This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.

Operation method

Transformation of EBV into lymphocytes

Materials and Instruments

Whole blood
Cutter Bean Agglutinin A
RPMI growth medium Centrifuge tubes

Move

1. Mix 10 ml of whole blood and 10 ml of RPMI growth medium.

Growth medium (RPMI1640 800 ml, FBS 200 ml, 2 mmol/L-glutamine, 5 μg/ml amphotericin, 50 μg/ml gentamicin)

2. Take a 15 ml sterile centrifuge tube, add 3 ml Histo-paque1077 (Sigma), add 4 ml diluted blood on it, and isolate the lymphocytes.

3. Centrifuge at 300 g for 30 minutes at room temperature and discard the upper layer of clear plasma.

4. Collect the opaque lymphocyte layer and pour the cell suspension into a 50 ml sterile centrifuge tube.

5. wash twice with 20 ml of RPMI 1640 growth medium and centrifuge at 300 g for 10 minutes at room temperature.

6. 10 ml of blood-separated cells are suspended in 1.0 ml of EBV-containing supernatant.

7. Place the cell suspension (1.0 ml) into a 25 ^cm2 tissue culture flask, place vertically and incubate at 37°C with 5% _CO2 for 1-3 hours.

8. After incubation, add 3 ml of RPMI 1640 Growth Medium containing 0.2 μg/ml of Spermophilus lectin A and place horizontally so that the cells are evenly distributed on the whole growth surface.

9. Add 1 ml of fresh RPMI 1640 Growth Medium containing Croton A every week without aspirating the original medium.

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