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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: gerhild.taimor@physiologie.med.uni-giessen.de
  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
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