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. 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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