293A (human embryonic kidney cells) resuscitation, passage and cryopreservation protocol [identified by STR]

293A (human embryonic kidney cells) [identified by STR]

Specifications: 1 × 106 cells / T25 flask

one,   recovery
1. Remove the cryotube from the liquid nitrogen and immediately put it into a 37 ° C water bath and shake it gently. After the liquid has melted (about 1-1.5 minutes), take out the alcohol and put it on the ultra-clean workbench.
2. Aspirate the above cell suspension into a 15 ml centrifuge tube containing 10 ml of medium (wash the cryotube with medium and wash the cells adhering to the wall) and centrifuge at 1000 rpm for 5 minutes.
3. Pour off the supernatant and add 1 ml of medium to suspend the cells. The cells in the culture dish were evenly distributed by sucking them into a 10 cm culture dish containing 10 ml of the medium and gently shaking them.
4. Mark the cell type and date, the name of the culture person, etc., put it in a CO2 incubator, and change the medium after the cells are attached.
5. Change the medium once every 3 days.
two,   pass on
1. Passage should be carried out when the cell coverage in the culture dish reaches 80%-90%.
2. Aspirate the original medium.
3. Add appropriate trypsin (can cover the cells) and digest for 1-2 minutes.
4. After the cells are rounded, add an equal volume of serum-containing medium to stop digestion.
5. Blow the cells with a pipette and suspend the cells.
6. Pipe the cells into a 15 ml centrifuge tube and centrifuge at 1000 rpm for 5 minutes.
7. Pour off the supernatant, add 1-2 ml of medium, and blow the cells.
8. Transfer the cells to several culture dishes depending on the cell type. Generally, there are 5 cancer cells and 3 normal cells. Continue to train.
three,   Cryopreservation
Digest the cells and centrifuge (ibid.). The cells were suspended in a prepared cryopreservation solution, dispensed into a sterile cryotube, and allowed to stand for a few minutes, indicating the cell type and the date of freezing. 4 ° C 30 min, -20 ° C 30 min, -80 ° C overnight, and then placed in liquid nitrogen irrigation.
Preparation of cryopreservation solution: 70% complete medium + 20% FBS + 10% DMSO. DMSO should be slowly added dropwise while shaking.
Pay attention to the aseptic operation!

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