- Take out 1 epitaph from liquid nitrogen tank, thaw it in 37oC water bath until only little icy cube left. Please stand by when thawing the cells because it takes <1 minute to finish. Take out the cell solution and Apply to 10% FBS DMEM.
- Collect cell solution into a tube and centrifuge 1000rpm for 3 min. (keep the balance of centrifuger).
- During the centrifuging period, take new tissue cultured dishes (if not tissue culture treated, coat the dishes with 2 % gelatin (just rinse), if not sterile, incubate with ethanol or light-bath with UV lamp for 30 min and then rinse with PBS for 3 times). Label the dishes with cell name, passage, date, initials of your name.
- Take out the centrifuged tube containing cells, you should be able to see a whitish pellet at the bottom of the tube. Tilt the tube and aspirate the supernatant with vacuum tip, resuspend the cell pellet with 10% FBS DMEM by pipetting up and down 20 times to break cell-cell aggregation. Apply cell solution to labeled dishes.
- Keep the cell dishes in the incubator supplemented with 5% CO2 at 37oC.