Recovery

1. Remove the frozen tube from liquid nitrogen and immediately into a 37℃ water bath, shaking slightly. After the liquid melts (about 1-1.5 minutes), take out some alcohol and put it on the ultra-clean work table.

2. Take the cell suspension into a 15ml centrifuge tube with 10ml medium (wash the frozen tube with the medium and wash all the cells stuck to the wall) and centrifuge at 1000 for 5 minutes.

3. The supernatant was reversed and 1ml of the medium was added to suspend the cells. Smoke into a 10cm dish with 10 ml of medium and gently shake left and right to evenly distribute the cells in the dish.

4. Mark the cell type and date, the human name, and so on, put it in the CO2 incubator, stick the cells and change the medium.

5. Medium was changed once every 3 days.

Passage

1. Plants were passaged to reach 80-90% coverage. 

2. Abup the original medium.

3. Add the appropriate trypsin (just cover the cells) and digest for 1-2 minutes.

4. The digestion was terminated by adding an equal volume of the serum-containing medium.

5. Blow the cells with a pipette gun and leave them all suspended.

6. Cells were aspirated into a 15ml centrifuge tube and centrifuged at 1000 for 5 min.　　

7. Pour the supernatant and add 1-2ml of medium to blow all the cells up.

8. Cells were passed to several culture dishes depending on the cell species. Generally, cancer cells are divided into 5 cells and three normal cells are transmitted. Continue to cultivate.

Free to digest the cells and centrifuge them (ibid. above).

Suspension the cells with the matched frozen storage solution, divide them into a sterilized freezer tube, rest for a few minutes, and specify the cell type and the frozen storage date.4℃ 30min, -20℃ 30min, -80℃ overnight, and then stored in liquid nitrogen perfusion.

Preparation of cryopreservation solution: 70% medium + 20% FBS + 10% DMSO. DMSO should be added slowly and shaken steadily.