Transfection of neural stem cells - Lonza AG

amaxa
› amaxa news 02
news
› Nucleofector technology and RNAi –
application note, ‘essentials’, data
› amaxa and QIAGEN ® cooperation –
joined forces in gene silencing
› New: nucleofection of cardiomyocytes
and natural killer cells
› New: nucleofection of neural stem
cells and astrocytes
gene transfer begins here
02

Integrated Solutions — Gene Silencing
Silence your genes in both
adherent and suspension
cells!
New
HUVEC cells transfected with siRNA
using RNAiFect Transfection Reagent
Jurkat cells transfected with siRNA using
Nucleofector technology
Use our new partnership for gene silencing in adherent and
suspension cells!
QIAGEN ® and amaxa have joined forces to offer you solutions
for RNAi from design to delivery. QIAGEN’s siRNA service
includes high-purity siRNA synthesis and an advanced design
algorithm. RNAiFect Transfection Reagent provides effective
transfection of adherent cells. amaxa’s Nucleofector technology
ensures success for transfection of suspension cells.
■ Effective gene silencing — combining our expertise in
siRNA design, synthesis, and delivery
■ Efficient siRNA delivery — typically 95% transfection
efficiency in adherent cells and 99% in suspension cells
■ Tested for many cell types — including HeLa, HEK293,
NIH/3T3, CHO, C6, AGS, Jurkat, HL-60, K562, U937,
THP-1, HUVEC, MEF, and primary human T cells
Find out more at
www.qiagen.com/siRNA/TransfectAllCells !
Trademarks: QIAGEN ® , RNAiFect (QIAGEN Group), Nucleofector (amaxa GmbH),
siRNA technology licensed to QIAGEN is covered by various patent applications,
owned by the Massachusetts Institute of Technology, Cambridge, MA, USA and others.
GSA1003AMAXA 10/2003 © 2003 QIAGEN, all rights reserved.
WWW.QIAGEN.COM
Editorial
We are proud to introduce the second issue of ‘amaxa news’. Your
feedback to our first issue was overwhelming and was a valuable
resource to further improve this newsletter. Thank you again for
your comments and your positive feedback!
At present, RNAi is one of the most exciting topics in the scientific
community and also of major importance to amaxa (pp. 6-9,
12-15). The Nucleofector technology addresses one of the key
bottlenecks in RNAi – highly efficient delivery into hard-to-transfect
cell lines and primary cells. In addition, amaxa has joined forces
with QIAGEN ® to provide you with an integrated solution for siRNA
design and delivery comprising QIAGEN’s siRNA design service
and amaxa’s Nucleofector technology. Read more about your
benefits from this co-operation in this issue of ‘amaxa news’ (p. 6).
Anything else besides RNAi? Quite a lot. ‘amaxa news’ will also
give an update on our latest R&D developments. Find out more
about our new Nucleofector Kits for cardiomyocytes, natural
killer cells (p. 4), astrocytes, neural stem cells (p. 5), mouse
embryonic stem cells and mouse embryonic fibroblasts (pp. 10-11).
And there is still more to learn … we wish you an interesting read.
Dr. Gregor Siebenkotten Rainer Christine
CSO CEO

[k]
q
d
New products
New products
amaxa insights
Application note
New products
Tips and hints
Product update
Backround information
amaxa insights
Highlighted paper
amaxa insights
amaxa worldwide
amaxa worldwide
FAQ
Ordering information
To order products contact our
Scientific Support Team (see back
cover for contact details).
Supplementary information
To order supplementary information
complete the reply card attached in
the center of this newsletter.
amaxa web information
To instantly find more detailed
information on certain subjects visit
the indicated website.
› Table of content
4 Nucleofector Kits for transfection of human natural killer cells
and rat cardiomyocytes
5 Transfection of neural stem cells (NSC) and astrocytes from
mouse and rat
6 amaxa and QIAGEN ® have joined forces in gene silencing
7 RNAi in suspension cells using amaxa’s Nucleofector
technology together with QIAGEN ® ’s HPP Grade siRNA duplexes
Elke Lorbach, Dietmar Lenz, Andrea Klaes, Katharina Hein, Bodo Ortmann, Tatjana Males,
Hanns-Martin Schmidt, Peter Hahn, Wolfgang Bielke and Günter Kraus
10 Nucleofection of mouse embryonic fibroblasts (MEF) and
mouse embryonic stem cells
12 Take a short cut for your RNAi experiments – the ‘essentials’ about
Nucleofector technology and RNAi
14 Nucleofector technology and RNAi – examples of successful
knockdown provided by Nucleofector users
16 Basics about nucleofection and the easiest way to transfect
your cell line
18 Fly amaxa competition winners announced
Highlighted paper: Messi et al. (2003)
19 amaxa user forum
20 WAKO Pure Chemicals - amaxa’s competent partner in Japan
21 Meet amaxa at your bench
amaxa’s distributors around the world
22 Frequently asked questions on Nucleofector technology in
neurobiology and immunology
Editor
Wolfgang Kintzel
Editorial board
Bernd Eschweiler, Katrin Höck,
Katja Thieke, Michael Nix
Production coordinator
Benno Limberg
amaxa news is published by
amaxa GmbH
Nattermannallee 1
50829 Koeln
Germany
amaxa_news@amaxa.com
Tel: +49(0)221-99199-0
Fax:+49(0)221-99199-111
› page 3 › www.amaxa.com
amaxa’s patent pending technologies
and information is the express and
proprietary information of amaxa GmbH.
amaxa, Nucleofection, Nucleofector,
“gene transfer begins here” are all
trademarks of amaxa GmbH.
Please note that to date amaxa’s
Nucleofector technology is intended for
research use or investigational use only.
Design
vierviertel – Agentur für
Kommunikationsdesign GmbH
info@vierviertel.com
© 2003 amaxa. All rights reserved.
Printed in Germany.

› New products
new
Nucleofector Kits for transfection
of human natural killer
cells and rat cardiomyocytes
Nucleofection of primary human natural killer cells
Cancer and immunology research was frequently hampered due
to the inability to transfect primary NK cells non-virally. With the
Human NK Cell Nucleofector Kit a major breakthrough has been
accomplished circumventing the use of rather artificial cell lines.
Due to the major obstacles encountered during NK cell transfection
so far an efficiency of up to 20% can be a major advancement
for your research.
natural
killer cells
› first non-viral transfection technology for primary NK cells
› transfection efficiencies of up to 20%
› viability post nucleofection: 50-60%
Nucleofection of primary neonatal rat cardiomyocytes
Cardiovascular research has to struggle with two constraints: the
lack of appropriate cell line models and an efficient transfection
method for primary cardiomyocytes. Nucleofection of rat cardiomyocytes
will help to overcome both limitations.
› first efficient non-viral transfection technology for primary
neonatal rat cardiomyocytes
› reproducible transfection efficiencies up to 40%
› viability post nucleofection: 50-60%
cardiomyocytes
plasmid encoding DsRed. Two days post nucleofection, the cells
[k] Ordering information
Human NK Cell Nucleofector Kit Cat. No.: VPA-1005 d
Rat Cardiomyocyte Nucleofector Kit Cat. No.: VPE-1002
› page 4 › amaxa news 02
SSC
NK cells -DNA NK cells +DNA
A B
eGFP
0.5%
SSC
eGFP
Nucleofection of neonatal rat cardiomyocytes
Primary neonatal rat cardiomyocytes were nucleofected with a
were analyzed by fluorescence microscopy. Fig A shows DsRed
expressing cells. Cardiomyocytes stained with FITC-labeled
tropomyosin antibody (a fibrous protein that is part of the muscle
fibers) are shown in fig. B. Fig. C is an overlay. (Photographs
courtesy of F. Engel and M. Keating, Cardiology Department,
Childrens’ Hospital, Havard Medical School, Boston, MA, USA)
amaxa web information
Cancer www.amaxa.com/cancer
Immunology www.amaxa.com/immunology
Cardiology www.amaxa.com/cardio
16.9%
Nucleofection of primary human NK cells
Primary human natural killer cells were nucleofected with a
plasmid encoding eGFP protein. Cells were analyzed by flow
cytometry 24 hours post nucleofection. eGFP expression in
natural killer cells is shown after nucleofection without (A) and
with plasmid DNA (B). (Courtesy of J. Sundbäck and K. Kärre,
Karolinska Institute, Microbiology and Tumor Biology Center,
Stockholm, Sweden)
A
C
B

[k]
% nucleofected cells
› New products
Transfection of neural stem
cells (NSC) and astrocytes from
mouse and rat
Recently amaxa introduced four new Nucleofector Kits for the transfection of neural stem cells and astrocytes to
further support your research in neurobiology. For the most important cell types in neurobiology Nucleofector Kits
are now available. These include kits for mouse, rat, and chicken neurons and for PC12 and C6 cell lines.
(Coming soon: SH-SY5Y).
Use the Nucleofector for your neurobiology research:
non-viral › first efficient non-viral method for neural and glial cells
high efficiencies › up to 70% transfection efficiency
functionality › cells remain functional after nucleofection
long-term survival › up to three weeks survival
no optimization › ready-to-use kits
100
90
80
70
60
50
40
30
20
10
0
new
eGFP
48h 24h 48h 24h
mouse mouse rat rat
NSC astrocytes astrocytes NSC
Average transfection efficiencies for neural stem cells and
astrocytes
Mouse and rat NSC and astrocytes were transfected with a
plasmid encoding the eGFP protein. Transfection efficiencies
are given as % positive cells. Analysis was performed by
fluorescence microscopy.
Ordering information
Mouse NSC Nucleofector Kit Cat. No.: VPG-1004
Rat NSC Nucleofector Kit Cat. No.: VPG-1005
Mouse Astrocyte Nucleofector Kit Cat. No.: VPG-1006
Rat Astrocyte Nucleofector Kit Cat. No.: VPG-1007
› page 5 › www.amaxa.com
A
Example for Nucleofection of rat neural stem cells (NSC)
Primary rat neural stem cells isolated from rat embryos (E14) were nucleofected with
a plasmid encoding the eGFP protein under the control of an EF1d promoter. After
nucleofection cells were cultured with bFGF for two days, then for five days without
bFGF to differentiate NSC into neurons and astrocytes. Cells were analyzed by fluorescence
microscopy two days (A) and seven days (B) post nucleofection. (Photographs
courtesy of Dr. S.H. Lee, College of Medicine, Dept. of Biochemistry, Hanyang
University, Seoul, South Korea)
q
Supplementary information
Neurobiology flyer – Nucleofection in neurobiology
WKB-1004
B
d amaxa web information www.amaxa.com/neurobiology

› amaxa insights
amaxa and QIAGEN ® have
joined forces in gene silencing
RNAi provides a rapid, efficient and powerful tool in functional genomics drawing widespread attention to this field. Although
RNAi has proven to be a powerful method for gene silencing some technical difficulties remain. QIAGEN and amaxa
have joined forces to address the two key bottlenecks in RNAi-mediated gene silencing i.e. siRNA design and delivery.
Take advantage of our partnership for siRNA
design and delivery
By combining QIAGEN’s knowledge of siRNA
design and synthesis and amaxa’s know-how on
efficient delivery, into virtually any cell, a com-
› page 6 › amaxa news 02
plete service for your gene silencing experiments is now available.
QIAGEN and amaxa’s technical services provide an invaluable
source of information for details on siRNA experiment set-up,
siRNA design and transfection protocols for optimal delivery. Call
today to discuss your experiment!
siRNA design made easy with QIAGEN
Optimal siRNA design › Use the convenient online QIAGEN siRNA Design Tool which
uses the sensitive Smith-Waterman algorithm.
High-purity, custom › HPP (High Performance Purity) Grade siRNA is > 90% pure.
synthesized siRNA
Minimal risk for gene silencing › 4-For-Silencing siRNA Duplexes are designed by QIAGEN
scientists to target your gene of interest. QIAGEN guarantees
70% knockdown or four additional duplexes will be provided.
Efficient siRNA delivery with amaxa’s Nucleofector technology
Efficient delivery › Up to 99% siRNA transfection efficiency already proven in
many different cells.
Low cytotoxicity › Typically over 85% cell viability post nucleofection regardless
of siRNA amount transfected.
No optimization › Ready-to-use kits and protocols for a wide range of cell types.
Flexibility of RNAi application › One nucleofection protocol for siRNA duplexes, vectors or
in vitro transcribed siRNAs.
d amaxa web information amaxa siRNA delivery www.amaxa.com/RNAi
QIAGEN web information QIAGEN siRNA synthesis www.qiagen.com/siRNA
contact information
Australia QIAGEN Pty Ltd • Orders 03-9489-3666 • Fax 03-9489-3888 • Technical 1-800-243-066 › Canada QIAGEN Inc.
Orders 800-572-9613 • Fax 800-713-5951 • Technical 800-DNA-PREP (800-362-7737) › France QIAGEN S.A. • Orders 01-60-920-920
Fax 01-60-920-925 • Technical 01-60-920-930 › Germany QIAGEN GmbH • Orders 02103-29-12000 • Fax 02103-29-22000
Technical 02103-29-12400 › Italy QIAGEN S.p.A. • Orders 02-33430411 • Fax 02-33430426 • Technical 02-33430414 › Japan
QIAGEN K.K. • Telephone 03-5547-0811 • Fax 03-5547-0818 • Technical 03-5547-0811 › Switzerland QIAGEN AG • Orders 061-319-30-30
Fax 061-319-30-33 • Technical 061-319-30-31 › United Kingdom QIAGEN Ltd. • Orders 01293-422-911 • Fax 01293-422-922
Technical 01293-422-999 › USA QIAGEN Inc. • Orders 800-426-8157 • Fax 800-718-2056 • Technical 800-DNA-PREP (800-362-7737)

› Application note
RNAi in suspension cells using amaxa’s
Nucleofector technology together with
QIAGEN ® ’s HPP Grade siRNA duplexes
Elke Lorbach*, Dietmar Lenz*, Andrea Klaes*, Katharina Hein*, Bodo Ortmann*, Tatjana Males*, Hanns-Martin Schmidt*,
Peter Hahn**, Wolfgang Bielke**, Günter Kraus*
Successful siRNA gene silencing depends on many different parameters such as mRNA turnover, mRNA abundance or
half-life of the protein. Two important factors that can positively influence your siRNA experiment are the design of
the siRNA oligo and the efficient delivery of the siRNA. Here we show that HPP Grade siRNA from QIAGEN works well
in combination with amaxa’s Nucleofector technology and can be used to efficiently silence genes in various
hard-to-transfect suspension cell lines as well as in primary cells.
* amaxa GmbH,
Köln, Germany
** QIAGEN GmbH,
Hilden, Germany
Introduction
RNA interference (RNAi) allows targeted
knockdown of gene expression by introducing
short interfering RNA (siRNA),
homologous to the mRNA of the target
gene, into cells. This method of gene
silencing has become a valuable tool in
functional genomics research, target
validation, and gene-specific therapeutic
research. Successful RNAi experiments
are dependent on two major parameters:
siRNA design and efficient delivery of
siRNA into cells.
QIAGEN and amaxa have joined forces
to provide a complete solution for RNAi
that overcomes the problems associated
with the key issues of siRNA design
and delivery into mammalian cells.
amaxa’s R&D team has demonstrated
efficient siRNA delivery in suspension
cells using QIAGEN’s HPP (High-Performance
Purity) Grade siRNA and amaxa’s
Nucleofector technology.
Figure 1: Easy siRNA design
Screenshot of the QIAGEN siRNA Design
Tool.
In this study, efficient silencing of the
housekeeping gene vimentin and the
T cell receptor genes, CD2 and CD4, is
shown in a variety of suspension cell lines,
such as JURKAT, U937, THP-1, HL-60 and
K562, as well as in non-dividing, unstimulated
primary human T cells.
siRNA design and synthesis
The design and quality of an siRNA has
a strong influence on the level and
specificity of gene silencing. QIAGEN
provides a siRNA Design Tool that uses
state-of-the-art design criteria for easy
design of siRNA (Figure 1).
To further minimize the risk that a particular
siRNA will not result in effective
silencing, 4-For-Silencing siRNA Duplexes
are available. These are a set of four HPP
Grade siRNA duplexes, designed by
QIAGEN scientists to target the gene of
choice. QIAGEN guarantees 70% mRNA
knockdown with at least one of these
1000
900
800
700
600
500
400
300
200
Figure 2: High-purity siRNA for
efficient gene silencing
QIAGEN performs MALDI-TOF mass
spectrometric analysis on every HPP
Grade siRNA to confirm its integrity.
› page 7 › www.amaxa.com
Intensity
100
0
5000 5500 6000 6500 7000 7500 8000 8500
Mass/Charge
siRNA duplexes, otherwise four additional
siRNA duplexes are provided without
additional cost.
siRNAs directed against a range of
endogenous and reporter genes are
available for developing and optimizing
knockdown experiments. In addition,
library sets are available for screening
large numbers of genes. A regularly
updated list can be found at
www.qiagen.com/siRNA. QIAGEN’s HPP
Grade siRNA is delivered >90% pure,
eliminating the time and expense needed
for HPLC or PAGE purification (Figure 2).
siRNA delivery with Nucleofector
technology
The Nucleofector technology is a nonviral
method for transfection of hard-totransfect
cell lines and primary cells.
It is based on two components, the
Nucleofector Device, which delivers
unique electrical parameters, and the
Nucleofector Kits which contain cell-specific
and optimized Nucleofector Solutions.
Ready-to-go protocols are available
for many cell lines and primary cell types.
The technology is suitable for the delivery
of different substrates such as DNA, RNA
or siRNA oligos without further optimization
of the nucleofection conditions.
Suspension cells are difficult to transfect
with conventional transfection
methods, e.g. lipid-based reagents, with
both plasmid DNA and siRNA oligos. In
this study, we show that siRNA oligos
can be efficiently delivered into many
different suspension cell types with the
Nucleofector technology and that
effective gene silencing can be achieved
as shown by different methods,
such as flow cytometry, western blot
and quantitative RT-PCR.

› Application note
JURKAT
JURKAT
HL-60
HL-60
K562
K562
THP-1
THP-1
Figure 3: Up to 99% siRNA delivery
into suspension cells
Different suspension cell lines were
nucleofected using the appropriate
Nucleofector Kit and Rhodamine-labeled
siRNA. Three hours post nucleofection
cells were analyzed by light and
fluorescence microscopy.
Gene silencing of various genes in
suspension cell lines
In order to determine the efficiency of
nucleofection with siRNA duplexes,
Rhodamine-labeled siRNA [QIAGEN] was
transfected into different suspension
cell types, such as JURKAT, HL-60, K562
and THP-1 cells (Figure 3). Cells were
analyzed by fluorescence microscopy
and siRNA delivery was achieved with up
to 99% efficiency.
JURKAT, HL-60, K562, THP-1, U937 and
primary human T cells were transfected
with siRNA targeted to the housekeeping
gene vimentin [QIAGEN, library
siRNA; Cat.No. 1022055]. 24 and 48 hours
after nucleofection, silencing of vimentin
gene expression was determined by
quantitative, real-time RT-PCR (Figure 4).
This experiment clearly demonstrates
that expression of vimentin mRNA was
downregulated with silencing effects
between 70 and 90%. Furthermore, it
% relative expression
› page 8 › amaxa news 02
100
75
50
25
0
shows that this level of silencing is
dependent on the cell type and time of
analysis due to different mRNA kinetics
in different cell types.
Knockdown of vimentin was also shown
at protein level using western blot.
JURKAT cells were transfected with
three different concentrations of siRNA
targeted to the vimentin gene (Figure 5).
Already after 24 hours, the amount of
vimentin protein was clearly reduced
using 0.7 µg siRNA. After 48 hours, the
expression was further downregulated
and with 0.7 µg the signal for vimentin
was no longer detectable. These data
indicate that silencing occurs also on the
protein level and that the silencing
effects are dose dependent.
Two other suspension cell lines, the
promyelocytic cell line HL-60 and the
monocytic leukemia cell line THP-1, were
used to show gene silencing of the CD4
Figure 4: Gene silencing of vimentin in various suspension cell types
Six different suspension cell types were transfected with 1.4 µg siRNA targeted to
vimentin or with 1.4 µg control siRNA using the cell-type specific Nucleofector
protocols. After 24 and 48 hours total RNA was prepared from cells and used for quantitative
real-time RT-PCR analysis. The expression level of vimentin mRNA was normalized
to the expression of a housekeeping gene and quantified using a standard curve.
kDa
62
Vimentin -
47
Actin -
32
Human T cells JURKAT THP-1 U937 K562 HL-60
M
Vimentin knockdown at protein level in JURKAT cells
24 hours 48 hours
No siRNA +
nucleofection
No nucleofection
1.4 µg siRNA
0.14 µg
0.7 µg
Figure 5: Vimentin knockdown at protein level in JURKAT cells
JURKAT (ATCC) cells were transfected using Nucleofector Kit V, program C-16 and
0.14 µg, 0.7 µg, or 1.4 µg siRNA targeted to vimentin. Western blot analysis was carried
out 24 and 48 hours post nucleofection. Control lanes show samples with siRNA
added without nucleofection and with nucleofection, but no addition of siRNA. The
level of actin expression is shown for comparison. M=markers.
1.4 µg
M
control siRNA
vimentin siRNA (24h)
vimentin siRNA (48h)
No siRNA +
nucleofection
0.14 µg
0.7 µg
1.4 µg
M

q
q
› Application note
counts
For further
info on siRNA
design and
synthesis go to
www.qiagen.com
/siRNA.
For further info
on siRNA
delivery go to
www.amaxa.com
/RNAi.
180
150
120
90
60
30
0
100 101 102 103 104 0.14 µg CD4 siRNA
10
CD4-PE
0 101 102 103 104 0.35 µg CD4 siRNA
10
CD4-PE
0 101 102 103 104 0.7 µg CD4 siRNA
10
CD4-PE
0 101 102 103 104 1.4 µg CD4 siRNA
10
CD4-PE
0 101 102 103 104 1.0 µg CD4 siRNA
CD4-PE
Figure 6: Optimization of siRNA
amounts in HL-60 cells
Five different amounts of siRNA duplexes
targeting human CD4 were nucleofected
T cell receptor gene. Both cell types
were transfected with siRNA targeted
to the CD4 gene [QIAGEN, library
siRNA; Cat.No. 1024675]. 48 hours after
delivery of siRNA, downregulation of
CD4 was analyzed by flow cytometry
(Figure 6 and 7). In HL-60 cells five
different amounts of siRNA duplexes
were transfected, and down regulation
of CD4 expression occurred in a dosedependent
manner. The most pronounced
gene silencing effects were achieved
with 1.4 µg siRNA (Figure 6). In THP-1
cells, 1.4 µg siRNA was transfected and
effective knockdown of CD4 expression
was achieved (Figure 7). Transfection
of non-specific control siRNA duplexes
did not influence the expression of
CD4.
Successful gene silencing in primary
human T cells
Efficient delivery of siRNA oligonucleotides
into primary cells is the key
180
120
60
0
counts 240
Fig. 7
10 0 10 1 10 2 10 3 10 4
CD4-PE
CD4 siRNA
control siRNA
no siRNA + nucleofection
Figure 7: CD4 Gene Silencing in
THP-1 Cells
THP-1 cells were transfected using
Nucleofector Kit V and program V-01
and 1.4 µg siRNA targeted to CD4.
48 hours after nucleofection, cells were
stained with anti-CD4-PE antibody and
analyzed by flow cytometry. Controls
included nucleofection of 1.4 µg control
siRNA and nucleofection without siRNA.
into HL-60 cells using Nucleofector Kit
V and program T-19. 48 hours after nucleofection,
cells were stained with anti-
CD4-Phycoerythrin (PE) antibody and
bottleneck before functional genomic
studies can be performed under in vivolike
conditions, such as those found in
primary cells. Here we show that primary
human T cells can be used for siRNA
experiments with the Nucleofector
technology. Unstimulated human T cells
were transfected with siRNA targeted to
the pan T cell receptor gene CD2 [siRNA
sequence published by D. Unutmaz in
amaxa application note: RNAi-mediated
CD2 suppression in primary human T
cells, WTB-1001]. 48 hours after nucleofection,
the downregulation of CD2
expression could be shown by flow cytometry
(Figure 8A). Expression of the
CD4 receptor was not influenced as
demonstrated by control staining with a
PE-coupled CD4 antibody and flow cytometric
analysis (Figure 8B). This clearly
indicates that the gene silencing effect
achieved was CD2 specific thus proving
the Nucleofector technology an efficient
tool to transfect siRNA duplexes
CD2 siRNA
control siRNA
no siRNA + nucleofection
Figure 8: High specificity of CD2
siRNA in primary human T cells
Primary human T cells were nucleofected
with 1.4 µg siRNA targeted to CD2
using the Human T Cell Nucleofector
Kit. Cells were stained with A anti
› page 9 › www.amaxa.com
counts
200
160
120
80
40
0
Fig. 8A
10 0 10 1 10 2 10 3 10 4
CD2-FITC
counts
analyzed by flow cytometry. siRNA targeted
against vimentin was used as control.
CD4 siRNA control siRNA
not only into suspension cell lines but
also into primary suspension cells.
Trademarks and disclaimers
Nucleofector,
Nucleofection (amaxa GmbH)
QIAGEN ® (QIAGEN GmbH)
siRNA technology licensed to QIAGEN is
covered by various patent applications,
owned by the Massachusetts Institute of
Technology, Cambridge, MA, USA and
others.
© 2003 amaxa, all rights reserved.
QIAGEN guarantees that under appropriate
transfection conditions at least
one of the 4-For-Silencing siRNA Duplexes
will reduce expression of your target
mRNA by 70%. If not, QIAGEN will
design and supply four additional siRNA
duplexes against the same gene target
at no additional charges.
300
240
180
120
60
0
Fig. 8B
10 0 10 1 10 2 10 3 10 4
CD4-PE
CD2-FITC antibody or B anti CD4-PE
antibody, and analyzed by flow cytometry
44 hours after transfection.
Controls included nucleofection with
1.4 µg control siRNA and nucleofection
without siRNA.

Step 1
Step 2
› New products
new
Nucleofection of mouse
embryonic fibroblasts (MEF) and
mouse embryonic stem cells
From cell differentiation to knock-out mice - amaxa introduces the new Nucleofector Kits for transfection of mouse
embryonic fibroblasts (MEF) and mouse embryonic stem cells (mouse ES cells). Transfection of MEF and mouse ES
cells has broad research potential. Both kits will be important for research on cell differentiation, organogenesis, cellbased
therapies, testing of new drugs, and gene function approaches like ‘loss-of-function’ and ‘gain-of-function’ studies.
The MEF strategy –
two simple steps for successful transfection
MEF display significant phenotypic variations
which depend on the mouse strain, the genetic
background of the donor mice as well as the
immortalization strategy. Due to these manifold
properties, each MEF type may behave differently
MEF Starter Kit
The MEF Starter Nucleofector Kit is designed to
determine the ideal nucleofection conditions for
each MEF cell type. This is accomplished in a total
MEF Kits 1 and 2
After you have determined the appropriate MEF
Nucleofector Kit for your MEF strain you can
› page 10 › amaxa news 02
with respect to the optimal conditions for transfection.
amaxa has addressed this by developing two MEF Nucleofector
Kits (1 and 2) and an easy-to-use MEF Starter Nucleofector
Kit. This strategy allows fast and individual nucleofection of your
MEF cells.
non-viral transfection › efficient transfection of MEF cells
simple optimization strategy › two step optimization with MEF Nucleofector Starter Kit
applicable for virtually any MEF strain › taking into account the significant phenotypic strain variations
high cell viability › viability from 60 - 80% (in strains tested so far)
1 Solution MEF 1 MEF 2
program 1 A-23 A-23
program 2 T-20 T-20
of just four reactions using MEF 1 and MEF 2 Nucleofector Solution
with two different programs. In the next step the appropriate MEF
Nucleofector Kit (1 or 2) can be used to perform the experiments.
>>>
2
3
Order MEF 1 or 2 Nucleofector TM Kit
+
>>>
nucleofect the cells with MEF Nucleofector Kit 1 or 2 and the
appropriate program.

› New products
60
50
40
30
20
10
0
% transfection efficiency
A B
MEF
protein. 24 hours post nucleofection, the cells were analyzed
Example for transfection efficiencies of primary MEF
MEF were nucleofected with a plasmid encoding the eGFP
by fluorescent microscopy. (A) Mouse strain C57BL/6, spontaneously
immortalized, passage 20, 1.5 µg eGFP (Courtesy
of Dr. H. Hermanns and Prof. P.H. Heinrich, University of
Aachen, Germany). (B) Mouse strain C57B6Jx129SV, SV40
transformed, passage 4, 7 µg eGFP (Courtesy of Prof. Saftig,
University of Kiel, Germany)
Research with mouse ES cells has already made
its way to a sophisticated assay system for
studying diverse biological questions. Transgenic
technology now allows to create mice with
various genotypes and assess the consequences
of this genotype in the context of developing and
adult animals. Transfection of mouse ES cells will
support the widespread adoption of the transgenic
system and the research on differentiation
of mouse ES cells.
Nucleofector technology is an efficient alternative
to standard electroporation to create
[k] [k]
Ordering information
MEF Starter Nucleofector Kit Cat. No.: VPD-1006
MEF Nucleofector Kit 1 Cat. No.: VPD-1004
MEF Nucleofector Kit 2 Cat. No.: VPD-1005
› page 11 › www.amaxa.com
Example for nucleofection of primary MEF
Spontaneously immortalized mouse embryonic fibroblasts (strain C57B6Jx129SV)
were nucleofected using the MEF Nucleofector Kit 1 and a plasmid encoding the
enhanced green fluorescent protein eGFP. 24 hours post nucleofection, the cells
were analyzed by fluorescence microscopy. (Photograph courtesy of Dr. H. Hermanns
and Prof. P.H. Heinrich, University of Aachen, Germany)
Nucleofection of Mouse ES cells
knockout mice. In addition, the cell-type specific nucleofection
conditions optimized for gentle DNA delivery in vitro support
differentiation of transfected mouse ES cells. Together with other
Nucleofector Kits for primary mouse cells, genetic manipulation
and analysis of various cell types is made possible.
Successfully tested ES cell lines:
› WW6
› R1
› CK-35
› H7
› U295V (an E14 subclone)
Ordering information
Mouse ES Cell Nucleofector Kit Cat. No.: VPH-1001

H
› Tips and hints
Selection of appropriate Nucleofector Kit
Each Nucleofector Kit contains a specific
Optimized Protocol for the respective cell type.
This protocol can be used for the nucleofection
of either siRNA oligos or vectors without any
further changes of electrical parameters.
Methods for generating siRNA
Several different methods for generating siRNA
are known; such as:
› synthetic siRNA oligos
› in vitro transcribed oligos
› siRNA expression vectors
Take a short cut for your
RNAi experiments –
the ‘essentials’ about Nucleofector technology and RNAi
The Nucleofector technology has proven its capability for efficient delivery of siRNA into hardto-transfect
cell lines and primary cells. To save your time and allow for a successful setup of RNAi
experiments in your lab we compiled some information on the ‘essentials’ about RNAi and the
Nucleofector technology. Some of the most crucial aspects are listed below. The opposite page
outlines how to establish RNAi for your particular cell type. For further questions on your RNAi
experiments contact our Scientific Support Team.
Tips for a successful
siRNA experiment
All methods can be used together with the
Nucleofector technology for efficient delivery
of siRNA into primary cells and cell lines.
› page 12 › amaxa news 02
siRNA oligo design
Design and selection of siRNA oligo target sequences play an
important role. In general, testing of 2-6 different siRNA oligos is
sufficient to find one or more siRNA oligos that display good silencing
effects. We recommend using Qiagen’s siRNA oligos and
design software.
siRNA oligo concentration
The amount of siRNA oligo required to efficiently downregulate a
specific gene has to be determined empirically and depends on
many parameters, such as efficiency of siRNA oligo sequence,
method of oligo preparation, cell type, mRNA abundance, mRNA
turnover etc. amaxa’s R&D team recommends to use between 0.14
and 1.4 µg siRNA.
Labeled siRNA oligos
Fluorescent labeled siRNA can be used to analyze transfection
efficiency. FITC or Rhodamine labeled siRNA oligos should be
analyzed after 0.5-3 hours. Cy5 labeled siRNAs can be detected
up to 24 hours post nucleofection.

d
› Tips and hints
eGFP
amaxa web information www.amaxa.com/RNAi
A simple strategy for RNAi in cell lines and primary cells
Follow the scheme below for RNAi experiments in your primary cell or cell line of interest.
primary cells
Optimized Protocol
available / yes
Choose the appropriate Nucleofector
Kit for cell type of interest.
Use the Optimized Protocol and an eGFP
plasmid to establish the Nucleofector
technology for your cell type.
Alternatively, fluorescence labeled
siRNA oligos can be used.
Establish RNAi experiments with positive
control siRNA oligos, e.g. vimentin or
lamin A/C.
Nucleofection of eGFP plasmid should
be included as a positive control for
transfection efficiency.
Efficient gene silencing in primary cells
› page 13 › www.amaxa.com
cell lines
Use Cell Line Optimization
Nucleofector Kit.
Optimize nucleofection conditions with
eGFP plasmid according to the optimization
strategy:
Use optimized parameters to establish
RNAi experiments.
V
Optimized Protocol
available / no
Solution R T V
R T
V
program 1 A-23 A-23 A-23
program 2 A-27 A-27 A-27
program 3 T-20 T-20 T-20
program 4 T-27 T-27 T-27
program 5 T-16 T-16 T-16
program 6 T-01 T-01 T-01
program 7 G-16 G-16 G-16
program 8 O-17 O-17 O-17
Efficient gene silencing in cell lines.
+

› Product update
Nucleofector technology and RNAi -
examples for successful knockdown provided by Nucleofector users
The following examples demonstrate an efficient targeted knockdown of gene expression by introducing siRNA via
the Nucleofector technology. In addition, the data show the suitability of the Nucleofector technology in answering
scientific questions by RNAi especially in hard-to-transfect cell lines and primary cells for your basic and applied
research needs.
* winner of publication award, see page 18
RNAi to study the caspase-8 mediated
activation of T cells
Chun et al. (2002) (1) studied the role of caspase-8
in T lymphocyte activation. Therefore, resting
T lymphoctes were nucleofected with siRNA
against caspase-8. Transfected cells were then
stimulated with CD3 and CD28, and the effect on
T cell activation markers CD25 and CD69 was
analyzed by FACS. The data nicely show a specific
downregulation. Caspase-8 siRNA leads to a
reduction in activation marker expression
indicating a role of caspase-8 not only in apoptosis
but also in T cell activation.
Downregulation of eGFP in primary mouse
neurons with siRNA
Primary mouse neurons were co-nucleofected
with GFP and siRNA against GFP. GFP expression
was significantly downregulated.
› page 14 › amaxa news 02
A CD69 (fold induction) B CD25 (fold induction)
60
50
40
30
20
10
NS C8
0 Pr2 Pr2 Pr3 Pr3 NS
20 40 20 40 50 nM
Figure 1: Downregulation of lymphocyte activation by nucleofection of caspase-8
siRNA oligonucleotides. Resting PBLs were transfected with the Nucleofector technology
with siRNA oligonucleotides in two different concentrations that were either nonspecific
(NS) or specific for caspase-8 (Pr2 and 3) and were then stimulated with CD3 and
CD28 for 18 hours. Induction of surface expression of CD69 is shown in (A) and CD25 in
(B). Insets show by RT-PCR that less caspase-8 mRNA is produced with siRNA pair 3 (C8)
than with non-specific siRNA (NS). (Data reproduced with permission from Nature)
A B
C D
30
25
20
15
10
5
0 Pr2 Pr2 Pr3 Pr3 NS
20 40 20 40 50 nM
Figure 2: Downregulation of eGFP in primary mouse cortical neurons with siRNA
oligonucleotides. Cortical neurons isolated from mouse embryos (E17) were co-nucleofected
using the Mouse Neuron Nucleofector Kit with a plasmid encoding the eGFP
protein (CMV promoter) and siRNA oligonucleotides against eGFP. Four days post
nucleofection, cells were fixed, mounted on a cover slide and analyzed by fluorescence
microscopy. Nuclei were stained with DAPI (C, D) to show comparable number of cells
per sample. Compared to control (A), overexpression of eGFP can be significantly
downregulated in co-nucleofection experiment (B). (Courtesy of H-J. Kim and T.
Vartanian, Beth Israel Deaconess Medical Center, Dept. of Neurology Boston, MA, USA)

d
› Product update
RNAi to study the early commitment phase of
apoptosis
Bidere et al. (2003) (2) analyzed the mitochondrial
apoptotic pathway activated by staurosporine
(STS). The publication reveals the role of Cathepsin
D (Cat D), Bax and AIF (apoptosis inducing
factor) in activating the mitochondrial pathway.
Cat D, Bax and AIF were efficiently knocked down
by nucleofection of specific siRNA in primary
human T cells. Subsequent stimulation of apoptosis
with STS showed that Cat D triggers Bax
activation and AIF release.
The examples shown here addressed the sequential
relationship between Cat D activity, Bax conformational
activation, and mitochondrial AIF
release. Optimum Cat D knockdown was found at
1.5 µM Cat D-siRNA (Figure A). Following exposure
to STS, these cells no longer displayed Bax
immunoreactivity (Figure B), nor AIF relocation
(data not shown). It was shown that the depletion
of Cat D protein by RNAi results in the
suppression of Bax activation and of AIF release
in STS-treated cells.
› page 15 › www.amaxa.com
Figure 3: Downregulation of Cat D expression by RNAi results in the
suppression of Bax activation in primary human T lymphocytes
Resting human T lymphocytes were nucleofected with Cat D-siRNA. Negative controls
consisted of untransfected cells, of mock transfected cells, and of cells transfected
with an inefficient siRNA (control siRNA). 16 hours later cells were stimulated
with the mitogenic CD2 mAb pair (GT2 + T11.1) for four days. (A) shows percentages
of cells displaying Cat D-associated immunofluorescence (cells are detected by DAPI
staining and conventional fluorescence microscopy). (B) Four days after stimulation,
Cat D-siRNA-transfected cells were incubated with 100 nM STS for 90 min. Cells were
counted for Bax-NT associated immunofluorescence by conventional microscopy.
(Data taken from Bidere et al. (2003); reproduced with permission from Journal of
Biological Chemistry)
References
1. Chun HJ et al. (2002) Nature; 419:395-399
2. Bidere N et al. (2003) J Biol Chem; 278:31401-31411
amaxa web information RNAi www.amaxa.com/RNAi
publications www.amaxa.com/citations
% Cat D positive cells
A Cat D knockdown B Suppression of Bax after
Cat D down regulation
100
90
80
70
60
50
40
30
20
10
0
not transfected
0 1.5 0.75 1.5(µM)
mock
control
Cat D
Cat D
% Bax-NT positive cells
100
75
50
25
siRNAs not transfected
0
0 1.5 1.5(µM)
mock
pretreatment
STS
no
control
siRNAs
Cat D

› Background information
Basics about nucleofection and
the easiest way to transfect your
cell line
Thank you for your remarks on our first newsletter. Due to the great response to the first edition of ‘amaxa news’ we
selected two of the most frequent requested topics to be further highlighted in this edition of amaxa news. Get information
on the Nucleofector technology basics and learn more about the easiest way to transfect your cell line of
interest. In addition, we set up the ‘amaxa user forum’ to give you the opportunity to discuss your topics of interest
with other Nucleofector users (see page 19 in this newsletter).
The Nucleofector technology - what is it all about?
The Nucleofector technology is a major breakthrough in transfection
since it delivers DNA straight into the nucleus of cell lines,
primary tumor cells and primary cells. This is achieved by an
optimization of the two components of nucleofection: electrical
parameters and cell type specific surroundings. The Nucleofector
Device delivers unique electrical parameters – already individually
optimized for each cell type. For successful transfection, you just
select the appropriate Nucleofector program and press ‘Start’.
The Nucleofector Kits contain Nucleofector Solution and
Supplement, specifically developed for each cell type. They provide
a cell-friendly environment during the nucleofection procedure
and support the delivery of DNA straight into the cell nucleus
yielding high efficiencies and cell viability.
This combination guarantees gene expression shortly after
transfection (see Figure 1) even in non-dividing primary cells.
The Nucleofection procedure does not impair cell functionality.
Nucleofected cells show long-term survival in culture and can
easily be differentiated or stimulated. Check our homepage
to view citations for the successful use of the Nucleofector
technology (www.amaxa.com/citations).
Nucleofector technology at a glance
› DNA delivery straight into the nucleus
› short time until analysis
› maintenance of cell functionality
› applicable for many different substrates
› ready to use protocols
› page 16 › amaxa news 02
Nucleofector technology – DNA delivery straight
into the nucleus
50
40
30
20
10
0
percentage of cells (n=3)
Expression of cytoplasmic eGFP and surface
molecule H-2Kk in unstimulated human T cells
10 30 100 300 1000
time after nucleofection (min)
Figure 1: Expression kinetics of two proteins showing the
early start of protein expression after nucleofection in
unstimulated primary human T cells.
Human PBMCs were nucleofected with either the cytoplasmic
protein eGFP or the surface protein H-2K k , a mouse MHC class I
heavy chain molecule. Due to the direct transport of DNA into
the cell nucleus, expression of eGFP can already be detected
after 40 min. Expression of H-2K k is delayed as the protein has
to be processed and transported to the cell surface.

d
› Background information
How do I nucleofect my cell line?
The easiest way to find the appropriate nucleofection
conditions for your cell line is to visit our
webpage at www.amaxa.com/celldatabase.
The amaxa cell database on our homepage gives
you extended information on the nucleofection
conditions of many different cell lines. One of the
following three cases may apply for your cell line
of interest:
1. Cell lines optimized by amaxa: These are cell
lines where amaxa’s R&D team has developed
a ready-to-use Optimized Protocol indicating
optimal nucleofection conditions such as best
Nucleofector programs and Nucleofector
Solution. These Optimized Protocols can be
downloaded from our webpage.
2. Customer cell line data: For a multitude of cell
lines we can offer preliminary data from other
scientists who used the Cell Line Optimization
Nucleofector Kit. These data may help you
with your research by providing basic nucleofection
conditions for your particular cell type.
This database is an increasing source of information
and will provide a platform to share
amaxa web information Nucleofector technology www.amaxa.com/technology publications www.amaxa.com/citations
overview www.amaxa.com/celldatabase user forum forum.amaxa.com
› page 17 › www.amaxa.com
your experience with other Nucleofector users. Therefore,
amaxa encourages you to submit your transfection results
including transfection efficiency and viability. Simply fill out
the optimization form and receive a gift for your contribution.
3. Any other cell line: For any other cell line not yet optimized by
amaxa or tested by other Nucleofector users, use our Cell
Line Optimization Nucleofector Kit to find the optimal
nucleofection settings.
www.amaxa.com/celldatabase
› Complete listing of all Nucleofector Kits for primary cells and
cell lines
› Overview of average transfection efficiencies for primary cells
and cell lines
› Links to Optimized Protocols
› Links to product-specific information
› Access to amaxa cell line database for obtaining and submitting
transfection data
Cell Line Optimization Nucleofector Kit
› Achieve transfection for virtually any cell line or primary
tumor cell.
› Contains three Nucleofector Solutions and gives eight
programs for a simple optimization within just two weeks.
› Constant support and fine tuning from amaxa’s Scientific
Support Team.

d
› Produkt amaxa insights News
› Highlighted paper
Fly amaxa competition
winners announced
amaxa is pleased to announce the winners of the 2003 “Fly amaxa” publication awards, established to reward published
studies with the highest scientific value that used Nucleofector technology. The high quality of publications submitted
for consideration made the selection process extremely difficult for our scientific committee and we would like to
congratulate and thank all of the participants. The prize money listed for each entry below is awarded to help fund
attendance at a scientific conference of the winner’s choice.
1 st prize $ 2.000
2 nd prize $ 1.500
3 rd prize $ 1.000
Hyung J. Chun, for his publication
”Pleiotropic defects in lymphocyte activation caused by caspase-8 mutations lead to
human immunodeficiency.” (Nature 2002; 419:395-399)
Julie Harriague, for her publication
”Imaging antigen-induced PI3K activation in T cells.”
(Nature Immunology 2002; 3:1090-1096)
Hyeseon Cho, for his publication
”Pericyte-specific expression of Rgs5: implications for PDGF and EDG receptor
signaling during vascular maturation.” (FASEB Journal 2003; 17:440-442)
Highlighted paper
Messi M, Giacchetto I, Nagata K, Lanzavecchia A, Natoli G, Sallusto F. (2003) Memory and flexibility of cytokine gene
expression as separable properties of human TH1 and TH2 lymphocytes. Nat Immunol. 4(1):78-86.
% Cytokine positive cells
40
30
20
10
0
HA -
HA +
IFN-g IL-4
= T H1 = T H2
› page 18 › amaxa news 02
The authors studied memory of flexibility of differentiated T lymphocytes an their
ability to switch between the T helper cell type 1 (TH1) and TH2 lineage.
In the experiment shown here, TH2 cells were nucleofected with a T-bet expression
vector (HA+, dark blue bars) or control vector (HA-, light blue bars) and analyzed
for cytokine production after stimulation. Overexpression of T-bet transcription
factors caused a re-commitment of the TH2 to the TH1 lineage. Between 12-22% of
TH2 cells expressing ectopic T-bet rapidly acquired the ability to produce IFN-g (TH1 specific response) indicating that T-bet expression plays a major role in commitment
to TH2 lineage.
amaxa web information citations www.amaxa.com/citations

d
› amaxa insights
new
Discuss your
topics around
nucleofection
Join now
amaxa user forum
An increasing number of laboratories worldwide
are using the Nucleofector and gaining
extensive experience on transfection with the
Nucleofector technology. This knowledge is an
important source of information for potential
users and those that are already applying the
technology for a variety of different research
questions. To give you the opportunity to share
Be part of the ‘Nucleofector community’! You
can access the user forum via the amaxa homepage
(www.amaxa.com). Just click on ‘user
forum’ in the horizontal navigation bar. Alterna-
amaxa web information user forum forum.amaxa.com
› page 19 › www.amaxa.com
and benefit from this information we set up the
amaxa user forum. You can post your questions
or comments in four categories:
› primary cells
› cell lines
› siRNA
› miscellaneous
tively enter ‘forum.amaxa.com’ in your browser
to access the forum directly. To post a message
you have to register. Simply follow the instructions
on the forum page.

[k]
› amaxa worldwide
WAKO Pure Chemicals –
amaxa’s competent partner in Japan
When Japanese scientists want to use the Nucleofector technology, they can trust in an outstanding
partner. WAKO Pure Chemicals has been distributing the Nucleofector technology and
the complete range of Nucleofector Kits to Japanese scientists since February 2002. WAKO has
established the Nucleofector technology as state of the art in primary cell and cell line transfection
in Japan. This is reflected by the increasing number of publications describing the successful use of
the Nucleofector technology in Japanese laboratories. Join us to learn more about amaxa’s partner
and the support WAKO provides to the scientific community in Japan.
One of WAKO’s missions is to
provide excellent support to the
Japanese scientists and to help
them in their daily workflow.
They aim to be a ”R & D oriented
corporation” and a ”corporation
trusted by customers”.
Construction of an information
network and complete scientific
WAKO research laboratory in Osaka, support is certainly one reason
Japan
for the great success in Japan.
In addition to the headquarters in Osaka, WAKO has a branch
office in Tokyo and 12 regional offices. Furthermore WAKO works
with 62 partners located in 218 places throughout Japan. As a
result of this, approximately 1100 sales assistants provide competent
customer support and are complemented by experienced
scientific support staff.
Founded in 1922, WAKO Pure Chemical Ind., Ltd. is well known in
Japanese labs as a supplier of laboratory chemicals and diagnostic
reagents with production facilities in Japan, Europe und the
United States and a proprietary distribution system and research
Ordering and scientific support ooga.yoshinobu@wako-chem.co.jp
› page 20 › amaxa news 02
laboratories. With amaxa’s Nucleofector technology
WAKO has extended its product portfolio
in the Life Sciences field.
As a matter of course WAKO has designed specific
catalogs, newsletters, hand-outs and brochures
in Japanese. Furthermore, WAKO generated
siRNA data in cooperation with Nippon Genetech,
a local supplier of chemically synthesized
siRNA oligonucleotides, demonstrating the
functionality of the Nucleofector technology
also in the area of RNAi.
Constant exchange of information is guaranteed
since WAKO participates in scientific meetings
and organizes workshops and seminars. At regular
intervals WAKO hosts seminars during major
scientific conferences where amaxa scientists as
well as Japanese users of the Nucleofector
technology share their extensive transfection
knowledge and hands-on experience.
d
WAKO web information www.wako-chem.co.jp

d
› amaxa worldwide
Meet amaxa at your bench
One of the most likely ways in which you first hear about amaxa is through one of our experienced European or US
sales managers. Not only do they demonstrate the Nucleofector technology in your lab and arrange a test use for
you, they normally act as the first contact person in answering your questions. To ensure that they give you the best
possible customer support amaxa continually provides each sales manager with trainings to keep them well informed.
Meet your amaxa sales representative on the
phone, in your lab or at our booth during a scientific
conference. Check amaxa’s web page to find
out who your local sales representative is or call
our Scientific Support Team for contact details.
Australia / New Zealand
Integrated Sciences Pty. Ltd.
phone +61-1800-252-204
email tech@integratedsci.com.au
Canada
ESBE Scientific
phone +1-800-268-3477
email custserv@esbe.com
China
Jingmei Biotech co. Ltd.
phone +86-7558-3546 194
email shenzhen@jingmei.com
Czech Republic
KRD - molecular technologies sro
phone +42-02-5162 6019
email viktor.k@krd.cz
Denmark
REACTIONLAB AS
phone +45-7027 9595
email info.denmark@reaction-lab.com
Finland
REACTIONLAB OY
phone +358-2-410 17 46
email info.finland@reaction-lab.com
India
Genetix Biotech Asia Pvt. Ltd.
phone +991-112 542 1714
email genetix@nda.vsnl.net.in
amaxa’s international sales team
amaxa’s distributors around the world
Israel
Tec-O-Pharm-Libra
phone +972-2-587 08 75
email itzik@toplibra.co.il
Italy
Instrumentation Laboratory spa
phone +39-0225-22 766
email beretta@itmil.ilww.com
Japan
Wako Pure Chemical Industries, Ltd.
phone +81-6-6203-2756
email ooga.yoshinobu@wako-chem.co.jp
Korea
Young In Scientific Co., Ltd.
phone +82-2-519 73 45
email shinhyuk@youngin.com
Malaysia
Research Biolabs Malaysia
phone +603 8070 3101
email biolabs@tm.net.my
Norway
REACTIONLAB AS
phone +47-66 98 97 42
email info.norway@reaction-lab.com
Poland
MEDianus
phone +48-12-665 31 31
email medianus@medianus.net
Singapore
Research Biolabs Pty. Ltd.
Singapore
phone +65-6273 1066
email sales@researchbiolabs.com
Slovakia
KRD - molecular technologies sro
phone +421-2-645 340 41
email krd@krd.sk
Spain / Portugal
IZASA, S.A.
phone +34-902-20 30 90
email CLC@izasa.es
techservice:
email consultasbiotec@izasa.es
Sweden
REACTIONLAB SVERIGE AB
phone +46-18-14 90 00
email info.sweden@reaction-lab.com
Taiwan
Taigen Bioscience Corporation
phone +886-2-2880 2913
email taigen@ms10.hinet.net
Thailand
Bio-Active Co., Ltd.
phone +662-295-4804
email psuwan@inet.co.th
amaxa web information For worldwide ordering details www.amaxa.com/ordering
› page 21 › www.amaxa.com

d
› FAQ
FAQ - frequently asked questions
on Nucleofector technology in neurobiology and immunology
› I noticed amaxa recommends a really
high cell density for plating of nucleofected
neurons. Is this advisable and how do I
get these cell numbers?
›› We recommend to prepare and transfect a
“mixed glial culture”. This provides you with
enough cells to get 4x106 cells per nucleofection
sample from just one mouse. By using of this
“high” amount of cells during nucleofection and
plating them as densely as recommended you
will increase cell survival significantly. The glial
cells support neuron recovery after nucleofection.
By changing the culture medium to a
medium that impairs glial cell growth you can
start depleting these cells 2-4 h after nucleofection
to get a pure neuron culture.
› I’m transfecting neurons in a mixed glial
culture. Is there an ’optimal’ way to
influence cell survival and selection for
neurons after nucleofection?
›› To improve survival it is crucial to change
medium two hours post nucleofection since any
debris present in the culture will impair cell
survival. If you are interested in getting a pure
neuronal cell population as fast as possible you
can switch to culture medium II including the
cytotoxin cytosine arabinoside (ARA-C) at this
time point. Alternatively, you can increase cell
survival by exchanging with culture medium I
(contains no ARA-C) two hours post nucleofection
for another 24 or 48 hours.
More information needed?
It takes just one e-mail, phone call or fax to
amaxa web information
www.amaxa.com/faq/
Find all Optimized Protocols at
www.amaxa.com/celldatabase
› page 22 › amaxa news 02
› Is there a special recommendation on T cell enrichment
before nucleofection?
›› For nucleofection it is preferable to use T cell populations
enriched by magnetic separation (e.g. MACS Microbeads by
Miltenyi Biotec) or by a rosetting method. For magnetic separation
we recommend negative selection or detaching beads after
enrichment. Do not apply protocols using hypo-osmolar buffers.
This may lead to high cell mortality after nucleofection.
› Are there any recommendations to avoid excessive
mortality during T cell nucleofection?
›› Try to keep the DNA amount for nucleofection quite low
(e.g. 1 µg plasmid DNA) since higher DNA amounts might cause
increasing toxicity and mortality. It is also important to use
highly purified DNA (e.g. QIAGEN ® EndoFree ® Plasmids Kits). The
A260:A280 ratio should be at least 1.8 for nucleofection.
› Is there a way to increase cell viability after
nucleofection for dendritic cell transfection?
›› LPS (lipopolysaccharides) has a strong negative effect on cell
viability. Please make sure that all components you use for
dendritic cell culture and transfection, e.g. PBS, FCS and especially
DNA, are LPS free. We recommend additionally purifying your
DNA by precipitating it twice with 20% PEG/2.5 M NaCl.
› I want to stimulate T cells post nucleofection.
Is there anything I have to bear in mind?
›› After nucleofection, just wait for at least four hours before
stimulation. Stimuli immediately after nucleofection may lead to
increased cell mortality.
obtain more information on the Nucleofector technology. Just
contact our Scientific Support Team.
Scientific Support Europe/World USA
phone +49(0)221-99199-400 (240) 632-9110
fax +49(0)221-99199-499 (240) 632-9112
e-mail techservice@amaxa.com techservice.US@amaxa.com

d
› › amaxa Produkt insights News
We are looking forward to
welcoming you at amaxa’s
booth at one of the following
meetings this year!
www.amaxa.com/meetings
RNAi Quest
› › page page 23 › › www.amaxa.com
www.amaxa.com
Meet amaxa in 2003 at:
Nov 14 - 17 European Society for Gene Therapy,
11th Annual Meeting, Booth #1, Edinburgh, Scotland
Nov 25 - 26 International Biotech 2003,
Booth #B102, London, England
Nov 26 - 28 French Society for Immunology – Annual Congress,
Antibes, France
Dec 2 - 5 British Society for Immunology – Annual Congress,
Booth #35, Harrogate, England
Dec 6 - 9 ASH - American Society for Hematology,
45th Annual Meeting, Booth #815, San Diego, USA
Dec 13 - 17 ASCB – American Society for Cell Biology,
43rd Annual Meeting, Booth #1533, San Francisco, USA
Dec 18 - 19 Dutch Society for Immunology – Annual Meeting,
Noordwijkerhout, The Netherlands
newGet the highscore!
amaxa’s new RNAi game
Meet ‘Electroman’ and ‘Solutiongirl’
on their challenging quest
to deliver siRNA into mammalian
cells and to knock out genes.
But watch for the evil mycoplasma
monsters and other traps on
your way!
www.amaxa.com/RNAiQuest
d www.amaxa.com/RNAiQuest

WAA-1001_02
amaxa GmbH
Nattermannallee 1
50829 Koeln
Germany
Scientific Support Scientific Support
Europe/World USA
phone +49(0)221-99199-400 phone (240) 632-9110
fax +49(0)221-99199-499 fax (240) 632-9112
e-mail techservice@amaxa.com e-mail techservice.US@amaxa.com
Nucleofection of rat
oligodendrocytes
Benefit from the Nucleofector
technology for rat oligodendrocytes
> first efficient non-viral transfection
technology for primary
rat oligodendrocytes
> for precursor cells with full
potential for differentiation
into oligodendrocytes
› www.amaxa.com
amaxa Inc.
814 West Diamond Avenue
Suite 110
Gaithersburg, MD 20878
USA
Nucleofection of rat oligodendrocytes
P0/1 rat oligodendrocyte precursor cells
(forebrain) were nucleofected with a
plasmid encoding the enhanced green
fluorescent protein eGFP. Subsequently
the cells were cultured for 7 days in
a chemically defined medium. (Photograph
courtesy of Dr. Wengbin Deng,
Children’s Hospital, Boston, USA)