( ...)H73C11

Multiporator Transfection Protocol Protocol No. 4308 915.024 11/2001 Cell line H73C11, mouse-human, heteromyeloma (suspension cell line) Transfection with Plasmid pEGFP-N1 (in bidistilled H₂O) Electroporation buffer Eppendorf Electroporation Buffer with 150 mOsmol/kg Culture medium RPMI 1640 / 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 Temperature RT (20-25 C) Reference Prof. Ulrich Zimmermann Lehrstuhl fr Biotechnologie Biozentrum Universitt Wrzburg
Am Hubland D-97074 Wrzburg Phone +49 931 888 4508 Fax +49 931 888 4509
e-mail: zimmerma@biozentrum.uni-wuerzburg.de

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 1640 / 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 150 mOsmol/kg Electroporation Buffer (= 68% Eppendorf Hypoosmolar Electroporation Buffer + 32% Eppendorf Isoosmolar Electroporation buffer). When doing so, set the cell concentration to 1 x 10⁶ cells/ml.
4. Add and mix plasmid DNA (10-20 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) 160 V Time constant (T) 40 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 5 ml RPMI 1640 / 10% FCS, and cultivate it in a 60 mm culture dish.
   Note: After pulsing, the cells should be incubated for 2-3 h at 37 C before any centrifugation is performed, to ensure resealing of the membrane.

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: 250% Transfection rate: 20% based on the initial number of cells used for the experiment Results were measured 48 hours after transfection.


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