( ...)Primary cells, rat heart muscle

Multiporator Transfection Protocol Protocol No. 4308 915.035 12/1999 Cell line Primary cells: Adult heart muscle cells from rat, 6 days in culture Transfection with Plasmid pCMV--galactosidase (in bidistilled H₂O) Electroporation buffer Eppendorf Isoosmolar Electroporation Buffer (PI) Culture medium M199 / HEPES (2 mM carnithin, 5 mM taurine, 10 M cytosine-arabinofur.)+ 20% FCS Outgrowth medium HEPES-buffer (140 mM NaCl, 3.6 mM KCl, 1.2 mM MgSO₄, 1 mM CaCl₂, 11 mM glucose, 100 U/ml penicillin, 100 g/ml streptomycin, 20 mM HEPES, pH 7.4) without phenol red + 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. Harvest the cells by collagenase treatment (0.4 mg/ml, 30 min., 37 C) and centrifuge them (for 2 minutes, 300 x g, at room temperature).
2. Resuspend the cells in M199 / 0.5% FCS, determine the number of cells and centrifuge them (for 2 minutes, 300 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 Isoosmolar Electroporation Buffer. When doing so, set the cell concentration to 1 x 10⁴ - 10⁵ 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) 100 V Time constant (T) 50 s No. of pulses (n) 1
   
7. After the pulse, allow the cell suspension to stand in the cuvette for 10 minutes at room temperature.
8. Carefully transfer the cell suspension from the cuvette to 3-5 ml HEPES medium without phenol red / 20% FCS, and cultivate them in a 35 mm culture dish.

Detection methods for transfection:

The expression of the plasmid pCMV--galactosidase can be detected clearly after 24-48 hours with X-gal-staining under a microscope.

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