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Primary cells, rat heart endothel

Multiporator Transfection Protocol Protocol No. 4308 915.036 12/1999 Cell line Primary cells: Microvascular endothelial cells from rat hearts, 4 days in culture Transfection with Plasmid pEGFP-C3 (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium M199 + 10% NCS + 10% FCS Outgrowth medium 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 + 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 l Temperature RT (20-25 C) Reference Dr. Gerhild Taimor Justus-Liebig-Universitt Physiologisches Institut Aulweg 129 D-35392 Gieen
Phone +49 641 9947243 Fax +49 641 9947239 mail:
  1. 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).
  2. 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). Remove supernatant.

    Note: The overall incubation time in the Eppendorf Electroporation Buffer must not exceed 30 minutes to guarantee a successful electroporation!

  3. Resuspend the cells in Hypoosmolar Electroporation Buffer. When doing so, set the cell concentration to 1 x 106 cells/ml.
  4. Add and mix plasmid DNA (5-20 g/ml final concentration, in bidistilled H2O).
  5. Transfer 400 l cell suspension into electroporation cuvettes (2 mm gap width). The cell suspension must be free of air bubbles.
  6. Electroporation:

    Mode Eukaryotes Voltage (V) 400 V Time constant (T) 100 s No. of pulses (n) 1
  7. After the pulse, allow the cell suspension to stand in the cuvette for 5-10 minutes at room temperature.
  8. 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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