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t-HUVEC

Multiporator Transfection Protocol Protocol No. 4308 915.049 11/2001 Cell line t-HUVEC, transformed human umbilical endothelial cells, EC304 (ATCC) Transfection with pcDNA3-EGFP (endotoxin-free DNA-preparation; in bidistilled H2O) Electroporation buffer Eppendorf Electroporation Buffer with 200 mOsmol/kg Culture medium RPMI 1640 + 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 l Temperature RT (20-25 C) Reference Ina Radtke, Edith Jasny Ruhr-Universitt Bochum Fakultt fr Chemie
Molekulare Neurobiochemie Universittsstrae 150 D-44780 Bochum
Phone +49 234 32 22067 Fax +49 234 32 14105 e-mail ina.radtke@ruhr-uni-bochum.de
  1. Harvest the cells in the exponential growth phase and centrifuge them (for 5 minutes, 160 x g, at room temperature).
  2. Resuspend the cells in RPMI 1640 with 1% FCS, determine the number of cells and centrifuge them (for 5 minutes, 160 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. < li>Resuspend the cells in Electroporation Buffer with 200 mOsmol/kg (= 42% Eppendorf Hypoosmolar Electroporation Buffer + 58% Eppendorf Isoosmolar Electroporation Buffer). When doing so, set the cell concentration to 1 x 106 cells/ml.
  4. Add and mix plasmid DNA (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) 670 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 10 minutes at room temperature.
  8. Carefully transfer the cell suspension with a pasteur pipette from the cuvette into 3 ml RPMI / 10% FCS, and cultivate it in a 55 mm culture dish.
Detection methods for transfection:
The expression of the plasmid pcDNA3-EGFP can be detected clearly after 24-48 hours under a fluorescence microscope. Result: Survival rate: > 95% Transfection rate: 70% based on the initial number of cells used for the experiment Results were measured 24 hours after transfection .


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