› Optimized Protocol DCL-1015 Vs.
08-2006
› for H9c2(2-1) [ATCC] page 1 of 7
Cell Line Nucleofector® Kit L
for H9c2(2-1) [ATCC]
Cell type Origin Rat heart myoblast [ATCC® CRL-1446™; frozen vial].
Morphology Myoblast.
Example for nucleofection® of H9c2 (2-1) cells.
A B
% transfection efficiency
100
80
60
40
20
24 h
Average transfection efficiencies of H9c2 (2-1) cells were nucleofected using the Cell Line Nucleofector Kit L, program C-20/C-020 and 2 µg of
H9c2 (2-1) cells. Cells were nucleo- pmaxGFP. 24 hours post nucleofection the cells were analyzed by fluorescence microscopy.
fected with program C-20/C-020 and
2 µg of pmaxGFP™. 24 hours post nuc-
leofection, the cells were analyzed by flow
cytometry. Cell viability (% PI negative
cells) is around 90% 24 hours post nuc-
leofection.
Chapter Contents
1 Procedure outline & important advice
2 Product description
3 Protocol
3.1 › Required reagents
3.2 › DNA preparation and quality
3.3 › Cell culture
3.4 › Important controls and vector information
3.5 › Nucleofection protocol
4 Recommended literature
5 5
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› Optimized Protocol DCL-1015 Vs. 08-2006
› for H9c2(2-1) [ATCC] page 2 of 7
1 Procedure outline & important advice
Procedure outline Important advice
1. Culturing of cells before › Replace medium every 2 - 3 days.
nucleofection. › Passage cells every 2 - 3 days.
(For details see 3.3.) › Subcultivation ratio of 1 : 4 - 1 : 5.
Subculture before confluency to avoid
differentiation.
› Passage 2 - 3 days before nucleofection.
2. Combine the cells of in- Contents of one nucleofection sample:
terest, DNA or siRNA and › 4 x 105 cells (optimal cell number)
the appropriate cell-type › 2 µg highly purified plasmid DNA
specific Nucleofector Solu- (in max. 5 µl) 2 nM - 2 µM siRNA (final
tion and transfer to an concentration, 3 ng - 3 µg/sample)
amaxa certified cuvette. › 100 µl Nucleofector Solution L
›
(For details see 3.5.)
Perform each sample separately to avoid
storing the cells longer than 15 min in
Nucleofector Solution L.
3. Choose the cell-type speci- › Optimal Nucleofector program:
fic program. Insert the C-20*/C-020**
cuvette into the Nucleofec-
tor and press the »X« but-
ton to start the program.
(For details see 3.5.)
› * for Nucleofector I Device
› ** for Nucleofector II Device
4. Rinse the cuvette with cul- › After program has finished, incubate
ture medium using an the sample in the cuvette at room tem-
amaxa certified pipette. perature for 10 min.
Transfer the cells into the › Take a 500 µl aliquot of the prewarmed
culture dish. medium and add it to the cuvette. Imme-
(For details see 3.5.) diately transfer the medium with cells
back to the culture dish and incubate at
37°C/5% CO2.
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› Optimized Protocol DCL-1015 Vs. 08-2006
› for H9c2(2-1) [ATCC] page 3 of 7
2 Product description
Cat. No. VCA-1005
Kit components 2.25 ml Cell Line Nucleofector® Solution L
0.5 ml Supplement 1
30 µg pmaxGFPTM (0.5 µg/µl in 10 mM Tris pH 8.0)
25 certified cuvettes
25 plastic pipettes
Size 25 reactions
Storage and stability Store Nucleofector Solution, Supplement and pmaxGFP at 4°C. For long term
storage pmaxGFP is ideally stored at -20°C.
The expiry date is printed on the Solution Box.
3 Protocol
3.1 › Required reagents
Medium Dulbecco`s modified Eagle`s medium with 4 mM L-glutamine
adjusted to contain 1.5 g/l sodium bicarbonate and 4.5 g/l glucose,
90% [ATCC Cat. No.30-2002]; fetal bovine serum, 10% [ATCC Cat.
No. 30-2020].
Trypsin 0.5 mg/ml Trypsin; 0.2 mg/ml EDTA in PBS.
Treatment
3.2 › DNA preparation and quality
The quality and the concentration of DNA used for nucleofection plays a central
role for the efficiency of gene transfer. We strongly recommend the use of high
quality products for plasmid purification like EndoFree ® Plasmid Kits
[QIAGEN Cat. No. 12391 Giga Kit, 12362 Maxi Kit, 12381 Mega Kit]. The purified DNA
should be resuspended in deionized water or TE buffer (10 mM Tris/HCl, 1 mM
EDTA, pH 8.0) with a concentration between 0.2 - 1 µg/µl. Please check the purity
of each plasmid preparation by measurement of the A260:A280 ratio, according
to QIAGEN protocol.
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› Optimized Protocol DCL-1015 Vs. 08-2006
› for H9c2(2-1) [ATCC] page 4 of 7
3.3 › Cell culture
Culture conditions Replace medium every 2 - 3 days.
Passage interval Cells should be passaged every 2 - 3 days.
Subcultivation ratio of 1 : 4 - 1 : 5 is recommended. Subculture
before confluency to avoid differentiation.
Seeding conditions Approximately 1.5 - 2 x 105 cells/T162.
Culture conditions before nucleofection
› The cells should be preferably passaged 2 - 3 days before nucleofection, with as Sub-
cultivation ratio of 1 : 4 - 1 : 5.
Note Contamination of cell culture with mycoplasma is a wide spread phenomenon that
might negatively influence experimental results. We recommend the use of NormocinTM
[Cat. No. VZA-1001], a new antibiotic formulation specifically developed to protect sensi-
tive cell lines from mycoplasma infection and microbial contaminations. For more infor-
mation and ordering info see www.amaxa.com/antibiotics.
3.4 › Important controls and vector information
Positive control We strongly recommend establishing the Nucleofector Technology with the positive
control vector pmaxGFP as provided in this kit. pmaxGFP encodes the green fluore-
scent protein (GFP) from copepod Pontellina p. Just like eGFP expressing cells, maxGFP
expressing cells can easily be analyzed by fluorescence microscopy or flow cytometry
to monitor transfection efficiency.
Esp 3l (7)
Eco 31l (18)
Nsil (27)
TM
Esp 3l (2667)
Kpnl (980)
Nhel (988)
Eco47lll (993)
Agel (997)
BspTl (1891)
Bglll (1676)
Eco31l (1896)
Xhol (1680)
Esp3l (1909)
Sacl (1687)
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› Optimized Protocol DCL-1015 Vs. 08-2006
› for H9c2(2-1) [ATCC] page 5 of 7
Negative control We recommend you always perform two control samples to assess the initial quality of
cell culture and the potential influences of nucleofection or amount/purity of DNA on
cell viabilty.
control 1 Recommended amount of cells in Nucleofector Solution with DNA
but without application of the program (alternatively: untreated cells)
(Cells + Solution + DNA - program)
control 2 Recommended amount of cells in Nucleofector Solution without DNA
with application of the program (Cells + Solution - DNA + program)
Vector information If using IRES sequences in your vectors, please remember that the gene encoded 3’ of
the IRES sequence is usually expressed to a lesser extent than the upstream gene, and
in some cell types may not be expressed at all. As alternatives we suggest either:
co-transfecting two (or more) plasmids, using one plasmid with each gene under the
control of its own promoter, or making a GFP fusion.
3.5 › Nucleofection protocol
Preparation of Add 0.5 ml Supplement to 2.25 ml Nucleofector Solution and mix gently.
Nucleofector Solution The Nucleofector Solution is now ready to use and is stable for 3 months at 4°C.
Note the date of addition on the vial.
One nucleofection › 4 x 105 cells
sample contains › 2 µg plasmid DNA (in 1 - 5 µl H2O or TE) or 2 µg pmaxGFP or 2 nM - 2 µM siRNA
› 100 µl Nucleofector Solution L
For more details about the nucleofection of siRNA:
www.amaxa.com/RNAi
Preparation of 1. Cultivate the required number of cells.
samples 2.Prepare 2 µg DNA or 2 nM - 2 µM siRNA (final concentration, 3 ng- 3 µg/sample) for
each sample.
3. Pre-warm the supplemented Cell Line Nucleofector Solution L to room temperature.
Pre-warm an aliquot of culture medium at 37°C in a 50 ml tube (500 µl per sample).
4. Prepare 6-well plates by filling appropriate number of wells with 1 ml of culture
medium containing supplements and serum. Pre-incubate plates in a humidified
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› Optimized Protocol DCL-1015 Vs. 08-2006
› for H9c2(2-1) [ATCC] page 6 of 7
37°C/5% CO2 incubator.
5. Remove the medium from the cell culture. Wash cells once with PBS.
6. Harvest the cells, e.g. with trypsin/EDTA and stop the trypsinization with supple-
mented culture medium or PBS/0.5% BSA (see Nucleofector Manual for details).
7. Take an aliquot of trypsinized cell suspension and count the cells to determine the
cell density.
8. Centrifuge the required number of cells (4 x 105 cells per nucleofection sample) at
90xg at room temperature for 10 min. Discard supernatant completely so that no
residual medium covers the cell pellet.
9. Resuspend the pellet in room temperature Cell Line Nucleofector Solution L to a
final concentration of 4 x 105 cells/100 µl. Avoid storing the cell suspension longer
than 15 min in Nucleofector Solution as this reduces cell viability and gene transfer
efficiency.
Important: Steps 10-15 should be performed for each sample separately.
Nucleofection 10. Mix 100 µl of cell suspension with 2 µg DNA or 2 nM - 2 µM siRNA (final concen-
tration, 3 ng- 3 µg/sample).
11. Transfer the nucleofection sample into an amaxa certified cuvette. Make sure that
the sample covers the bottom of the cuvette, avoid air bubbles while pipetting. Close
the cuvette with the blue cap.
12.Select the appropriate Nucleofector program, C-20/C-020 (see Nucleofector Manu-
al for details). Insert the cuvette into the cuvette holder (Nucleofector I : rotate
carousel to final position) and press the »X« button to start the program.
13. After the program has finished (display showing »OK«) take the cuvette out of the
holder and incubate the sample in the cuvette for 10 min at room temperature.
14.After the post nucleofection incubation step, add 500 µl of the pre-warmed cul-
ture medium to the cuvette and gently transfer the sample into the prepared
6-well plates. To transfer the cells from the cuvettes, we strongly recommend
using the plastic pipettes provided in the kit to prevent damage and loss of cells.
15. Press the »X« button to reset the Nucleofector.
16. Repeat steps 10-15 for the remaining samples.
Cultivation after 17. Incubate cells in a humidified 37°C/5% CO2 incubator. Following nucleofection, gene
nucleofection expression should be analyzed at different times. Depending on the gene, expres-
sion is often detectable after 3 - 8 hours. If this is not the case, the incubation period
may be prolonged up to 24 hours.
amaxa GmbH amaxa Inc.
Europe/World USA
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+49 (0)221-99199-400 (240) 632-9110
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› Optimized Protocol DCL-1015 Vs. 08-2006
› for H9c2(2-1) [ATCC] page 7 of 7
4 Recommended literature
For an up-to-date list of all Nucleofector references, please refer to:
www.amaxa.com/citations
* amaxa’s Nucleofector® process, Nucleofector® device and Nucleofector® Solutions are covered by PCT appli-
cations PCT/EP01/07348, PCT/DE02/01489, PCT/DE02/01483 and other pending patents and domestic or
foreign applications corresponding thereto.
* amaxa, Nucleofector, nucleofection and maxGFP are trademarks of amaxa GmbH.
* This kit contains a proprietary nucleic acid coding for a proprietary copepod fluorescent protein intended to
be used as a positive control with this amaxa product only. Any use of the proprietary nucleic acid or protein
other than as a positive control with this amaxa product is strictly prohibited. USE IN ANY OTHER APPLICA-
TION REQUIRES A LICENSE FROM EVROGEN. To obtain such a license, please contact Evrogen at
license@evrogen.com.
* 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.
* QIAGEN and EndoFree are trademarks of QIAGEN.
* All other product and company names mentioned herein are the trademarks of their respective owners.
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