Spin cells for 10 min at 1000 g (1200 rpm)

Resuspend in 90% Medium/10% DMSO at >4x10e6 cells/ml

Dispense into cryovials on ice

Place at -20°C for 1 hour

Transfer vials to -80°C over night

Transfer and store vials to liquid Nitrogen

Thaw frozen cells quicklyby gentle agitation in a 37°C water bath. When thawed (30-40 sec), quickly rinse the outside of the vial with 70% ethanol and proceed in the hood. Transfer the cells to a centrifuge tube, add 20 ml of medium and spin at 600xg for 2-5 min to pellet the cells. Remove the supernatant and resuspend the cells in 5-10 ml fresh medium. Seed the entire suspension into a single flask (25-75 cm2) and incubate at 27°C. After 12-24 hrs, remove the old medium and replace with fresh medium. The cells should be dividing within two days.