LCL721.221 cell line細胞株

BioVector NTCC質粒載體菌種細胞基因保藏中心

Cell line LCL721.221, EBV transfected B cell line, MHC class I A, B, C negative,MHC class II positive

Culture medium RPMI / 10% FCS

Transfection protocol:

Transfection with Plasmid pEGFP-N1 (in bidistilled H2O). (Example)

Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH)

Cuvette Eppendorf, 2 mm gap width, 400 μl

Temperature RT (20-25 °C)

1. Harvest the cells in the exponential growth phase and centrifuge them (for 5 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 minutes, 200 x g,

at room temperature). Remove supernatant.

3. Resuspend the cells in medium, set the cell number to 1 x 106 cells/ml and centrifuge them (for 5 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!

4. Resuspend the cells in Hypoosmolar Electroporation Buffer. Add and mix plasmid DNA (20 μg/ml final concentration,

in bidistilled H2O).

5. Transfer 400 μl cell suspension into electroporation cuvettes (2 mm gap width). The cell suspension must be free of

air bubbles.

6. Electroporation:

Mode Eukaryotes “¤“

Voltage (V) 200 V

Time constant (t) 40 μs

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 with a pasteur pipette from the cuvette into 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 by viable 721.221 cells can be detected after 24 hours with a flow cytometer

by costaining with propidium iodide (final concentration 1 μg/ml). Incubate for 10 min. at RT.

Result:

Survival rate: 86.18%

Transfection rate: 13.28% based on the number of surviving cells.

Results were measured 24 hours after transfection.