PriCells: The basic method of primary cell resuscitation
First, the experimental preparation
(1) Instruments: purification workbench, centrifuge, constant temperature water bath, inverted phase contrast microscope, incubator, liquid nitrogen refrigerator
(2) Glassware: straw (elbow, straight), culture bottle, glass bottle (250ml, 100ml), waste tank
(3) Plastic utensils: tip, tip, rubber stopper, pipette (10ml), 15ml centrifuge tube, cryotube (1~2ml)
(4) Other items: micro sample gun, red blood cell counting board, marker pen, medical adhesive plaster, pipetting gun
(5) Reagents: PBS, calf serum, culture medium, double antibody (penicillin, streptomycin), trypsin (0.08%)
Second, the operation steps
(1) Remove the cryotube from the liquid nitrogen container, directly immerse it in warm water at 37 ° C, and shake it as occasionally to melt it as soon as possible.
(2) Remove the cryotube from the 37 ° C water bath, disinfect the 75% alcohol, open the lid, and aspirate the cell suspension with a pipette and add to the centrifuge tube. PriCells recommends inoculation of culture flasks and static culture in a 37 °C incubator .
(3) Centrifugation, 1000 rpm, 5 min. PriCells recommends not centrifuging .
(4) Discard the supernatant, resuspend the cells by adding 10% fetal calf serum, count, adjust the cell density, inoculate the culture flask, and incubate in a 37 °C incubator. PriCells recommends calf serum concentrations based on primary cell types .
(5) Replace the 1/2 medium once a day and continue the culture.
Third, matters needing attention
(1) When removing the cryotube from the liquid nitrogen container, it is necessary to do a good job of protection against frostbite.
(2) Immediately after taking out the cryotube, put it into a water bath and melt it as soon as possible (within 1-2 minutes).
(3) It is best to use a newly prepared culture solution for resuscitation.
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