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Multiporator Transfection Protocol Protocol No. 4308 915.037 11/1999 Cell line U937, human leukemic monoblasts Transfection with Plasmid pEGFP-N1 (in bidistilled H₂O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium RPMI / 10% FCS Cuvette Eppendorf, 4 mm gap width, 800 l Temperature RT (20-25 C) Reference Dr. Steven Grant Box 980230 Medical College of Virginia Richmond VA 23 298 USA
Phone +1 804 828 5168 Fax +1 804 225 3788 e-mail: stgrant@hsc.vcu.edu

1. Harvest the cells in the exponential growth phase and centrifuge them (for 5-10 minutes, 200 x g, at room temperature).
2. Resuspend the cells in RPMI / 0.5% FCS, determine the number of cells and centrifuge them (for 5-10 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!
   
   
3. Resuspend the cells in Hypoosmolar Electroporation Buffer. When doing so, set the cell concentration to 2 x 10⁶ cells/ml.
4. Add and mix plasmid DNA (25 g/ml final concentration, in bidistilled H₂O).
5. Transfer 800 l cell suspension into electroporation cuvettes (4 mm gap width). The cell suspension must be free of air bubbles.
6. Electroporation:
   
   Mode Eukaryotes Voltage (V) 620 V Time constant (T) 60 s No. of pulses (n) 1
   
7. After the pulse, allow the cell suspension to stand in the cuvette for 15 minutes at room temperature.
8. Carefully transfer the cell suspension from the cuvette to 3 ml RPMI / 10% FCS, and cultivate them in a 55 mm culture dish.
   

Detection methods for transfection:
The expression of the plasmid pEGFP-N1 can be detected clearly after 24-48 hours with the aid of FACS analysis or under a fluorescence microscope. Result: Survival rate: 50% Transfection rate: 24% based on the initial number of cells used for the experiment Results were measured 48 hours after transfection.


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