Instrumentation 1. purification bench 2. centrifuge 3. constant temperature water bath 4. refrigerator (4°C, -20°C, -70°C) 5. inverted phase contrast microscope 6. incubator 7. liquid nitrogen refrigerator
Glassware 1. pipette (curved head, straight head) 2. culture flasks 3. glass vials (250 ml, 100 ml) 4. waste tank.
Plasticware 1. pipette tip 2. gun tip 3. glue plug 4. pipette (10 ml) 5. 15 ml centrifuge tube 6. cryopreservation tube (1 to 2 ml).
Other items 1. microfuge gun 2. red blood cell counting plate 3. marker 4. medical eraser 5. pipette gun (v) Reagents 1. D-Hanks solution 2. calf serum 3. culture solution 4. double antibiotic (penicillin, streptomycin) 5. trypsin (0.08%) 6. 1NHCl 7. 7.4% NaHCO 38. DMSO (analytically pure) or colorless fresh glycerol.
Cell freezing 1. Prepare freezing culture medium containing 10% DMSO or glycerol and 10-20% calf serum;
2. Take the cells in logarithmic growth phase, and use trypsin to digest the monolayer growth, and transfer the cells in suspension growth directly to 15 ml centrifuge tubes;
3. Centrifuge at 1000 rpm for 5 min;< /p>4.
4. Remove trypsin and old culture medium, add appropriate amount of prepared frozen culture medium, gently blow the cells with a pipette to make them homogeneous, count them, and adjust the final density of cells in the frozen culture medium to be 5×106/ml~1×107/ml;
5. Dispense the cells into freezing tubes of 1~1.5 ml each;
6. Label the freezing tubes with the name of the cells, the time of freezing and the duration of freezing. Label the cryopreservation tubes with the name of the cells, the time of freezing and the operator;
7. Freezing: The standard freezing procedure is to reduce the temperature at a rate of -1 to -2°C/min; when the temperature reaches -25°C or below, the temperature can be increased to -5°C to -10°C/min; and when the temperature reaches -100°C, the cells can be rapidly immersed in liquid nitrogen. You can also put the frozen tube with cells into the -20℃ refrigerator for 2h, and then put it into the -70℃ refrigerator overnight, take out the frozen tube and move it into the liquid nitrogen container.