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Nucleofector® technology
your link to transfection
> N u c l e ofe c to r ® te c h n o l o g y
The Nucleofector® technology
is the only reliable non-viral transfection method for a wide
range of difficult-to-transfect cell lines and primary cells.
Established in leading labs
The Nucleofector® technology has become an established transfection technology in many labs worldwide. A growing number
of publications illustrates the importance of the technology for
day-to-day research in addressing issues of formerly difficultto-transfect cell lines and primary cells.
One technology for cell lines and primary cells
Using the Nucleofector technology, cell lines, as well as
primary cells, can be reliably transfected. DNA delivery
straight into the nucleus ensures transfection efficiencies of
up to 90% in both, cell lines and primary cells.
Use of many different substrates
With the Nucleofector technology various nucleic acids
can be easily transfected such as DNA, RNA or siRNA. Transfection parameters remain the same for DNA, RNA and siRNA
so that no further optimization is required.
Maintenance of functionality
The nucleofection ® procedure has no influence on the cell
functionality. Nucleofected cells show long-term survival in
culture and can easily be differentiated or stimulated.
DNA delivery straight into the
nucleus
Normal human dermal fibroblasts –
neonatal were nucleofected with 2.5 μg
TMR-labeled plasmid DNA encoding
eGFP. After 2 hours, cells were fixed
with 3.5% PFA and analyzed by confocal microscopy. TMR label is shown
in (A), GFP fluorescence in (B), DAPI
nuclear staining in (C) and a merge of
all three fluorescent labels in (D).
page 2
www.amaxa.com
A
B
C
D
Nucleofector ® technology
> N u c l e ofe c to r ® te c h n o l o g y
The two components of the
Nucleofector® technology
new
The Nucleofector® II Device
The Nucleofector II Device delivers unique electrical parameters
that are different from other commercially available electroporation instruments. Electrical settings are pre-programmed for
each optimized cell type. The integrated chipcard reader allows
quick program and software updates for future applications.
> easy handling
motor-driven carousel for automatic cuvette handling
> high convenience
large graphic display for easy day-to-day use
> great flexibility
software enables the filing of individual programs
> future proof
suitable to use together with the 96-well Shuttle™
The Nucleofector® Kits
The Nucleofector Kits contain amaxa specified cuvettes, pipettes,
pmaxGFP™, Nucleofector Solution and Supplement. Each
solution and supplement is individually developed for every
primary cell type. For cell lines, five different Nucleofector
Solutions, C, L, R, T and V, are available. All solutions provide a
cell-friendly environment that ensure the highest transfection
results and cell viability. Nucleofector Kits are only functional
in combination with the Nucleofector Device.
Ka
n
Positive controls available
in
yc
am
PC
M
V
Introducing maxGFP™
All Nucleofector Kits contain a plasmid encoding maxGFP, a
green fluorescent protein from the copepod Pontellina p.
Int ron
pmaxGFP
(3486 bp)
i
or
GF
P
pu C
ax
m
SV 4
0 pA
The new siRNA Test Kit offers the opportunity to easily establish siRNA in cell lines or adherent primary cells. The kit contains
Plasmid map of pmaxGFP
Cells transfected with pmaxGFP can be easily analyzed for
transfection efficiency by flow cytometry or fluorescent
microscopy.
pmaxGFP and an siRNA duplex directed against maxGFP together
with a detailed protocol. Gene silencing can thus be easily
monitored by decrease of maxGFP expression.
siRNA Test Kit
page 3
www.amaxa.com
amaxa – your link to transfection
Cat. No. VSC-1001
> ce l l l i n es
The ideal tool for
transfection of cell lines
The Nucleofector technology is the ideal tool for transfection of
virtually any type of cell line, such as, commonly-used cell lines
or difficult-to-transfect cell lines. High transfection efficiencies
are combined with high cell viability and reproducibility of transfection results.
> high efficiencies
up to 90% gene transfer efficiency with high cell viability
> ideal for siRNA
up to 99% transfection efficieny with siRNA duplexes even
> safe and easy procedure
easy and convenient handling
> fast experiments
expression within hours - from transfection to analysis in
in suspension cells
one day
How to find your cell line of interest
Visit the amaxa
cell line database
Enter your cell line
of interest or view
Your cell line of interest
complete list
Screen results
Cell type listed
Cell type not listed
Click on cell name
Use Cell Line Optimization
Nucleofector Kit or contact
amaxa’s Scientific Support
Use recommended
Nucleofector Kit in combination
with the respective Optimized
Protocol or use the settings
optimized by other users.
page 4
www.amaxa.com
Nucleofector ® technology
for further advice.
> ce l l l i n es
Optimization of virtually any cell line in just one experiment
Difficult-to-transfect
The advanced Cell Line Optimization Nucleofector Kit is
cell lines
an ideal tool for the transfection of your difficult-to-transfect
cell line. It enables you to conveniently determine the optimal
nucleofection parameters of your cell line of interest within one
experiment.
1
Solution
V
program 1
A- 0 2 0
A- 0 2 0
program 2
T- 0 2 0
T- 0 2 0
program 3
T-030
T-030
program 4
X- 0 0 1
X- 0 0 1
program 5
X- 0 0 5
X- 0 0 5
program 6
L- 0 2 9
L- 0 2 9
program 7
D-023
D-023
Step 1 The cell line of interest is transfected with the Nucleofector Solutions
L and V in combination with seven
different Nucleofector programs.
2
Step 2 The Nucleofector Solution and
program which result in highest transfection efficiencies with lowest mortality are selected.
V Solution
> > >
+
3
> > >
L
Step 3 A further fine tuning of the
nucleofection conditions can be performed with the help of our Scientific
Support Team.
Examples of amaxa optimized cell lines
293 ATCC® CRL-1573™
293 (DSMZ)
32D ATCC® CRL-11346™
3T3-L1 (pre-ad) ATCC® CL-173™
A-10 ATCC® CRL-1476™
A20 ATCC® TIB-208™
A-431 ATCC® CRL-1555™
A549 ATCC® CCL-185™
AGS ATCC® CRL-1739™
BA/F3 (DSMZ)
BHK-21 ATCC® CCL-10™
BJ ATCC® CRL-2522™
C2C12 ATCC® CRL-1772™
C6 ATCC® CCL-107™
Caco-2 ATCC® HTB-37™
CCRF-CEM ATCC® CCL-119™
CHO-K1 (DSMZ)
CHO-K1 ATCC® CCL-61™
CHO-suspension (ECACC)
COS-1 ATCC® CRL-1650™
COS-7 (DSMZ)
DU 145 ATCC® HTB-81™
EL4 ATCC® TIB-39™
FDC-P1 ATCC® CRL-12103™
HaCaT (DKFZ)
HCT 116 ATCC® CCL-247™
d
84%
57%
79%
73%
64%
74%
45%
72%
73%
88%
85%
52%
82%
95%
59%
68%
84%
94%
92%
49%
85%
47%
65%
82%
43%
78%
HeLa ATCC® CCL-2™
HeLa (DSMZ)
HepG2 ATCC® HB-8065™
HL-60 ATCC® CCL-240™
HT-29 ATCC® HTB-38™
HT-1080 ATCC® CCL-121™
HuT 78 ATCC® TIB-161™
IMR-90 ATCC® CCL-186™
JURKAT ATCC® TIB-152™
JURKAT (DSMZ)
K562 ATCC® CCL-243™
K562 (DSMZ)
KG-1 ATCC® CCL-246™
LNCaP ATCC® CRL-1740™
MCF-7 ATCC® HTB-22™
MDA-MB-231 ATCC® HTB-26™
MDA-MB-453 ATCC® HTB-131™
MDA-MB-468 ATCC® HTB-132™
MDCK ATCC® CCL-34™
MDCK II (ECACC)
MOLT-4 ATCC® CRL-1582™
NALM-6 (DSMZ)
NB-4 (DSMZ)
NCI-H1299 ATCC® CRL-5803™
Neuro-2a ATCC® CCL-131™
NIH/3T3 ATCC® CRL-1658™
70%
57%
64%
50%
51%
74%
53%
51%
54%
63%
89%
76%
70%
70%
71%
80%
54%
60%
73%
80%
55%
64%
71%
99%
76%
84%
For an updated cell list and to view customer derived data for approximately 300 additional cell types please go to
www.amaxa.com/celldatabase.
page 5
NIH-3T3 (DSMZ)
NRK ATCC® CRL-6509™
NS0 (ECACC)
P19 ATCC® CRL-1825™
PC-12 ATCC® CRL-1721™
PC-3 ATCC® CRL-1435™
PANC-1 ATCC® CRL-1469™
Raji ATCC® CCL-86™
Ramos ATCC® CRL-1596™
RAW 264.7 ATCC® TIB-71™
RBL-1 ATCC® CRL-1378™
S49 ATCC® TIB-36™
Saos-2 ATCC® HTB-85™
SH-SY5Y ATCC® CRL-2266™
SK-OV-3 ATCC® HTB-77™
SK-N-SH ATCC® HTB-11™
SW480 ATCC® CCL-228™
T-47D ATCC® HTB-133™
THP-1 ATCC® TIB-202™
U-2 OS ATCC® HTB-96™
U-937 ATCC® CRL-1593.2™
U-87 MG ATCC® HTB-14™
U266BI ATCC® TIB-196™
Vero ATCC® CCL-81™
WEHI-231 ATCC® CRL-1702™
www.amaxa.com
amaxa – your link to transfection
68%
44%
83%
85%
34%
81%
68%
58%
27%
65%
84%
81%
82%
82%
89%
85%
60%
51%
67%
98%
56%
43%
86%
79%
77%
> p r i m a r y ce l l s
Nucleofection of primary cells
In the past, the lack of efficient and easy transfection methods
for primary cells restricted experiments mainly to cell lines.
However, the artifical nature of cell lines limits the significance
of experimental results. Today, the isolation and culture of primary cells is well-established. With introduction of the unique
Nucleofector technology the last obstacle, the efficient non-viral
transfection of such cell types, was overcome in 2001. The
technology is now a well-established method worldwide.
Researchers are now able to address open questions of their
specific research area in many primary cell types which reflect
a much more relevant physiological system than any cell line
could ever be.
> high efficiencies
up to 90% gene transfer efficiency with high cell viability
> no optimization
ready-to-use kits for a multitude of cell types
> reproducible results
Optimized Protocols for each cell type with details on cell
> ideal for primary cells
direct transport of nucleic acids into the nucleus thus
isolation and culture
enabling transfection even of non-dividing cells
siRNA transfection in primary cells up to 99% transfection efficiency
The Nucleofector technology ensures efficient delivery of siRNA
even in primary cells enabling strong knock-down effects. High
cell viability of more than 85% is maintained even with higher
amounts of siRNA.
d
page 6
200
300
A
240
120
180
80
120
40
60
counts
160
counts
Functional CD2 silencing in primary
human T cells
Primary human T cells were transfected with 1.4 μg siRNA targeted to
CD2 using the Human T Cell Nucleofector Kit. 44 hours post nucleofection, cells were stained with anti
CD2-FITC antibody (A) or anti CD4PE antibody (B) and analyzed by
flow cytometry. Controls included
nucleofection of 1.4 μg control siRNA
and addition of CD2 siRNA without
nucleofection.
0
100
101
102
103
104
CD2-FITC
CD2 siRNA
B
0
100
101
102
103
control siRNA
no siRNA + nucleofection
For further information about nucleofection of siRNA in primary cells and cell lines, go to www.amaxa.com/RNAi
or contact our Scientific Support Team.
www.amaxa.com
Nucleofector ® technology
104
CD4-PE
> p r i m a r y ce l l s
Transfection results you can trust
Examples for primary cell
Transfection efficiency*
Nucleofector Kits
cancer research
Dendritic cells, human
49%
HUVEC
90%
MEF
43%
etc.
cardiovascular research
Cardiomyocytes, rat
35%
HMVEC-L
52%
MSC, human
47%
etc.
dermatology
Dermal fibroblasts, human
89%
Keratinocytes, human
53%
etc.
immunology
B cells, human
36%
Macrophages, mouse
47%
Macrophages, human
59%
T cells, human
64%
T cell, mouse
39%
etc.
neurobiology
Neurons, mouse
58%
Neurons, rat cortical
59%
Neural stem cells, mouse
60%
etc.
gastroenterology
Hepatocytes, mouse
54%
Hepatocytes, rat
52%
* Data show examples of average transfection efficiencies given as % positive viable cells. Cells were either transfected with a plasmid encoding maxGFP, eGFP
or H-2K k , a mouse MHC class I molecule. Analysis was performed by either flow cytometry or fluorescence microscopy. Cell viability varies between 50-90%.
Protect your cells from mycoplasma
amaxa offers new antibiotic formulations specifically developed to protect cell lines or sensitive
primary cells against mycoplasma, gram+ and gram- bacteria as well as fungi.
d
page 7
Primocin™
for primary cells
Cat. No. VZA-1021
Normocin™
for cell lines
Cat. No. VZA-1001
Plasmocin™
for mycoplasma cure
Cat. No. VZA-1012
For more information about the new cell culture antibiotics, see www.amaxa.com/antibiotics.
www.amaxa.com
amaxa – your link to transfection
Ordering information
Cat.No.
AAD-1001
Nucleofector® device
VPG-1006
Astrocytes, mouse
Nucleofector® Kits for primary cell types
VPG-1007
Astrocytes, rat
VPA-1001
B cells, human
VPE-1002
Cardiomyocytes, rat neonatal
VPA-1003
CD34+ cells, human
VPF-1001
Chondrocytes, human
VPA-1004
Dendritic cells, human
VPD-1001
Dermal fibroblasts, human
VPD-1006
Embryonic fibroblasts, mouse, Starter Kit
VPD-1004
Embryonic fibroblasts, mouse, Kit I
VPD-1005
Embryonic fibroblasts, mouse, Kit II
VPH-1001
Embryonic stem cells, mouse
VPB-1001
Endothelial cells, human coronary artery (HCAEC)
VPB-1003
Endothelial cells, human microvascular (HMVEC-L)
VPB-1002
Endothelial cells, human umbilical vein (HUVEC)
VPK-1001
Epithelial cells, human coronary artery
VPK-1002
Epithelial cells, human mammary
VPK-1003
Epithelial cells, human prostate
VPL-1002
Hepatocytes, mouse
VPL-1003
Hepatocytes, rat
VPD-1002
Keratinocytes, human
VPA-1008
Macrophages, rat
VPA-1009
Macrophages, mouse
VPA-1007
Monocytes, human
VPD-1003
Melanocytes, human epidermal neonatal (NHEM)
VPE-1001
Mesenchymal stem cells, human
VPA-1005
Natural killer cells, human (NK)
VPG-1004
Neural stem cells, mouse
VPG-1005
Neural stem cells, rat
VPG-1002
Neurons, chicken
VPG-1001
Neurons, mouse
VPG-1003
Neurons, rat
VPG-1009
Oligodendrocytes, rat
VPC-1001
Smooth Muscle Cells, human aortic (AoSMC)
VPA-1002
T cells, human
VPA-1006
T cells, mouse
VPI-1001
Mammalian endothelial cells
VPI-1002
Mammalian fibroblasts
Basic Nucleofector® Kits
VPI-1003
Mammalian neural cells
VPI-1004
Mammalain smooth muscle cells
VPI-1005
Mammalian epithelial cells
VCO-1001
Cell Line Optimization Nucleofector® Kit
VCA-1004
Cell Line Kit C
VCA-1005
Cell Line Kit L
WKB-1001_01.2006
Nucleofector® Kits for cell lines
d
For an up-to-date
overview or more
detailed information
about amaxa products, please see
www.amaxa.com/
productlist.
VCA-1001
Cell Line Kit R
VCA-1002
Cell Line Kit T
VCA-1003
Cell Line Kit V
amaxa's Nucleofector ® Process, Nucleofector ® Device and Nucleofector ® Solutions are covered by PCT Applications PCT/EP01/07348,
PCT/DE02/01489, PCT/DE02/01483, and other pending patents, and domestic or foreign applications corresponding thereto.
amaxa, Nucleofector, nucleofection, maxGFP and 96-well Shuttle are trademarks of amaxa GmbH.
The CMV promoter is covered under U.S. Patents 5,168,062 and 5,385,839 and its use is permitted for research purposes only. Any other use of
the CMV promoter requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA 52242.
ATCC® and the ATCC Catalog Marks are trademarks of ATCC used under License.
amaxa GmbH, Europe/World
Scientific Suppor t
+49 (0)221-99 199-400
scientific-suppor t@amaxa.com
www.amaxa.com
amaxa Inc., USA
Scientific Suppor t
(240) 632-9 110
scientific-suppor t.US@amaxa.com