Specific steps and requirements for cell culture, and precautions

First, the recovery

1. The frozen tube from the liquid nitrogen removed, immediately put 37 ℃ water bath, slightly shake. After the liquid has melted (about 1-1.5 minutes), take out the spray of alcohol into the clean bench .

2. The above cell suspension was aspirated in a 15 ml centrifuge tube containing 10 ml of medium (the tube was washed once with the culture medium, and the cells on the wall were washed) and centrifuged at 1000 rpm for 5 minutes.

3. Remove the supernatant and add 1 ml of the medium to suspend the cells. The cells in the Petri dish were evenly distributed in a 10 cm dish containing 10 ml of medium, gently shaking left and right before,

4. Standard cell type and date, culture name, etc., into the CO2 incubator culture, cell adherent for medium.

5. 3 days for a culture medium.

Second, passaged

1. Petri dish in the cell coverage rate of 80% -90% to pass on.

2. Absorb the original medium.

3. Appropriate trypsin (can cover cells on the line), digestion 1-2 minutes.

4. Cells are rounded and added to an equal volume of serum-containing medium to terminate the digestion.

5. Blow cells with pipettes and suspend cells.

6. The cells were aspirated into a 15 ml centrifuge tube and centrifuged at 1000 rpm for 5 minutes.

7. Remove the supernatant, add 1-2ml of medium, the cells are blowing up.

8. Transfer cells to several petri dishes according to cell type. In general, cancer cells are divided into five, normal cells pass 3. Continue to develop.

Three, frozen

The cells are digested and centrifuged (ibid.). Suspension of the cells with a good cryopreservation solution, dispensing into a sterile cryopreservation tube, resting for a few minutes, indicating the cell type, the date of freezing. 4 ℃ 30min, -20 ℃ 30min, -80 ℃ overnight, and then placed in liquid nitrogen irrigation preservation.

Preparation of cryopreservation: 70% complete medium + 20% FBS + 10% DMSO. DMSO should be slowly dripping,

Should pay attention to the aseptic operation !


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