1. First of all, observe whether the cell culture bottle is in good condition, whether the culture medium has leakage, turbidity and other phenomena. If yes, please take photos and contact technical support in time (the photos will be used as the basis for subsequent services).

2. Wipe the surface of the cell culture bottle with 75% alcohol and observe the cell state under the microscope. Due to transportation problems, some adherent cells will fall off the wall of the bottle; do not open the culture bottle cap first, and put the cells in the cell incubator for 2-4 hours, so as to stabilize the cell state.

3. Read the cell instructions carefully, and understand the cell related information, such as attachment characteristics (attachment / suspension), cell morphology, basic medium used, serum proportion, required cytokines, passage proportion, fluid change frequency, etc.

4. After the completion of the standing, take out the cell culture bottle, carry out microscopic examination, take photos, and record the cell status (the photos taken will be used as the basis for subsequent services); it is recommended to take photos and record the cell growth status regularly after the cell subculture.

5. Adherent cells: if the cell growth density is more than 80%, it can be passed on normally; if it is less than 80%, remove the culture medium in the cell culture bottle, reserve about 5ml for further culture, until the cell density is about 80%, then carry on the passage operation, and the bottle cap can be slightly loosened.

6. Suspension cells: transfer all the liquid in the cell culture bottle to a 50ml sterile centrifuge tube, centrifugation at 1200rpm for 5min, after centrifugation, the supernatant culture medium can be collected for standby, and the cell sedimentation at the bottom of the tube is added to 5ml culture medium for blowing and re suspension. In microscopic examination, if the cell density is more than 80%, the cell suspension can be divided into two cell culture bottles for culture, and the culture medium can be added to 5ml; if the cell density is not more than 80%, the cell suspension can be transferred to the original bottle for further culture until the cell density reaches about 80%, and then the passage operation can be carried out.

Warm reminder:
1. The redundant medium in the culture bottle can be transferred to a 50ml sterile centrifuge tube for standby; when the cell is first passaged, the medium can be mixed with the culture medium prepared by the customer according to a certain proportion to make the cell gradually adapt to the culture conditions.

2. After confirming that the cells are in good condition, part of the cells should be frozen in time, and then follow-up experiments should be carried out to avoid the loss of cells caused by cell pollution or death due to the mistakes in later experiments, which may affect the follow-up experiments.

3. It is recommended that the customer take 3 cell photos of 100x, 200X and 400X respectively in the first 3 days after receiving the cell to record the cell status, so as to facilitate the communication with technical support.

Cell processing:
1) Resuscitated cells: thaw the cryopreservation tube containing 1ml cell suspension in 37 ℃ water bath, add 4ml medium and mix evenly. Centrifugation at 1000 rpm for 4 minutes, discarding the supernatant, adding 1-2 ml of medium, and blowing. Then all cell suspensions were added to the culture bottle for overnight culture (or cell suspensions were added to a 10cm dish and about 8ml cell culture medium for overnight culture). The next day, change the fluid and check the cell density.

2) Cell subculture: if the cell density reaches 80% - 90%, subculture can be carried out.

For adherent cells, the following methods can be used for subculture:
Discard the culture supernatant and wash the cells with PBS without calcium and magnesium ions for 1-2 times.

1. Add 2 ml of digestion solution (0.25% typsin-0.53mm EDTA) to the culture flask, put it in a 37 ℃ incubator for digestion for 1-2 minutes, and then observe the cell digestion under the microscope. If most of the cells become round and fall off, take them back to the operation platform quickly, tap the culture flask for a few times, and then add a small amount of culture medium to stop digestion.

2. Add the culture medium according to 6-8ml / bottle, gently beat and suck it out, centrifugate for 4 minutes at 1000rpm, discard the supernatant, add 1-2ml culture medium and blow it up.

3. Divide the cell suspension into a new dish or bottle containing 8ml medium according to the ratio of 1:2 to 1:5.

Cell cryopreservation: cell cryopreservation can be carried out when the cell is in good condition.

Take T25 bottle as an example:

1. When the cells are frozen, after discarding the culture medium, add 1ml pancreatin after PBS cleaning bottle bottom for 1-2 times, after the cells become round and fall off, add 2ml of complete culture medium to stop digestion, and the blood cell counting plate can be used for counting.

2. Centrifuge at 1000 rpm for 5 minutes to remove the supernatant. The final concentration was 10%. After adding DMSO, mix it up quickly and distribute it to the freezing storage tube according to the quantity of 1ml. Pay attention to the marking of the freezing storage tube. The number of cells in each cryopreservation tube is more than 1x106.

3. Put the frozen storage tube in the program cooling box, put it in the - 80 degree refrigerator, and transfer it to the liquid nitrogen tank for storage at least 2 hours later. Record the location of the frozen storage tube for next retrieval.

4. Transportation and preservation of cells: transportation and delivery of resuscitated viable cells with dry ice can be selected: (1) transportation with dry ice and transfer to liquid nitrogen cryopreservation or direct resuscitation immediately after receiving; (2) survival cells shall continue to grow after receiving, and cryopreservation shall be carried out when the cells are in good growth condition. See the cell culture procedure for the specific operation.