Protocol No. 4308 915.036 12/1999
Primary cells: Microvascular endothelial cells from
rat hearts, 4 days in culture
Plasmid pEGFP-C3 (in bidistilled H2
Eppendorf Hypoosmolar Electroporation Buffer (PH)
M199 + 10% NCS + 10% FCS
HEPES-buffer (140 mM NaCl, 3.6 mM KCl, 1.2 mM MgSO4
1 mM CaCl2
11 mM glucose, 20 mM HEPES, pH 7.4) without phenol red + 10% NCS +
Eppendorf, 2 mm gap width, 400 l
RT (20-25 C)
Dr. Gerhild Taimor Justus-Liebig-Universitt
Physiologisches Institut Aulweg 129 D-35392 Gieen
Phone +49 641 9947243 Fax +49 641 9947239 mail: firstname.lastname@example.org
- Cultivate the cells for four days and harvest them by treatment with
trypsin/EDTA (10 min., 37 C). Cultivate the cells again overnight,
harvest them by treatment with trypsin/EDTA (10 min., 37 C) and centrifuge
them (for 5 minutes, 1,000 x g, at room temperature).
- Resuspend the cells in M199 / 10% NCS / 0.5% FCS, determ
ine the number
of cells and centrifuge them (for 5 minutes, 1,000 x g, at room temperature).
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. When doing
so, set the cell concentration to 1 x 106 cells/ml.
- Add and mix plasmid DNA (5-20 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 5-10 minutes at room temperature.
- Carefully transfer the cell suspension from the cuvette to 3-5 ml
HEPES medium without phenol red / 20% NCS/FCS, and cultivate them in
a 35 mm culture dish
Detection methods for transfection:
The expression of the plasmid pEGFP-C3 can be detected clearly after
24-48 hours with the aid of FACS analysis or under a fluorescence microscope.
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