(5) Recovery of cryopreserved cells (6) Passage (7) Frozen storage (8) Precautions
(1) Prepare and install filters
The filter was cleaned, dried, and placed in a microporous membrane with a pore size of 0.22 Î¼m. It was packed in a cloth and autoclaved at 15 lbs. 20 min. Open the filter holder in the clean bench. One end of the hose is connected to the filter pump and inserted into the liquid to be sterilized. The outlet end hose is deep into the sterilized bottle. Filter with a pump and check if the filter is intact after filtration.
(2) Preparation of synthetic medium
1. According to the instructions in the cell catalogue, select the appropriate dry powder of the culture medium according to the following table and use the appropriate amount of ultrapure water to dissolve.
2. Add sodium bicarbonate, sodium glutamate, HEPES, etc. as required, and stir well to dissolve.
3. Adjust the pH to about 7.2.
4. Add water to the final volume.
5. Filter and sterilize the solution in a clean bench, place it in a 250 mL or 500 mL glass vial, and tighten the bottle with a flip cap.
6, the bottle mouth is sealed, stored in a refrigerator at 4 Â°C.
(3) Treatment of calf serum
The calf serum sold on the market is generally sterilized, but it should be heat inactivated before use, that is, the method of heating destroys the complement (there is also a view that heat inactivation treatment is unnecessary). Fetal bovine serum does not have to be inactivated.
1. Heat the serum to 56 Â° C for 30 min, and gently shake it from time to time to make the heat even and prevent precipitation.
2. The treated serum was stored at 4 Â°C.
3, calf serum is best screened before use to master the quality of serum.
(4) Preparation of growth medium
In addition to serum-free culture, various synthetic media require the addition of a certain amount of calf serum or fetal bovine serum and antibiotics prior to use.
The adherent cells were digested, collected by centrifugation, and the suspended cells were directly collected by centrifugation, and the cells were resuspended in complete medium or fetal bovine serum to a final concentration of about 106/ml. Add 10% DMSO. Pack 1 to 2 ml per tube into the cryotube. Wrap in a refrigerator at -70 Â° C overnight with a heat-insulating material. Save to liquid nitrogen the next day.
(eight) matters needing attention
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