Treatment of different cell lines with the same drug

1) Cultivation and passaging of cell lines

Culture:

Cell lines used for cytokine detection were usually cultured in culture medium containing 10% calf serum and antibiotics, and appropriate amounts of cytokines were added to culture cytokine-dependent cell lines. The cells were cultured in a saturated water vapor carbon dioxide incubator at 37°C with 5% CO2 and passaged once every 3-4 days.

Suspension of growing cells:

Directly sucked off or centrifuged after sucking off the culture supernatant, suspended cells with fresh culture medium, 1:3 or 1:5 dilution of the cells, flasks continue to cultivate.

Passage of wall-growing cells:

Aspirate the culture medium, wash the cells once with PBS, add digestive solution, and digest the cells at 37℃ for about 3-10 minutes, when the cells start to fall off, pour off the digestive solution, add fresh culture medium, and suspend and blow the dispersed cells. You can also wait until the cells are completely digested and detached, centrifuge at 500×g for 5 minutes, remove the digestive solution, and suspend the cells with fresh culture medium. 1:3 or 1:5 dilution of the cells was performed, and the culture was continued in separate flasks.

2) Freezing of cell lines:

1. Take the cells that have grown vigorously in culture for 2~3 days, and prepare the cells to 2×106~2×107/ml with the cell culture solution.

2. Add 0.5 ml of cell suspension, 0.4 ml of calf serum, and 0.1 ml of dimethylsulfoxide (or glycerol) to a 1 ml cell-freezing tube, and then seal it after mixing well. Place at 4℃ for 1 hour, -20℃ for 2 hours, then directly into liquid nitrogen or place on liquid nitrogen vapor overnight and then immersed in liquid nitrogen.

3) Resuscitation of cell lines:

When resuscitating, take out the cryopreservation tube from liquid nitrogen, and immediately melt the cells in a 37℃ water bath. The cells were directly added to 10 ml of RPMI-1640 culture medium containing 15% calf serum, and incubated at 37℃ in a 5% CO2 saturated water vapor carbon dioxide incubator. Suspended cells were carefully aspirated to remove the supernatant after all the cells had settled, replaced with fresh culture medium, and continued to be cultured; walled cells were cultured for 2 to 3 days.