Basic Operations of Cell Culture

　　1. Recovery

　　1.1 Open the water bath and adjust it to 37°C; spray the 50ml EP tube and T75 culture bottle with alcohol and put them in a biological safety cabinet for sterilization, and put the culture medium into the water bath to preheat;

　　1.2 Spray alcohol from the preheated culture medium and place it in the biosafety cabinet. Take 10ml of the culture medium in a 50ml EP tube, and then add 15ml of the culture medium to the T75 culture bottle;

　　1.3 Take out the cells from the liquid nitrogen tank, immediately put them into a 37°C water bath to thaw them quickly so that they are completely melted within 2 minutes, spray them with alcohol and quickly take them to the biological safety cabinet;

　　1.4 Transfer the cells to a 50ml EP tube with 10ml of culture medium, add slowly and shake well, 800rpm/5min (centrifuge at room temperature);

　　1.5 After centrifugation, spray alcohol and discard the supernatant. Take an appropriate amount of culture medium from the T75 culture bottle and pipette the precipitate to suspend the cells. Transfer to the culture bottle, cap it tightly, and place it in a CO2 incubator for culture. Transfer to the culture bottle and label the cell name, time, and operator;

　　2. Digestion and passage

　　2.1 Open the water bath and adjust it to 37°C, sterilize the biological safety cabinet, and put the culture medium, trypsin, and PBS in the water bath to preheat;

　　2.2 Take out the culture bottle from the CO2 incubator and place it in a biological safety cabinet. Pour out the culture medium in the culture bottle and add 10 ml of PBS to wash it twice;

　　2.3 Add an appropriate amount of trypsin to the culture bottle and place it in a CO2 incubator for digestion for about 3 minutes;

　　2.4 Take out the culture bottle from the incubator and observe it under the microscope. If the cells become round and flowing, add culture medium and pipet to stop digestion. Centrifuge and discard the supernatant. Add new culture medium and mix evenly by pipetting. Transfer to the culture bottle and add culture medium. Smooth the bottom of the culture bottle;

　　2.5 Passaging needs to be based on cell growth and density, usually at a ratio of 1:2 to 1:6. Mark the cell name, passage number, passage time, and operator’s name on the cell culture bottle;

　　3. Count with a cell counter

　　3.1 Mix the digested cells with 0.2% trypan blue at a volume of 1:1, and drop 20 μl into the groove of the counting plate;

　　3.2 The groove and the indicator light are opposite, open the counting software, edit the name and time, select the parameters, and start measuring after one minute;

　　3.3 Sampling 3 times, the system automatically calculates the average value and saves it;

　　3.4 After the measurement, take out the counting plate and put it in the original tape for next use. If all 5 samples have been measured, discard it, and then turn off the computer and instrument;

　　4. Cryopreservation

　　4.1 Before cryopreserving cells, first observe the growth of the cells, referred to as whether the cells still retain their unique properties. Cells to be cryopreserved should grow well and have a high survival rate, and the coverage rate of adherent cells should be about 80%-90%. The cell concentration is 1～5 ×106 cells/ml;

　　4.2 Prepare cell cryopreservation solution: Prepare the cryopreservation solution according to the freezing conditions recommended by ATCC for each type of cell. Common cryopreservation solutions are 75% medium + 20% FBS + 5% DMSO or normal growth medium + 10% DMSO. Mix thoroughly by inverting and keep at 4 degrees until use. Add 250ml of isopropyl alcohol to the freezer box and place it at 4°C until use;

　　4.3 After counting the digested cells, calculate the number of cryopreservation tubes, prepare cryopreservation solution, and make cell suspension;

　　4.4 Transfer the cell suspension into cryovials (1ml/tube). Mark the cell name, passage number, freezing time and operator;

　　4.5 Immediately place the cryopreservation tube in a freezing box with isopropyl alcohol and place it at -80°C overnight. The next day, transfer the cells to a liquid nitrogen tank for storage, and record the cryopreservation location;

　　4.6 In order to ensure the survival rate of cryopreserved cells, take out a tube from liquid nitrogen on the third day for recovery and observe the cell viability. If the cell condition is good, the cryopreserved cells can be used;