Protocol No. 4308 915.044 11/2001
721.221, EBV transfected B cell line, MHC class I A,
B, C negative,
MHC class II positive
Plasmid pEGFP-N1 (in bidistilled H2
Eppendorf Hypoosmolar Electroporation Buffer (PH)
RPMI / 10% FCS
Eppendorf, 2 mm gap width, 400 l
RT (20-25 C)
Dr. Kirsten Falk and Dr. Olaf Rtzschke
Max Delbrck Center for Molecular Medicine Robert-Rssle-Strasse
10 D-13125 Berlin-Buch
Detection methods for transfection:
- Harvest the cells in the exponential growth phase and centrifuge
them (for 5 minutes, 200 x g, at room temperature).
- Resuspend the cells in RPMI / 0.5% FCS, determine the number of cells
and centrifuge them (for 5 minutes, 200 x g, at room temperature). Remove
- Resuspend the cells in medium, set the cell number to 1 x 106
cells/ml and centrifuge them (for 5 minutes, 200 x g, at
room temperature). Remove supernatant.
Note: The overall incubation time in the Eppendorf Electroporation
Buffer must not exceed 30 minutes to guarantee a successful electroporation!
- Resuspend the cells in Hypoosmolar Electroporation Buffer. Add and
mix plasmid DNA (20 g/ml final concentration, in bidistilled
- Transfer 400 l cell suspension into electroporation cuvettes (2 mm
gap width). The cell suspension must be free of air bubbles.
Time constant (T)
No. of pulses (n)
- After the pulse, allow the cell suspension to stand in the cuvette
for 15 minutes at room temperature.
- Carefully transfer the cell suspension with a pasteur pipette from
the cuvette into 3 ml RPMI /10% FCS, and cultivate
them in a 55 mm culture dish.
The expression of the plasmid pEGFP-N1 by viable 721.221 cells can be
detected after 24 hours with a flow cytometer
by costaining with propidium iodide (final concentration 1 g/ml).
Incubate for 10 min. at RT.
13.28% based on the number of surviving cells.
Results were measured 24 hours after transfection.
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