM, Tryps in
Figure 3. AdipoRed® data from hMSCs
differentiated into adipocytes in a
48 well multidish. The cells were
assayed at days 3, 7, and 18 post
change to differentiation media.
Data displayed as relative fluorescence
units.
Advance S TEM ,
undifferentiated control
1500
1000
500
0
Day 3
Day 7
Day 18
Growth curves: To develop the protocol, growth
curves of hMSC cultivated in AdvanceSTEMTM
and α-MEM growth media were established.
hMSCs in passage 2 were seeded at 100,
350, 1000 and 4000 cells/cm2 in NunclonTM
Delta treated T25 flasks. The cultures were
placed in an IncuCyte™ Plus and incubated
at 37 °C in a humidified atmosphere of 5%
CO2. The IncuCyteTM Plus is an automated
imaging platform, configured to fit inside a CO2
incubator, and designed to provide kinetic, non
invasive live cell imaging by acquiring phase
contrast images of the cells at user-defined
times and locations within the cultures. The
primary metric of the instrument is culture
confluence, that is, the fraction of the surface
that is covered by cells. The cells were cultivated
for 12 days with media change every 4 days.
Expansion protocol: A single cryo vial with
5 x 105 cells was thawed (cells in passage
2), and the percentage of viable cells was
established. The cells were divided and seeded
in two NunclonTM Delta treated TripleFlasks
with a final seeding density of 350 cells/cm2
with either AdvanceSTEMTM Mesenchymal Stem
Cell Basal Medium or α-MEM growth media.
The cells were cultivated for 8 days with media
change on days 3 and 7. The expanded cells
were re-seeded in a NunclonTM Delta treated
TripleFlasks at seeding density of 350 cells/cm2.
The cells were cultivated for 8 days with media
change on day 4.
Differentiation protocol: After the expansion
protocol cells were harvested with trypsin
and re-seeded in AdvanceSTEMTM or α-MEM
in 48 well multidishes at a density of 5000
cells/cm2 for differentiation into osteoblasts
and adipocytes. The cells were incubated
Figure 4.
Composite OsteoImage/Hoechst stained
hMSCs differentiating into osteoblasts.
The cells are photographed at day 18 post
induction. The hMSC were expanded in
AdvanceSTEMTM Mesenchymal Stem Cell
Basal Medium and passaged using trypsin.
Composite AdipoRed®/Hoechst stained
hMSCs differentiating into adipocytes.
The cells are photographed at day 18 post
induction. The hMSC were expanded in
AdvanceSTEMTM Mesenchymal Stem Cell
Basal Medium and passaged using trypsin.
Composite AdipoRed®/Hoechst stained
hMSCs differentiating into adipocytes.
The cells are photographed at day 18 post
induction. The hMSC were expanded
in α-MEM medium and passaged using
trypsin.
April 2012 I VWRbioMarke Issue 28 I VWR International I
21
the market source for life science
NunclonTM Delta treated TripleFlasks an effective format for the cultivation of hMSC. The growth of hMSC in α-MEM
medium and AdvanceSTEMTM Mesenchymal Stem Cell Basal Medium TripleFlasks using our in-house developed protocol was
effective in generating a large population of hMSCs for either differentiation or cryogenic storage.
for 48 hours at which time the media in the
48 well multidishes were changed to either
AdvanceSTEMTM Adipogenic or osteogenic
differentiation media with AdvanceSTEMTM
Growth Supplement. The cells were incubated
for 18 days with the media being changed
every 4 - 5 days. The cells were assayed for
differentiation using the following commercial
kits: For Osteogenic differentiation, the
OsteoImage PA-1501 kit was used. The kit
measures specific staining of the hydroxyapatite
portion of the bone like nodules deposited
by cells. For adipogenic differentation, the
AdipoRed® PT-7009 was used. The kit utilises
Nile Red to dye the intracellular lipid droplets
formed inside the differentiating adipocytes.
The differentiation cultures were assayed on
days 3, 7 and 18.
Results
The lowest seeding concentration displaying
good exponential growth was 350 cells/cm2.
In consequence a seeding density of 350 cells/
cm2) were chosen for the expansion protocol. In
our experiment we expanded the original hMSC
population. Differentiation into adipocytes and
osteoblasts were possible with cells expanded
in HyClone AdvanceSTEMTM Mesenchymal
Stem Cell Basal Medium, but only osteogenic
differentiation, at a lower yield, was possible
with cells expanded in α-MEM medium.
Conclusions
•
NunclonTM Delta treated surfaces including
TripleFlasks are an effective format for the easy
and rapid expansion of hMSC
•
Expansion of hMSC on NunclonTM Delta using
AdvanceSTEMTM Mesenchymal Stem Cell Basal
Medium does not compromise the osteogenic
and adipogenic potential of the hMSCs
•A
substantially higher yield of osteoblast
is achieved when cells are expanded in
AdvanceSTEMTM Mesenchymal Stem Cell Basal
Medium compared to α-MEM medium
References:
1. Koelling S, Miosge N. Stem cell therapy for cartilage
regeneration in osteoarthritis. Expert Opin Biol Ther. 2009
Nov;9(11):1399-405.
2. Aggarwal S, Pittenger MF. Human mesenchymal stem cells
modulate allogeneic immune cell responses. Blood. 2005
Feb 15;105(4):1815-22. Epub 2004 Oct 19.
3. Baldazzi F, Ripa RS, Jørgensen E et al. Release of biomarkers of myocardial damage after direct intramyocardial
injection of gene and stem cells via the percutaneous
transluminal route. Eur Heart J 2008;29:1819-26.
4. Sotiropoulou PA, Perez SA, Salagianni M, Baxevanis
CN, Papamichail M. Characterization of the optimal
culture conditions for clinical scale production of human
mesenchymal stem cells. Stem Cells. 2006 Feb;
24(2):462-71.
5. Reger RL, Wolfe MR. Freezing harvested hMSCs and
recovery of hMSCs from frozen vials for subsequent
expansion, analysis, and experimentation. Methods Mol
Biol. 2008;449:109-16.
www.thermoscientific.com
© 2011 Thermo Fisher Scientific Inc. All rights reserved. IncuCyte is a registered trademark of Essen Instruments; and OsteoImage and AdipoRed are registered trademarks of
Lonza Walkersville, Inc. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.
22
I VWR International I VWRbioMarke Issue 28 I April 2012
Cell biology
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
Simple solutions for biospecimen management
The development of large scale
screening abilities for genomics,
proteomics, kinomes, lipidomes,
secretomes, and transcriptomes
has led to an increase in the value
of large and small biobanks or
biorepositories. Biobanks collect,
organise, store and distribute
biological and environmental
specimens, such as cells, DNA,
RNA, proteins, blood, tissues and
urine from humans, animals or
plants or dirt, dust and air from
the environment for clinical,
research and taxonomical
purposes. Standardisation of
biobanking best practices and
regulations are ongoing, leading
to the development of minimum
guidelines to ensure biospecimen
reliability. For a biobank to
be useful to researchers,
large numbers of high quality
samples with related associated
information are required.
These samples must be reliable
and reproducible. As a result,
cryopreservation of the samples is
essential. Cryostorage is best for
many sample types, as it allows
storage of the samples below the
temperature at which all biological
activity occurs (-132 °C). To do this,
samples are stored in the vapour
phase of liquid nitrogen (-150 °C).
The challenge
While cryopreservation is best for sample
storage, it leads to the development
of specific issues. Samples need to be
stored in sealed, sterile containers, with
appropriate labelling to allow sample
retrieval and tracking. The harsh condition
of storage in the vapour phase of liquid
nitrogen has led to the development of
novel solutions - permanently labelled
cryo tubes and ampoules. However,
these labelling solutions also need to be
married to the requirement for linking to
a large amount of information related to
sample handling and identification. As a
result, barcoding of samples has become
an industry standard. Additionally, the
increase in sample volume numbers leads
to a requirement for better tracking of
samples. Barcoding of samples allows for
the use of barcode readers for sample
tracking and traceability.
Sample labelling
There are many challenges and difficulties
related to cryogenic storage. One of the
most preventable ways in which vital
samples get lost is poor labelling and poor
tracking of vials. If vials are not marked
with a durable, long-term labelling system,
the label can become unreadable or worse
come off, and the sample is lost. The
loss of vital samples not only affects the
sample itself, but adds to the long-term
cost of the biobank. Samples without
labels occupy valuable storage space,
increasing the overhead cost with no
return on value. April 2012 I VWRbioMarke Issue 28 I VWR International I
23
the market source for life science
Multiple different methods are used for
marking cryogenic vials, from marking pens
to adhesive labels to digital barcoding. Let’s
look at the pros and cons of each method.
Marking pens,
€ 0,01 – 0,05 per vial
Pros - Manual, inexpensive, readily available
Cons - Time-consuming, unreliable, limited
space, error rate of 1 in 200
Summary - Useful for temporary storage
of midstream processing of samples
Adhesive labels,
€ 0,035 – 0,30 per vial
Pros - Manual, ease of pre-labelling,
versatile
Cons - Time-consuming, unreliable, limited
space, special labels required, error rate of 1
in 200, required to match label to vial
Summary - Useful for short-term storage
of samples
Digital barcoding,
€ 0,12 – 0,50 per vial
Pros - Automated, uniformity, reliable,
permanent, unique, UV and cryo-resistant,
high data density, integration with LIMS/
easier to track, customisable/flexible
Cons - Upfront cost, upfront design, 1-D error rate of 1 in 2 million, 2-D - error rate
of 1 in 10 million
Summary - Useful for long-term samples,
correlating clinical, demographic and
laboratory data
1-D barcodes
These are familiar to anyone who has ever
bought anything in a retail store. Look at
the UPC code on nearly any product and
you have an example of a 1-D barcode. 1-D
barcodes have low information density and
capacity and are only able to relay numbers
and English letters. Basically, they are
useful for including an object label and the
corresponding index to match the label to
(i.e., this is sample 1249 in database A333).
24
2-D barcodes
These allow for the inclusion of high
amounts of information in a small
overall area. The data encoded can be
numbers, English and Chinese characters,
pictures, voice, and any other binary
information. Basically, they are useful
for not only including an object label
and the corresponding index to match
to the label as above, but also include
descriptive information on the sample itself.
Furthermore, due to the large capacity, data
can be duplicated leading to redundancy
and increased reliability.
Barcode scanning
The use of barcodes allows for the use
of scanners for sample vial tracking and
traceability. Currently, single, multi and
cordless scanners that link to various LIMS
and software systems are available. The
ability to scan a vial, or an entire open
bottom cryostorage box, decreases both
time and technician data entry error.
Furthermore, the ease of scanning at
multiple steps during sample processing
increases the amount of processing
information that can be stored with a
sample for later referral. Additionally, the
ability to link this information to individual
sample demographics (be it individual
demographics, therapeutic markers,
drug discovery isolates, microbiological/
virological mutants, etc.) in external
databases allows for greater ownership of
sample processing and greater potential
for downstream end use value.
The benefit of linking to external
databases is that these databases can be
expanded as required in the future.
While markers and adhesives may be
inexpensive, when it comes to reliably
labelling samples, digital barcoding
offers realistic abilities without breaking
the bank.
I VWR International I VWRbioMarke Issue 28 I April 2012
Key points:
1.Manual sample identification increases
error 10 000-fold
2.Digital barcoding is an affordable solution
3.Good sample tracking is essential
Sample tracking
and traceability
are necessities for
all biobanking
resources, large
and small
Samples need to be tracked from
the site of collection to receipt at the
repository. The first step is setting up a
reliable tracking system with uniform and
permanent labelling. Furthermore, even
established labs have “biobanks”. These
can include virology and microbiology
labs, immunology, cancer and rare disease
research labs, drug discovery labs, etc.
Even in these sometimes smaller labs,
it is essential to have proper, reliable
sample tracking and retrievability.
Tracing or tracking of sample processing
and storage, including environmental
conditions, thaw cycles, distribution and
loss, all must be maintained and easily
linked to the samples as well.
During sample collection or processing, it
is essential to ensure that the samples are
labelled appropriately. First, the material
used for labelling the samples should
be resistant to all common laboratory
solvents and conditions. This includes
ethanol, methanol, phenol, chloroform,
and acetone, as well as extreme heat and
cold. Additionally, the information must
reliably match the database allowing
Cell biology
proper identification of the sample and
correlation with collected information.
Prior to collecting and storing samples,
a trial run should be conducted with the
chosen labelling system. The container
and label should be able to survive
all permutations of conditions during
transport, processing and storage.
Each sample requires a unique ID that
links it to the data collected for that
sample. Once this unique ID is generated,
it should be linked to the sample and the
data at all points of processing, sample
distribution, and data dissemination. This
unique ID should be generated at the
time of sample collection, and should be
attached to the sample and the collection
sheet.
Inventory
For each sample, there should be a
minimum amount of data collected and
maintained. This information should be
maintained in an inventory database,
which includes the following information
and has the following characteristics:
Tracking of location and status of samples:
•Tracking of events (freeze/thaw,
processing, loss, destruction, distribution)
•Unique ID allows linkage to additional
data sets for individual sample
demographics (patient ID, study ID,
collection ID, etc.)
•Audit of all changes
•Secure system
•Location of sample in freezer/Dewar,
location in unit, unit#, sample box,
rack, etc.
Best practice – include the ability to link to
other database for additional information,
ensuring that all electronic information is
readily shareable.
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
information and has the following
characteristics:
•Material (specimen type, vial type,
volume, date of collection, date of
receipt, date of processing, processing
method, storage temperature, etc.)
•Subject information (ID, diagnosis,
procedure and date, type of treatment,
medical history, family history, smoking
history, vitals, clinical lab values,
availability of other biological specimens)
•Linked to digitally scanned documents
(clinical lab reports, pathology reports,
questionnaires, consent forms, MTAs)
•Audit of all changes
•Secure (following HIPAA or other
regulatory laws)
• Built-in system for updating/adding
information if required
Best practice - store basic, essential
information in main database, with link
to external database for study specific
information.
As a result, labelling and tracking of all
biospecimens needs to be correlated with
all relevant sample information. Basically,
if it happens to the sample, it should be
recorded in the database.
Key points:
1.ISBER guidelines - http://www.isber.org/
bp/BestPractices2008.pdf
2.NCI guidelines - http://biospecimens.
cancer.gov/global/pdfs/NCI_Best_
Practices_060507.pdf
Conclusion
The banking of biospecimens is the
foundation for future research and
development. Investing in a system
for proper tracking, tracing and data
management ensures the future of your
life’s work. Wheaton is here to partner you
on your investment, because it’s our life’s
work too.
Database
For each sample, there should also be a
minimum amount of demographic data
collected and maintained. This information
should be maintained in a linked external
database, which includes the following
April 2012 I VWRbioMarke Issue 28 I VWR International I
25
the market source for life science
The use of Spectra/Por® Regenerated
Cellulose membrane tubing
By Peter Haggie, Ph.D.
Post-Doctoral Fellow University of California,
San Francisco USA
At one time or another most scientists working
in ”biochemical” sciences will have to dialyse
something. Dialysis is a simple technique to exchange
a solute’s solution or to separate/purify differently
sized molecules. Macromolecules are dialysed by
placing them in size-selective permeable tubing and
subsequently equilibrating the sample with large
volumes of new buffer: efficient dialysis relies upon
appropriate selection of dialysis tubing and effective
“washing” that results from large volumes, multiple
changes and full equilibration with the new buffer.
I have recently been generating many fluorescent
analogues of proteins and have used dialysis to
remove ammonium sulphate from protein suspensions
prior to labelling and to remove unincorporated
succinimidyl esters of various fluorophores after
labelling. For these steps I have been using Spectra/
Por® dialysis tubing from Spectrum® Laboratories, Inc.
Spectra/Por® 1 - 7 dialysis tubing is made of
Regenerated Cellulose (RC). Cellulose has long been
used for dialysis as it is uncharged and does not readily
absorb solutes. Furhermore, the selectivity of cellulose
membranes is not altered greatly by many chemicals
or reasonable pH and temperature ranges. Processed
cellulose has crystalline regions and these regions
26
I VWR International I VWRbioMarke Issue 28 I April 2012
cross-link chains to introduce structural integrity to
the cellulose. Depending upon how the cellulose is
processed the number of crystalline areas varies and
the resultant regions between the cross-links can act
like size-selective pores.
Many Spectra/Por® Regenerated Cellulose (RC) basic
membranes (Spectra/Por® 1, 2, 3, 4 and 5) come
in 3,5, 6 - 8 and 12 - 14 kDa MWCO’s and more
expensive RC membranes (Spectra/Por® 6 and 7)
have a range of MWCO’s from 1 - 50 kDa. MWCO
values are established by Spectrum® and represent
the size at which a solute is 90% retained during a
test period. The small pore sizes available mean that
relatively small molecules can easily be processed
and I have used dialysis to buffer exchange samples
of ~10 kDa with only minimal sample loss. Certain
parameters such as pH may however alter MWCO’s
and so appropriate tubing needs to be established for
a particular situation.
That said, with my application using simple buffers
the MWCO’s have proven to be robust. Spectrum®
suggest that efficient separation can be achieved if the
size ratio of the moieties is at least 25, this ratio is easy
to achieve for buffer exchange of a protein or removal
PROTEOMICS
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
of unincorporated fluorescent dyes when labelling
macromolecules. Equilibration of the membranes is
relatively quick, especially for the larger pore sizes that
I typically use, for instance, after labelling I can detect
unincorporated dye molecules in my equilibration
buffer within 2 - 4 hours during the first wash. This
equilibration time means that 4 buffer changes can
easily be performed in 24 hours.
Spectra/Por® is supplied in an essentially readyto-go format, no boiling in bicarbonate buffer is
needed. Spectra/Por® 1 - 6 merely need soaking in
distilled water for 30 minutes whereas other tubing
is supplied pre-hydrated and must be stored at 4
°C. One concern is that these membranes contain
trace amounts of heavy metals and sulphur although
Spectrum® do also produce specific cleaning solutions
and supply Spectra/Por® 7 which comes with minimal
contaminants.
Membranes can be sterilised and also have two year
shelf lives. Finally, Spectra/Por® tubing is supplied in
different widths depending upon the scale of dialysis
you plan.
For me dialysis essentially provides a ”low
maintenance” way to perform buffer exchange or
purify a sample if there is a large size differential.
Other methods do exist to perform the same task
including ultra-filtration units that are faster and
extremely convenient. Dialysis is however, much
more cost effective and so if I am not in a hurry for a
sample I tend to use it. Further, dialysis does not rely
on concentration of a sample and so may be more
”gentle” for your sample.
The Spectra/Por® range of tubing definitely proves
reliable, cost effective and widely applicable.
New!
TRIAL KIT with shorter tubing length
Spectra/Por® Regenerated Cellulose (RC) tubing 1, 2 and 3
are now also available in 5 m lengths instead of 15 or 30 m,
including a pair of tubing closures! Ideal for small volume
dialysis or evaluation of membranes for an application.
Take a look at the
VWR bioMarke Shop for
this introductory offer!
REVIEW
Products
The good
The bad
The bottom line
Spectra/Por® 1, 2, 3, 4, 5, 6, and 7 Dialysis Tubing from Spectrum® Laboratories, Inc.
Extensive range of tubing to cover all your dialysis needs. Almost ready-to-use.
MWCO’s are typically reliable.
Cost effective.
Contamination from sulphur or heavy metals (except for Spectra/Por® 7) may be an issue, although
this is accounted for with cleaning solutions and tubing that has low levels of contamination.
Simply reliable dialysis tubing, comes in a wide range of MWCO’s (making it widely applicable) and it
is a simple technique.
April 2012 I VWRbioMarke Issue 28 I VWR International I
27
the market source for life science
The Rapid Translation System (RTS) – a cell-free protein
biosynthesis platform for simplified protein research
Cell-free protein synthesis
accelerates and simplifies
protein expression in
comparison to traditional
cell-based methods, showing
clear advantages in speed,
throughput and flexibility. The
scalable system with unique
features meets the demands
of both screening as well as
preparative protein synthesis
with yields up to 50 mg.
Supply of sufficient amounts of pure
protein can be a major bottleneck for
structural and functional protein analyses. The Rapid Translation System combines a unique
set of innovative technologies for efficient and optimised protein expression.
RTS provides:
1.Coupled transcription-translation reaction
2.Continuous Exchange Cell-Free (CECF) technology
3.Advanced lysate and buffer technology for high yields
4.Flexible PCR-based system for the rapid generation of linear templates for direct protein
expression without the need for time-consuming cloning procedures
5.Tools for incorporation of labelled amino acids and improving functionality (e.g.
optimised folding, formation of disulphide bonds, etc.)
6.Modular and scalable workflow
Coupled transcription/translation reaction
RTS lysates contain the whole machinery to drive coupled transcription and translation in
one reaction vessel in the presence of a given DNA template. This allows for a streamlined
process as mRNA has not to be generated separately (Figure 1).
Figure 1. Coupled transcription and translation
Besides the gene of interest, a T7 promotor (T7P), a ribosomal binding
site (RBS), a start codon (ATG) and a T7 terminator (T7T) are prerequisite
as proper template.
Figure 2. Schematic illustration of the CECF reaction principle
1aTemplates, that are suitable for use in the RTS E. coli and RTS Wheat
Germ systems.
1bTemplates that are suitable for use in the RTS Wheat Germ system.
2 The semi-permeable membrane of the RTS reaction device allows
the continuous exchange of substrates and energy components, and
the dilution of inhibitory by-products.
Continuous Exchange Cell-Free (CECF) technology
In traditional batch mode expression, a plateau is reached after approximately 2 - 4 hours
as substrates become exhausted and inhibitory reaction by-products accumulate. The
unique RTS CECF reaction devices extend the duration of expression, maximising protein
yield. The enzymatic machinery required for coupled transcription and translation is
separated in the reaction chamber of the CECF device (Figure 2).
The semi-permeable membrane between reaction and feeding chamber facilitates
continuous supply of substrates and removal of inhibitory by-products. This allows cell-free
protein synthesis reactions to continue for up to 24 hours, significantly increasing the yield
(Figure 3).
CECF devices are available in three sizes for 50 µl, 1 ml and 10 ml reactions allowing for
protein yields from 20 µg to 50 mg per reaction.
28
I VWR International I VWRbioMarke Issue 28 I April 2012
PROTEOMICS
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
Advanced buffer and lysate biochemistry for high yields
RTS E. coli HY Kits allow for protein expression yields of up to 400 μg per ml reaction in
a 2 hour batch reaction (RTS 100 E. coli HY) and up to 6 mg per ml reaction in the CECF
formats (1 ml or 10 ml). This has been achieved by extensively optimising substrates, the
energy regenerating system and factors required for regulation of the coupled reactions.
Figure 3. Yield comparison for batch and
CECF technology. Comparison of protein yields
obtained with the RTS 100 E. coli HY and RTS
500 ProteoMaster E. coli HY Kits after 1, 2, 4, 6,
8, and 24 hours. GFP (Green Fluorescent Protein,
27 kDa) expression was performed in 50 μl batch
reactions (RTS 100 E. coli HY Kit) or 1 ml CECF
reaction devices (RTS 500 ProteoMaster E. coli HY
Kit). With the RTS 500 ProteoMaster E. coli HY
Kit, incorporating CECF and HY technologies, the
reaction continues for up to 24 hours and yield
reaches up to 6 mg/ml; the batch reaction plateaus
after only 4 hours, with yields of up to 0,4 mg/ml.
Protein engineering without cloning
The use of either linear or circular DNA templates is possible in RTS 100 E. coli HY and RTS
100 Wheat Germ reactions. However, direct expression from linear templates, generated
by PCR, gives access to substantial amounts of protein without cloning and cell culturing, as
a result making the RTS 100 platform the ideal screening and optimisation platform. Rapid
engineering of proteins by established PCR methods offer the flexibility to easily design
proteins that have the desired functionality and express at high yields. RTS Linear Template
Generation sets are designed to use two consecutive PCR reactions to generate linear
templates containing all necessary regulatory elements.
Open system for easy adaptation
Reaction conditions can be easily adapted in a protein specific manner by adding chemicals
and factors to the reaction mixture (e.g. adding chaperones for increased solubility and correct
folding). Also incorporation of labelled amino acids into the protein of interest is possible.
Modular and scalable workflow
Figure 4. Expression functional
disulphide‑bonded protein
Urokinase, a proteolytically active protein with
six disulphide bonds was expressed using the RTS
500 E. coli system. After 24 hours of expression
1 µl samples of the reactions were analysed in
Western blot with anti-His6 antibody. Expression
with the RTS 500 E. coli Disulfide Kit resulted
in soluble urokinase protein in the supernatant
(lane 1) indicative of correct folding, whereas
no protein was found in the pellet (lane 2). In
contrast, expression with the standard RTS 500
ProteoMaster E. coli HY kit yielded no soluble
protein in the supernatant (lane 3) but only
incorrectly folded protein in the pellet (lane 4).
M Molecular weight marker
Description
RTS 100 E. coli LinTempGenSet, His6-tag RTS pIVEX His6-tag, 2nd Gen. Vector set
RTS DnaK supplement
RTS GroE supplement
RTS 100 E. coli HY Kit (24 react.)
RTS 100 E. coli HY Kit (96 react.)
RTS 100 E. coli Disulfide Kit
RTS 500 ProteoMaster E. coli HY Kit
RTS 500 E. coli HY Kit (5 react.)
The RTS product family offers three lysate systems and three reaction sizes to meet the
different requirements defined by target proteins and downstream applications.
RTS E. coli HY Kits enable expression of a wide range of proteins. Linear template
generation kits and optional supplements allow for rapid screening and optimisation at
small scale and subsequent scale-up.
RTS E. coli Disulfide Kits employ activated lysates and CECF technology to enable
milligram expression of functional disulphide bonded protein e.g., antibody fragments
(Figure 4).
Using a eukaryotic lysate, RTS Wheat Germ Kits have been shown to express eukaryotic
proteins up to 220 kDa in milligram yields without optimisation.
RTS 100 Kits are ideal for optimisation and screening and can be easily adapted for high
throughput applications, such as protein array production.
RTS 500 Kits provide sufficient protein for many applications and enable efficient labelling
e.g., for structural analysis.
The RTS 9000 E. coli HY Kit can yield up to 50 mg of protein e.g., for structural analyses.
With the Rapid Translation System one can yield substantially more functional protein
than with classical cell-free expression systems not employing the CECF technology. Even
disulphide bonded proteins and large eukaryotic proteins can be expressed successfully.
This makes RTS an attractive and convenient alternative to in vivo expression, whenever
pure proteins are needed for functional or structural analysis.
Cat.No.
PRME2401000
PRME2401010
PRME2401020
PRME2401030
PRME2401100
PRME2401110
PRME2401120
PRME2401500
PRME2401510
Description
RTS 500 E. coli Disulfide Kit
RTS Amino Acid Sampler
RTS 500 Adapter
RTS 9000 E. coli HY Kit (10 ml) RTS Wheat Germ LinTempGenSet, His6-tag
RTS pIVEX Wheat Germ His6-tag Vector set
RTS 100 Wheat Germ CECF kit
RTS 500 Wheat Germ CECF kit
Cat.No.
PRME2401520
PRME2401530
PRME2401540
PRME2401900
PRME2402000
PRME2402010
PRME2402100
PRME2402500
April 2012 I VWRbioMarke Issue 28 I VWR International I
29
the market source for life science
High throughput screening for antibodies
and histidine tagged proteins using Mag
Sepharosetm magnetic beads
G. Risberg and N. Norrman
GE Healthcare Bio-Sciences AB, Uppsala, Sweden
Mag SepharoseTM
magnetic beads have
been developed to
simplify handling
in protein sample
preparation.
The beads are an
excellent choice for
enrichment, small
scale purification,
and screening of
target proteins. In
this study, automated
screening methods
for purification of
monoclonal antibodies
and histidine tagged
proteins are described.
A combination of Mag
SepharoseTM magnetic
beads in a 96 well
microplate, MagnaBotTM
96 Magnetic Separation
Device and Tecan
Freedom EVOTM liquid
handling workstation
was used.
deviation for 96 samples was only 3.0% when
omitting the 12 samples from wells H 1-12.
The Mag SepharoseTM platform combines
well established enrichment and purification
methods with the magnetic bead format to
provide high quality and reproducible results.
The magnetic beads are scalable and provide
simple capture of target protein in small or
large sample volumes, from low microlitre to
high millilitre scale. Mag SepharoseTM magnetic
beads are suitable for enrichment, small scale
purification and screening of target proteins
such as in screening for optimal purification
conditions.
Flexible purification
Reproducible purification
Combining Tecan Freedom EVOTM, MagnaBotTM
96 Magnetic Separation Device, and Protein
G Mag SepharoseTM Xtra ensures high
reproducibility. To demonstrate this, 96
replicate runs on a 96 well microplate filled
with Protein G Mag SepharoseTM Xtra beads
were performed to purify a monoclonal human
IgG expressed in CHO cells. The load was 60%
of the total binding capacity and the yield
of the eluted fractions was determined via
absorbance measurements.
Figure 1 shows the yield of the monoclonal
human IgG. The results show good well-towell reproducibility with low relative standard
deviation (RSD) of 4,6%. All 12 samples from
wells H 1 – 12 in the 96 well microplate had
a lower amount of eluted monoclonal human
IgG. This reduced amount of eluted human
IgG indicates a systematic error but no further
investigation was made. The relative standard
Adapting the purification scale to different
sample volumes is one advantage with the
magnetic bead format. In this study, different
volumes of magnetic beads were scaled down
from 5 µl magnetic beads to 1 µl. Human IgG
was purified using Tecan Freedom EVOTM,
MagnaBotTM 96 Magnetic Separation Device,
and Protein G Mag SepharoseTM Xtra filled in
96 well plates. The yield and recovery of the
eluted fractions was determined by absorbance
measurements. The purification showed good
well-to-well variation with relative standard
deviations (Figure 2).
Screening for optimal IMAC
purification conditions
Purification of histidine tagged proteins
by immobilised metal ion adsorption
chromatography (IMAC) is a balance between
yield and purity, modulated by the imidazole
concentration in the sample and binding/wash
buffer. The optimal imidazole concentration is
protein dependent and can be determined for
each histidine tagged protein.
A screening study for optimal sample loading
and wash conditions was performed with
eight different imidazole concentrations and
four different sample loads, varying from
25 to 100% of the total binding capacity of
40
RSD
4.6%
35
Amount (µg)
30
25
20
15
10
5
0
1
A 1-12
B 1-12
C 1-12
D 1-12
E 1-12
F 1-12
G 1-12
Well number
Figure 1. Elution reproducibility over 96 runs of monoclonal human IgG purified
from CHO cells using Protein G Mag Sepharose Xtra magnetic beads.
30
I VWR International I VWRbioMarke Issue 28 I April 2012
H 1-12
96
PROTEOMICS
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
80
5 µl Protein G Mag SepharoseTM Xtra
medium (RSD = 3,7%)
4 µl medium (RSD = 3,5%)
His Mag Sepharose Ni. Histidine tagged green
fluorescent protein, GFP-(His)6, was purified
from Escherichia coli lysate using Tecan Freedom
EVOTM, MagnaBotTM 96 Magnetic Separation
Device, and His Mag SepharoseTM Ni filled in
96 well plates. The yield and purity of the
eluted fractions was determined by absorbance
measurements and SDS-PAGE analysis,
respectively.
Eluted monoclonal human IgG (µg)
3 µl medium (RSD = 2,0%)
60
2 µl medium (RSD = 1,7%)
1 µl medium (RSD = 3,4%)
40
20
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16
Sample number
Figure 2. Human IgG was loaded with 50% of the total
binding capacity for each magnetic bead volume. The yield and
recovery of the eluted fractions was determined by absorbance
measurements. The results showed good well-to-well variation
with low relative standard deviations.
The results showed that during sample
application and wash, a good balance between
yield and purity was obtained with 40 mM
imidazole (Figure 3) and a sample load of 50 to
100% of the total binding capacity (Figure 4).
100
Yield
Purity
Mag SepharoseTM magnetic beads filled in
96 well plates and combined with robotics
provide an excellent platform for screening
of protein constructs and conditions that can
be tested in drug development and structural
studies.
Using screening methodology with Mag
SepharoseTM beads combined with robotics
enables highly reproducible results, as well as
a fast and cost effective screening with limited
effort.
For more information on the Mag SepharoseTM
platform, visit www.gelifesciences.com/
sampleprep
To see an animation demonstrating the practical
use of His Mag SepharoseTM Ni beads, visit http://
www.gelifesciences.com/aptrix/upp01077.nsf/
content/sample_ preparation~news~mag_sepha
rose?OpenDocument&intcmp=ibc000005
Purity and relative yield (%)
Conclusions
80
60
40
20
0
0
20
40
60
Conc. imidazole (mM)
80
100
Figure 3. Purity and relative yield of GFP- (His) 6 with a 50%
sample load relative to the total binding capacity of His Mag
Sepharose Ni.
Increasing imidazole,
0-100 mM
Mr × 10
25% sample load
50% sample load
75% sample load
100% sample load
3
97
66
GST-GFP- (His) 6
45
30
20.1
14.4
M SM FT
Description
Protein G Mag SepharoseTM Xtra
Protein G Mag SepharoseTM Xtra
His Mag SepharoseTM Ni
His Mag SepharoseTM Ni
His Mag SepharoseTM Ni
MagRackTM 6
Pk (ml)
2x 1
5x 1
2x 1
5x 1
10x 1
1
Cat. No.
28-9670-66
28-9670-70
28-9673-88
28-9673-90
28-9799-17
28-9489-64
M
Figure 4. SDS-PAGE of GFP-(His) 6 enriched from a background
of E. coli protein using His Mag SepharoseTM Ni beads. GFP-(His) 6
was detected using Deep Purple™ Total Protein Stain and Ettan™
DIGE Imager. The gel image was analysed with ImageQuant™ TL
software. M = molecular weight markers, SM = start material,
FT = flowthrough from a sample load of 75% of the total binding
capacity, 40 mM imidazole.
April 2012 I VWRbioMarke Issue 28 I VWR International I
31
the market source for life science
Thermo Scientific
Human SRM
ATLAS Peptide
Library
Since the middle of the last decade, quantitative
proteomics has gained more and more interest, as
researchers want to know how much of given protein
or peptide is present in serum, urine or other samples.
Quantitative proteomics data support a better
understanding of dose/effect relationships and of
interactions of proteins with each other in particular
and finally of the entire human proteome in general.
32
I VWR International I VWRbioMarke Issue 28 I April 2012
PROTEOMICS
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
Quantitative Mass Spectrometry (MS) workflows involve
three steps:
(a) In-silico selection of unique peptide identifier of the protein of interest (so called
proteotypic peptides) with adequate mass spectrometry properties
(b)Selection of the proteotypic peptides checking ionisation and fragmentation
properties, e.g. optimising single reaction monitoring (SRM) and multiple reaction
monitoring (MRM) assays on appropriate MS instrumentation
(c) Synthesis of the best proteotypic peptides as isotopically labelled peptides and use
of those as internal standards (HeavyPeptide AQUA) for absolute quantitation
Due to its absolute specificity, sensitivity3
and high multiplexing potential4 the well
established method of isotopic dilution
has been quickly adopted for peptide
quantitation5,6,7 and absolute protein
quantitation8,9 using mass spectrometry in
complex matrices.
The increasing interest in quantitative
proteomics was documented at the 2010
Annual HUPO meeting in Sydney, where
scientists from the Institute for Systems
Biology (ISB) and the Swiss Federal Institute
of Technology announced that they have
completed an initial mass spectrometry-based
map of the entire human proteome. Thermo
Fisher Scientific supplied peptides used in
this work.2
The Thermo Scientific Human SRM ATLAS
Peptide Library* will support the efforts of
the scientific community to generate targeted
proteomics assays using mass spectrometry
to quantify almost every protein of the entire
human proteome, including proteins present
in subproteomes with individual pathways of
interest.
The use of this peptide library offers the
advantage of eliminating the upfront insilico selection of proteotypic peptides
and therefore will contribute to an overall
reduction of development time of SRM assays.
Consequently is can be expected that targeted
quantitative assays for the complete human
proteome will be established faster.
The highlights of the Thermo Scientific Human SRM ATLAS
Peptide Library are:
• 93 140 unique proteotypic peptides
• 24 524 unique access numbers (UniProtKB/SWISS-PROT®)
• More than 90% coverage of the human proteome
The purification of the individual peptides is not
included; peptide quantity >10 µg for 97% of
the peptides (based on weighing). The complete
library consists of more than 1000 plates in 96
well formats. Resynthesis of peptides failing
to work in SRM assay development is possible
at discounted PEPotec™ SRM prices. There is
a limited availability of the Thermo Scientific
Human SRM ATLAS Peptide Library, which will
be sold as supply lasts.
Potential application areas for the Thermo
Scientific Human SRM ATLAS Peptide Library
are the fields of biomarker discovery, verification
and validation, protein expression monitoring,
pathway validation and cell signal profiling,
as well as general functional quantitative
proteomics and systems biology.
Further reading
1 www.mrmatlas.org
2 Adam Bonislawski, ISB, ETH Zürich
complete initial draft of SRMAtlas
covering entire human proteome.
Proteomonitor, p.2, September 24, 2010
3 Anderson et al., The human plasma
proteome: History, character, and
diagnostic prospects. Mol Cell
Proteomics 1: 845-67 (2002)
4 Junichi Kamiie et al., Quantitative atlas
of membrane transporter proteins:
Development and application of… Pharm
Res. 2008 June; 25(6):1469-83 (2008)
5 Tuthill, C. W et al., Quantitative analysis
of thymosin alpha1 in human serum by
lc-ms/ms. AAPS PharmSciTech 1, E11
(2000)
6 Desiderio, D. M. Et al., Highperformance liquid chromatographic
and field desorption mass spectrometric
measurement of picomole amounts… J
Chromatogr 217, 437-52 (1981)
7 Cristian G. Arsene et al., Protein
quantification by isotope dilution mass
spectrometry of proteolytic fragments:
cleavage rate and accuracy. Anal Chem.
Jun 1,80 (11): 4154-60 (2008)
8 Barr, J. R., Maggio., Isotope dilutionmass spectrometric quantification of
specific proteins: Model application with
apolipoprotein a-i. Clin Chem 42: 167682 (1996)
9 Gerber, S. A., Gygi et al., Absolute
quantification of proteins and
phosphoproteins from cell lysates by
tandem MS. PNAS 100 (12): 6940-5
(2003)
© 2011 Thermo Fisher Scientific Inc.
All rights reserved. SWISS-PROT is
a trademark of Institut Suisse De
Bioinformatique (SIB) Foundation
Switzerland. AQUA is a trademark
of Harvard Medical School. All other
trademarks are the property of Thermo
Fisher Scientific Inc. and its subsidiaries.
Specifications, terms and pricing are
subject to change. Not all products are
available in all countries. Please consult
your local sales representative for details.
For more information, including delivery times,
further specifications and price, please contact
your local sales representative or visit and
register at www.thermoscientific.com/SRMATLAS
(*) This library was developed at the Institute for System
Biology (ISB)1, Seattle, WA, USA. Approx. 90% of the
peptides are reported to be usable in highly multiplexed
SRM development assays (personal communication, Rob
Moritz, Institute for System Biology; information at time of
submission of this article).
April 2012 I VWRbioMarke Issue 28 I VWR International I
33
the market source for life science
Pall Life Sciences new PRC and LRC
chromatography columns
Pall Life Sciences have
recently introduced Pall
PRC chromatography
columns into their range
of chromatography tools.
PRC chromatography
columns have been
developed for fast
selectivity screening
of “gel-in-a-shell”
ion exchangers and
new mixed-mode
chromatography sorbents.
Convenient – ready-to-use 1 and 5 ml pre-packed columns
Easy-to-use – direct connection to commonly used laboratory chromatography systems
Efficient – high packing efficiency (≥2500 plates/m)
Consistent – screen Pall ion exchange and mixed-mode chromatography sorbents
under reliable and reproducible conditions, plus guaranteed performance
run after run
Functionalities – Ion exchange: Q and STM; Q and CM Ceramic HyperD® F sorbents
– Mixed-mode: MEP, HEA and PPA HyperCelTM sorbents
Rapid screening and condition optimisation in
the 1 ml PRC column enables the rapid selection
of the appropriate chromatography chemistry.
Once the optimisd chemistry has been selected
on a 1 ml PRC column, the conditions of use can
be optimised in a 5 ml PRC column by simply
doubling the height.
Two or more columns can be connected in series
to increase the column height and therefore
closely model real conditions in pilot scale or for
scale down applications.
Further scale up can be achieved with minimal
re-optimisation by packing new Pall LRC empty
glass columns.
a screw-lock system to allow a rapid adjustment
of the plunger. Each column is supplied
assembled and ready for use, with tubing and
fittings to connect the column to a standard
system.
Robust design
•Robust inlet and outlet connections made at
the exterior of the column provide a more
reliable and visible connection
•Linear motion of plunger avoids torsional load
on the packed bed and assures true linear
compression
•True frits pressed into the plunger assure even
flow distribution across the bed
Pall Life Sciences LRC chromatography columns
have been designed to meet the requirements of
most laboratory applications (e.g. ion exchange,
affinity, hydrophobic interaction, mixed-mode
chromatography). Four internal diameters from
10 to 50 mm and four lengths provide a range
of column volumes up to 900 ml and bed
heights up to 750 mm. The columns are made
of a borosilicate glass tube, and are equipped
with one adjustable and one fixed plunger, and
Easy-to-use
Column: 100 μl ovalbumin (10 mg/ml), ß-lactoglobulin (10
mg/ml), cytochrome C (2,5 mg/ml) and lysozyme (5 mg/ml).
For 5 ml
Column: 500 μl of same proteins. Equilibration: 50 mM Na
acetate, pH 4,5. Elution: 50 mM Na acetate, pH 4,5 + 0,5 M
NaCl by linear gradient from 0 to 100%.
•Screw-lock system allows a rapid adjustment
of the plunger
•Compatible with any liquid chromatography
system due to standard fittings 1/4–28 ULS
• Easy open ends: Easily removable threaded
end fittings make column disassembly
effortless
• Easy adjustment of O-ring seals
Figure 1. Separation of
model proteins (Ovalbumin,
ß-lactoglobulin, Cytochrome C and
Lysozyme) on 1 ml PRC S HyperCelTM
pre-packed columns and scale up
on 5 ml column, constant residence
time of 2 minutes
Columns: Pall S HyperCelTM PRC
pre-packed columns of 1 ml
(5 mm I.D. x 50 mm) and 5 ml
(8 mm I.D. x 100 mm).
Load: For 1 ml
34
I VWR International I VWRbioMarke Issue 28 I April 2012
PROTEOMICS
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
Mustang® XT Acrodisc® ion exchange chromatography units for
laboratory use and process development
Mustang® XT Acrodisc® units, available in Q and
S chemistries, are ideal for scale down process
development work in a variety of downstream
process applications. A membrane volume of
<1 ml reduces the amount of sample required
for evaluation, and the female Luer lock inlet
and outlet simplifies connection to typical low
pressure chromatography systems. The open
pore structure eliminates diffusion limitations of
conventional macroporous sorbents, allowing
biomolecules to access all binding sites by
convective flow. In addition, the large pores
eliminate the size exclusion effect allowing full
access to the binding chemistry for very large
proteins, plasmids and viruses.
In many applications, the dynamic binding
capacity of Mustang® Q and S membrane are
equivalent to or greater than the equivalent
sorbent chemistries. Unlike sorbents, the
dynamic binding capacity is independent of flow
rate. In polishing applications, for the removal of
low levels of contaminants, this means high flow
rates (more than 10 MV/min) and low pressure
drops that can be fully exploited with minimum
membrane volume. In capture applications,
good resolution with high yields and minimal
elution volumes are achieved, due also to the
characteristics of the membrane, the uniform
flow path and the very low hold up volume to
membrane volume ratio.
Each unit contains 16 layers of Mustang®
membrane, exactly the same as larger Mustang®
units, allowing accurate scale up from process
development to pilot and process scale,
especially with regards to flow/differential
pressure and dynamic binding capacity.
Mustang® XT Acrodisc® units are disposable membrane
chromatography devices for fast, easy-to-use, scale down
Mustang® membrane unit facilitates process development work.
• Device volume of <1 ml minimises sample volume requirements
• 16 layers of Mustang® membrane allows easy scale up to Mustang®
membrane pilot and process scale capsules
•Units achieve flow rates of up to 10 membrane volumes per minute
(MV) without affecting performance
•Female Luer lock inlet and outlet simplifies use with typical low pressure
chromatography systems
• Disposable 25 mm units
•Available in Q and S chemistries
For further information and ordering details of these new Pall Life Sciences products please
contact your local VWR customer services department or e-mail labsupport@pall.com.
April 2012 I VWRbioMarke Issue 28 I VWR International I
35
the market source for life science
DNA quantification in microlitre volumes
UV photometry is a common
way to quantify nucleic acids
in a sample.
Both DNA and RNA absorb UV
light very efficiently, making
it possible to detect and
quantify the concentrations.
Typical applications for this
include, for example, the
quantification of template
prior to sequencing or PCR.
With Thermo Scientific µDrop
Plate it is possible to measure
DNA concentrations from a
few nanograms to thousands
of nanograms per microlitre.
introduction
The photometric method is based on
Lambert-Beer’s equation and it is based
on fact that the nitrogenous bases in
nucleotides have an absorption maximum
at about 260 nm.
For dsDNA 1,0 Abs at 260 nm corresponds
to a concentration of 50 μg/ml. As a result
the amount of DNA can be calculated by
using the formula:
DNA concentration (µg/ml) =
Abs 260 x 50 µg/ml
Unlike the nucleic acids, proteins have
a UV absorption maximum at 280 nm,
mostly due to the tryptophan residues.
Therefore, the Abs260/Abs280 ratio gives an
estimate of the protein contamination of
the sample. For a good quality sample,
the value should be between 1,8 and 2,0.
A value smaller than 1,8 indicates the
presence of proteins and a value higher
than 2,0 indicates probable contamination,
such as phenols.
The fixed path length of the
plate enables direct
calculation of the nucleic acid
concentrations.
Figure 1. µDrop plate and Multiskan GO
spectrophotometer. The low sample volume
measurement area with 16 measurement
locations on the left and a cuvette location on
the right part of the plate.
36
I VWR International I VWRbioMarke Issue 28 I April 2012
Another common parameter used to
describe the quality of DNA is the 260
to 230 nm ratio. It is used to estimate
chemical contamination, such as phenols,
carbohydrates or a high salt concentration.
The ideal 260/230 nm ratio is around 2.
A possible background caused by
impurities in the sample can be corrected
by a measurement made at a wavelength,
at which the absorption level for nucleic
acids and proteins is really low. The
wavelength most commonly used for
this background subtraction is 320 nm;
(Abs260-Abs320 / Abs280-Abs320). For example,
magnetic beads are now commonly used
in the nucleic acid purification process,
and when bead-based purification is
used, the 320 nm subtraction is always
recommended.
The amount of sample available for the
analysis is quite often really low, as a
result there is a need for a tool such as
the µDropTM Plate that enables these
measurements at a microlitre scale.
GenoMICS
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
Figure 2. The theoretical difference
between the detection ranges of the
cuvette and µDrop Plate.
The μDrop Plate consists of two separate
measurement locations, one for measuring
low sample volumes and the other for
cuvettes (Figure 1).
The low volume measurement area
consists of two quartz slides, the top
clear quartz slide and the bottom partially
Teflon®-coated quartz slide. The bottom
slide contains 16 sample positions,
arranged in a 2 x 8 matrix, onto which
samples can be pipetted. The cuvette
slot of the plate is used to perform
photometric measurements with standard
cuvettes.
With a µDrop Plate
and a photometer
with high precision and
a wide linear range, it
is possible to measure
DNA concentrations from
a few nanograms to
thousands of nanograms
per microlitre.
µDrop plate is compatible with Thermo
Scientific Multiskan GO microplate
spectrophotometer and Thermo Scientific
Varioskan Flash multimode reader.
Blank
Any photometric measurement device,
cuvette, microplate or µDrop Plate, always
has certain background absorption.
Therefore, blank subtraction is always
necessary when photometric quantification
of the sample concentrations is performed.
Path length
Path length of the µDrop Plate is the
distance between the quartz glass
surfaces, i.e. the length of the light beam
in the liquid sample.
Absorbance always depends on the path
length and when pathlenght is shorter, it is
possible to measure bigger concentrations.
Therefore it is possible to measure higher
concentrations with the µDrop plate than
with e.g. a 10 mm cuvette.
Detection range
With a µDrop Plate type of a measurement
device, the maximum measurement range
is always determined by the instrument.
The lower part is determined by the
precision of the blank (LOD) and the upper
part by the linear range of the instrument.
A shorter path length also increases the
requirements of the photometer used
as it reduces the measured absorbance.
For photometers the upper part of the
absorbance area (high concentrations) is
especially difficult, because less and less
photons reach the detector.
Both the theoretical minimum and
maximum concentrations that can
be measured on the µDrop Plate are
instrument dependent and can be
calculated from the given instrument
specifications. For example, with Multiskan
GO, which has a given precision
specification of 0,003 Abs and a linearity
up to 2,5 Abs, the theoretical maximum
concentration range is from 3 x 0,003 x
50 µg/ml x (10 mm/0,5 mm) = 9 µg/ml
to 2,5 x 50 µg/ml x (10 mm/0,5 mm)
= 2500 µg/ml
A theoretical comparison of a dsDNA
measurement with a 10 mm cuvette and
the µDrop Plate low volume area with
an instrument with the performance
described above is shown on Figure 2.
April 2012 I VWRbioMarke Issue 28 I VWR International I
37
the market source for life science
EZ-Vision® DNA Dye featured in AMRESCO’s line
of People-Planet-Safe products
Electrophoresis and
subsequent detection
of resolved DNA are
commonly performed to
analyse the size, purity
and quantity of DNA
samples. Despite its status
as a toxic, mutagenic and
potentially carcinogenic
substance, the most
frequently used dye for
DNA visualisation since
the 1970’s has been
ethidium bromide. Health
and safety concerns
for people and the
environment, however,
have increased demand
for safer alternatives.
To meet that demand,
AMRESCO developed
EZ-Vision® DNA Dye, as a
non-toxic, non-mutagenic
DNA visualisation dye
that eliminates hazardous
ethidium bromide use in
DNA gels.
EZ-Vision® DNA Dye is available as a component
of three convenient loading buffers, which differ
only by the number of tracking dyes included:
EZ-Vision® One, EZ-Vision® Two and EZ-Vision®
Three DNA Dye as Loading Buffer, 6X. EZ-Vision®
One contains a single, fast running, tracking dye
that migrates at approximately 10 bp in a 1%
agarose gel. EZ-Vision® Two contains two tracking
dyes that migrate at 4000 and 400 bp, while EZVision® Three has three tracking dyes that migrate
at 4000, 400 and 10 bp. DNA samples mixed
with any of the three EZ-Vision® DNA Dyes, may
be applied directly to agarose or polyacrylamide
gels, without using ethidium bromide in the gel,
running buffer or sample. Samples are visualised
immediately after electrophoresis, without the
need for post-staining or destaining. Visualisation
is achieved using standard UV illumination with a
green filter (500 - 600 nm), although an ethidium
bromide filter (550 - 640 nm) may be used with
less sensitivity.
Figure 1. EZ-Vision® One, Two and Three. Left
image: 1% TAE agarose gel showing fluorescence of
AMRESCO’s 1 kb Ladder stained with EZ‑Vision® One
(lane 1), EZ-Vision® Two (lane 2) and EZ‑Vision® Three
(lane 3) captured using UV transillumination and a 500 600 nm filter. Right image: A digital photograph of the
same gel on the left to show the tracking dyes included
in EZ‑Vision® One (lane 1), EZ‑Vision® Two (lane 2) and
EZ‑Vision® Three (lane 3).
Methods and results
Sharp, bright DNA bands detected using EZVision® One, Two or Three
5 µl of AMRESCO’s DNA MW Marker, 1 kb ladder
was mixed with 1 µl of either EZ-Vision® One,
Two or Three and was loaded onto a 1% agarose
gel with TAE buffer. The gel was resolved for
1,25 hours at 130 V in 1X TAE running buffer and
then imaged with UV transillumination and a 500
- 600 nm filter using the Syngene G:Box HR gel
documentation system to detect DNA bands. The
same gel was then photographed with a digital
camera using visible light to show the colours and
migration position of the tracking dyes included
in each EZ-Vision® loading buffer. As shown, each
of the EZ-Vision® loading buffers stains DNA at
equivalent levels (Figure 1, left). The different
tracking dye combinations accommodate
individual user preferences and are sufficiently
bright for easy monitoring of sample migration
during electrophoresis (Figure 1, right). Image
capture may be performed immediately after
electrophoresis, without additional staining or
destaining and no specialised imaging equipment
is required.
Sensitivity of EZ-Vision® is similar to that of
ethidium bromide
A 2% agarose gel with TAE buffer was loaded
with decreasing concentrations of AMRESCO’s
PCR DNA Marker mixed with EZ-Vision® Two
DNA Dye as Loading Buffer, 6X on one half with
identical concentrations of the marker mixed with
6X loading buffer containing 0,5 µg/ml ethidium
bromide. The gel was resolved at 130 V for 1,5
hours and then imaged with UV transillumination
and a 500 - 600 nm filter on a Syngene G:Box
HR gel documentation system. Sensitivity of both
EZ-Vision® Two and ethidium bromide is down to
6 ng of DNA for bands at 1500 bp and 500 bp
(Figure 2). At 50 bp, sensitivity is approximately
12,5 ng for EZ-Vision® Two and 6,25 ng for
ethidium bromide (Figure 2). Overall, the
sensitivity of EZ-Vision® Two is similar to that of
ethidium bromide and is an excellent alternative
for routine staining of DNA for electrophoresis.
Figure 2. EZ-Vision® sensitivity compared to ethidium
bromide. 1500, 500 and 50 bp DNA bands from
AMRESCO’s PCR DNA Marker resolved on a 2% TAE
agarose gel were compared at decreasing concentrations
for EZ-Vision® Two (left) and ethidium bromide (0,5 µg/
ml in 6X loading buffer, right) stained samples. The
gel was imaged immediately after electrophoresis with
standard UV transillumination and a 500 - 600 nm filter.
38
I VWR International I VWRbioMarke Issue 28 I April 2012
Genomics
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
monitors the ability of various mutant strains of S.
typhimurium, which are histidine dependent, to
revert to histidine independent bacteria. Five mutant
strains that detect different types of mutagens were
used with and without an S-9 activation system,
which mimics mammalian metabolic conditions to
determine the potential mutagenicity of metabolites
of the original substance. The data shown as the
percentage increase in revertants over controls for
each of the S. typhimurium strains demonstrate
the absence of a twofold increase in the number
of revertants (Figure 4, left), a criteria for mutagen
status. There also was no sign of a dose dependent
response resulting from exposure to various
Figure 3. Environmental hazard
testing of EZ-Vision®. EZ-Vision®
environmental hazard testing was
determined by the CCR Title 22
Fathead Minnow Hazardous Waste
Screen Bioassay. Both EZ-Vision®
Two and EZ-Vision® Three were
deemed non hazardous with LC50
>750 mg/l.
Figure 4. Mutagenicity testing of
EZ-Vision®. EZ-Vision® does not
meet the criteria for classification
as a potential mutagen as
determined by the Ames test.
Exposure to EZ-Vision® did
not increase the percentage
of revertants of mutant S.
typhimurium over controls either
without (top) or with S-9 activation
(bottom).
EZ-Vision® DNA Dye as a Loading Buffer
is safe
EZ-Vision® Two and Three were submitted to
Aquatic Testing Laboratories (Ventura, CA)
for independent environmental safety testing
according to the CCR Title 22 Fathead Minnow
Hazardous Waste Screen Bioassay. The assay
gauges the potential hazard of releasing a specific
substance to the environment by monitoring
its toxicity in aquatic organisms, specifically
fathead minnows. The results were measured
as the percent survival of the fish at specified
concentrations of EZ-Vision® Two, EZ‑Vision® Three
or a control substance (CaCO3) after 96 hours of
exposure. For designation as a non hazardous
substance, the LC50 must be greater than 500
mg/l. EZ-Vision® Two and EZ-Vision® Three are non
hazardous as determined by their LC50 of greater
than 750 mg/l (Figure 3).
Mutagenicity of EZ-Vision® was assessed by
Nelson Laboratories (Salt Lake City, UT) using the
Salmonella typhimurium reverse mutation assay,
otherwise known as the Ames test. The Ames test
Description
EZ-Vision® One DNA Dye as Loading Buffer, 6X
EZ-Vision® One DNA Dye as Loading Buffer, 6X
EZ-Vision® Two DNA Dye as Loading Buffer, 6X
EZ-Vision® Three DNA Dye as Loading Buffer, 6X
Size (ml)
5x 1
0,5
5x 1
5x 1
Cat. No.
AMREN472-KIT
AMREN472-Q-0,5ML
AMREN650-KIT
AMREN313-KIT
dilutions of EZ‑Vision® (data not shown), indicating
EZ-Vision® is not a potential mutagen. The similar
results obtained using the S-9 activation system
indicate that metabolic by-products of EZ-Vision®
are also non mutagenic (Figure 4, left).
Conclusions
Researchers worldwide are already benefiting from
the switch to EZ-Vision® products for DNA1. The EZVision® DNA dye is safe, enabling gels to be handled
without exposing lab personnel or the environment
to hazards. Switching to EZ‑Vision® not only eases
regulatory concerns regarding gel disposal, it also
increases cost savings since gels can be safely
discarded in common rubbish bins. EZ-Vision® is
simple to use and optimised to work with standard
UV gel documentation systems already present in
most labs. The sensitivity of EZ-Vision® DNA dye is
similar to that of ethidium bromide and it is fully
compatible with downstream applications, including
gel purification of DNA.
References
Tissue Antigens, 77 (2), 143-148.
Nature, 468, 67-71.; Grassland Science, 55 (4), 216-220.
Veterinary Microbiology, 141 (3-4), 332-341.
Parasitology International, 59 (3), 407-413.
Peptides, 32 (3), 483-492.
Journal of Plant Physiology, 168 (2), 181-187.
The EMBO Journal, 30, 205-220.
April 2012 I VWRbioMarke Issue 28 I VWR International I
39
the market source for life science
ART® Essentials kits
A new variety of packaging
The ART® Essentials kit contains four trays of the most popular ART® barrier tip volumes (ART® 10 REACH,
ART® 200, and ART® 1000XL) allowing the researcher the ability to conduct a wide variety of applications
with a single pack. The ART® Essentials kit is also available for the ART® Reload System™.
•ART®
self-sealing barrier: Proprietary barrier guarantees 100% protection against carry‑over
contamination. The ART® self-sealing barrier pipette tip seals when exposed to potential
aerosol and liquid contaminants, trapping them inside the barrier, providing the ultimate in
contamination protection.
•ART
barrier tip reloads with 60% less waist: Environmentally friendly packaging
•Recycle
•
logo moulded onto all components
Easy traceability and reordering: Ink jetting on each insert includes lot and part number
•Hinged
racks: One handed open and closing of top cover
•Reversible
lid option: Lift off or hinged lid versatility
•Stackable
racks: Prevents sliding around when stacked
•
8 x 12 matrix/96 well matrix: Easy multichannel pipetting
•
Empty hinged rack part numbers allow for easy conversion to the ART® Reload SystemTM
•Manufactured
according to the industry’s highest quality standards, ART® tips are certified as
RNase, DNase and pyrogen-free, and are ideal for the pipetting of radioactive samples, nucleic
amplification procedures, or any applications where critical sample handling is required.
Description
Cat. No.
ART® Essentials kit - hinged racks MLBP2921-HR
732-1140
4x racks of ART® 10 REACH
732-1145
4x racks of ART® 200
732-1146
4x racks of ART® 1000XL
40
I VWR International I VWRbioMarke Issue 28 I April 2012
Description
Cat. No.
ART® Essentials kit Reload inserts MLBP2931-RI
732-1148
4x inserts of ART® 10 REACH
732-1153
4x inserts of ART® 200
732-1154
4x inserts of ART® 1000XL
Supporting products
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
Optimising high sensitivity protein
assays with Biotix X-RESINTM
The high cost of rare biological samples, coupled with the increasing
sensitivity of today’s protein assays, demands high precision pipetting
by laboratory technicians. Even minor variations in sample delivery can
impact data quality in high sensitivity ELISAs1. Multi-analyte protein
detection systems further challenge technicians with their typically
low sample volume requirements2. For a number of years, pipette tip
manufacturers have tried to market the efficiency of their tips for
sample delivery through gravimetric studies using water, viscous dye
comparisons, and even high resolution photos showing “smoothness”
of the inner pipette tip wall. These methods, however, fail to provide
quantitative assessment of tip efficiency for delivery of molecules in a
given substrate. The question remains, are protein molecules left behind
in tips due to inherent retention of plastic?
In an study performed by MRIGlobal, a major U.S. non profit research
institute, BiotixTM tips with X-RESINTM technology proved to be the most
effective in preventing protein sample loss during pipetting as compared
against leading pipette tip brands on the market.
Protein challenge
X-Resin™
Delta filter 10 µl
Delta filter 20 µl
Delta filter 100 µl
Delta filter 300 µl
Delta filter 1250 µl
10 µl
10 µl XL
200 µl
300 µl
1250 µl
Sterile
+
+
+
+
+
+
+
+
+
+
Pk
960
960
960
960
880
960
960
960
960
880
Cat. No.
732-1430
732-1432
732-1433
732-1434
732-1435
613-1376
613-1377
613-1378
613-1379
613-1380
Methods
Results
Bovine Serum Albumin (BSA) at 5 mg/ml
was used in the protein study. The BSA
solution was labelled with fluorescent dye
as per Invitrogen’s Qubit® Protein Assay Kit
protocol. An electronic pipettor was used
to ensure equivalent handling of liquids
for each brand of tip, thereby limiting
user introduced variance. Fluorescent
BSA was pipette up and down three full
times, with final dispense back to the
original tube. Next, 100 µl of molecular
grade dH2O was pipetted up and down
three times in the tip. The procedure was
repeated in triplicate for each brand of
tips. The Qubit® 2,0 fluorometer was used
to measure any residual fluorescent signal
associated with retention of the protein
solutions on the pipette tips.
There was measurable sample loss due
to residual protein solution left in tips
following dispensing of the sample
(Table 1). The average measured loss of
sample due to retention was <12,5 µg/
ml (below the detection limit) for BiotixTM,
56,2 µg/ml for Brand A, and 77,4 µg/ml
for Brand M. BiotixTM demonstrated the
least amount of protein sample loss, and
is ideally suited for use in high sensitivity
protein expression assays.
For more information on BiotixTM tips,
visit www.vwr.com
(1)eBioscience, “Immunoassay Product Guide”,
Q111020, Jan. 2011, pp. 3, 5.
(2)R&D Systems, “ELISA Reference Guide & Catalog”,
2011, pp. 15-16.
table 1. Graph of residual
protein carry-over
April 2012 I VWRbioMarke Issue 28 I VWR International I
41
the market source for life science
Detection of light absorbing
leachable chemicals from
disposable filter tips
Quality differences
BRAND is known as one of the leading manufacturers of disposable
plastics for life science applications. These products include pipette tips,
filter tips, PCR disposables, microplates (BRANDplates®), etc.
In addition to products like microtubes and PCR tubes, the quality of
filter tips is crucial to obtain optimal results for critical applications such
as enzymatic tests, PCR etc.
Filter tips are well suited for performing
PCR techniques, and fully meet the
requirements for microbiology and
radioisotope work. The integrated
hydrophobic polyethylene filter serves as
a barrier against aerosols formed during
pipetting. BRAND filter tips protect
the pipette shafts from contamination,
and as a result lessen the risk of cross
contamination and consequential
erroneous results.
a considerable number of additives
including, but not limited to chemical
antioxidants, slipping agents and radiation
stabilisers. These additives can move to
the surface of the disposable product
and affect biological tests leading to false
positive or negative results.
Filter tips are made from polypropylene
for the tips and polyethylene for the
filter. Both components can contain these
additives.
Plastic disposables are frequently made
from polymer materials that contain
This is a comparison between the BRAND filter tips and filter tips of a direct
competitor using an extremely easy to follow procedure.
• Materials and methods
The idea behind this method is to establish a quick and easy test to detect leachable
products from the filter of filter tips using UV/Vis measurement in the wavelength range
between 230 and 800 nm.
• Filter tips
BRAND filter tips 5 – 100 µl
Competitor A filter tips 100 µl
• Chemicals
Distilled water
• Procedure
Twenty filters were removed from 20 filter tips and incubated for 12 hours at room
temperature with 1 ml distilled water.
UV/Vis absorbance scans were performed on this liquid using a Biochrom WPA
spectrophotomer. Note that the scale on the Y axis varies to present the best graph for
each scan.
42
I VWR International I VWRbioMarke Issue 28 I April 2012
Supporting products
For more information on these products contact your local VWR sales office,
send an email to vwrbiomarke@eu.vwr.com or visit our website www.vwr.com
Results
Extinction
0,10
1
Extinction
2,40
Extinction
0,10
2
0,06
1,92
0,06
0,02
1,44
0,02
-0,02
0,96
-0,02
-0,06
0,48
-0,06
-0,10
240
352
464
576
688
800
nm
0,00
-0,10
240
352
464
576
688
800
nm
240
3
352
464
576
688
800
nm
Figure 1: Spectrum of distilled water
Figure 2: Spectrum of leachable products from competitor A’s filter after incubating 20 filters in 1 ml of water for 12 hours.
Figure 3: Spectrum of leachable products from the BRAND filter after incubating 20 filters in 1 ml of water for 12 hours.
Conclusion
The direct comparison between the filter
from the BRAND filter tips and the one
from the direct competitors shows that
the BRAND filter does not contain any
photometrically detectable leachable
products. The filter of competitor A
exhibits leaching of chemicals that can be
detected via
UV/Vis measurements absorbing at
280 nm, 400 nm and 620 nm. These
extractable chemicals can be a source of
error during the evaluation of results.
Leachable products are an important
source of error in biological test systems.
The filter from BRAND filter tips are third
party tested by analytical, clinical, and life
cycle microbiology testing laboratories and
do not contain additives or contaminants,
heavy metals or cytotoxic agents. For the
PP component of the filter tips, specially
selected transparent PP types, free
from the additives di-(2-hydroxyethyl)
methyldodecylammonium (DiHEMDA) and
9-octa-decenamide (oleamide), are used.
Both of these additives are frequently
found in PP granules, and can interfere
with biological tests, leading to spurious
results*.
* G. R. McDonald, A. L. Hudson, S. M. J. Dunn,
H. You, G. B. Baker, R. M. Whittal, J. W. Martin,
A. Jha, D. E. Edmondson, A. Holt (2008).
Bioactive Contaminants Leach from Disposable
Laboratory Plasticware. Science, 322 (5903),
917-917.
Tip-Box N, sterile, BIO-CERT®
0,1 - 1 µl
0,5 - 10 µl
2 - 20 µl
5 - 100 µl
5 - 200 µl
50 - 1000 µl
BRAND racked filter tips
Type
Standard
Low Retention
Standard
Low Retention
Standard
Low Retention
Standard
Low Retention
Standard
Low Retention
Standard
Low Retention
Volume (µl)
0,1 - 1
0,1 - 1
0,5 - 10
0,5 - 10
2 - 20
2- 20
5 - 100
5 - 100
5 - 200
5 - 200
50 - 1000
50 - 1000
Pk
10x 96 (960)
10x 96 (960)
10x 96 (960)
10x 96 (960)
10x 96 (960)
10x 96 (960)
10x 96 (960)
10x 96 (960)
10x 96 (960)
10x 96 (960)
10x 100 (1000)
10x 100 (1000)
Cat. No.
613-1695
732-1456
613-0591
732-1457
613-1768
732-1458
613-0593
732-1459
613-0594
732-1460
613-0595
732-1461
April 2012 I VWRbioMarke Issue 28 I VWR International I
43
Austria
Ireland / Northern Ireland
Spain
VWR International GmbH
Graumanngasse 7
1150 Wien
Tel.: 01 97 002 0
Fax: 01 97 002 600
E-mail: info@at.vwr.com
VWR International Ltd /
VWR International (Northern Ireland) Ltd
Orion Business Campus
Northwest Business Park
Ballycoolin
Dublin 15
Tel.: 01 88 22 222
Fax: 01 88 22 333
E-mail: sales@ie.vwr.com
VWR International Eurolab S.L.
C/ Tecnología 5-17
A-7 Llinars Park
08450 - Llinars del Vallès
Barcelona
Tel.: 902 222 897
Fax: 902 430 657
E-mail: info@es.vwr.com
Italy
VWR International AB
Fagerstagatan 18a
163 94 Stockholm
Tel.: 08 621 34 00
Fax: 08 621 34 66
E-mail: info@se.vwr.com
Belgium
VWR International bvba
Researchpark Haasrode 2020
Geldenaaksebaan 464
3001 Leuven
Tel.: 016 385 011
Fax: 016 385 385
E-mail: customerservice@be.vwr.com
Denmark
VWR - Bie & Berntsen
Transformervej 8
2730 Herlev
Tel.: 43 86 87 88
Fax: 43 86 87 90
E-mail: info@dk.vwr.com
Finland
VWR International Oy
Valimotie 9
00380 Helsinki
Tel.: 09 80 45 51
Fax: 09 80 45 52 00
E-mail: info@fi.vwr.com
France
VWR International S.A.S.
Le Périgares – Bâtiment B
201, rue Carnot
94126 Fontenay-sous-Bois cedex
Tel.: 0 825 02 30 30 (0,15 EUR TTC/min)
Fax: 0 825 02 30 35 (0,15 EUR TTC/min)
E-mail: info@fr.vwr.com
Germany
VWR International GmbH
Hilpertstrasse 20a
D - 64295 Darmstadt
Tel.: 0180 570 20 00*
Fax: 0180 570 22 22*
E-mail: info@de.vwr.com
*0,14 €/Min. aus d. dt. Festnetz,
Mobilfunk max. 0,42 €/Min.
Hungary
VWR International Kft.
Simon László u. 4.
4034 Debrecen
Tel.: (52) 521-130
Fax: (52) 470-069
E-mail: info@hu.vwr.com
VWR International PBI S.r.l.
Via San Giusto 85
20163 Milano (MI)
Tel.: 02-3320311/02-487791
Fax: 800 152999/02-40090010
E-mail: info@it.vwr.com
info@internationalpbi.it
The Netherlands
VWR International B.V.
Postbus 8198
1005 AD Amsterdam
Tel.: 020 4808 400
Fax: 020 4808 480
E-mail: info@nl.vwr.com
Norway
VWR International AS
Haavard Martinsens vei 30
0978 Oslo
Tel.: 02290
Fax: 815 00 940
E-mail: info@no.vwr.com
Poland
Sweden
Switzerland
VWR International AG
Lerzenstrasse 16/18
8953 Dietikon
Tel.: 044 745 13 13
Fax: 044 745 13 10
E-mail: info@ch.vwr.com
UK
VWR International Ltd
Customer Service Centre
Hunter Boulevard
Magna Park
Lutterworth
Leicestershire
LE17 4XN
Tel.: 0800 22 33 44
Fax: 01455 55 85 86
E-mail: uksales@uk.vwr.com
Labart Sp. z o.o.
A VWR International Company
Limbowa 5
80-175 Gdansk
Tel.: 058 32 38 200 do 204
Fax. 058 32 38 205
E-mail: labart@pl.vwr.com
Portugal
VWR International Material de Laboratório, Lda
Edifício Neopark
Av. Tomás Ribeiro, 43- 3 D
2790-221 Carnaxide
Tel.: 21 3600 770
Fax: 21 3600 798/9
E-mail: info@pt.vwr.com
Go to vwr.com for the latest news, special offers
and details of your local VWR distributor.
EN-03-2012-IJ