5-Freezing cells


After steps 11 from the protocols “splitting and passing cells”


1)      Centrifuge cells 5 minutes at 1000 rpm (=117xg)

2)      Aspirate supernatant.

3)      Disperse pellet.

4)      Add freezing medium (10% glycerol, 15% fetal calf serum, 75% DMEM/F12) to a concentration of 1.5X106 S1 cells/ml or 0.75X106 T4 cells/ml (that is, 1X density for a T75 flask per ml freezing media).

5)      Pipette up and down to mix cells into media, then add 1 ml to each cryogen tube.

6)       Place cells in freezing chamber and store at –800C for 4 to 24 hours, then transfer to either liquid nitrogen or –140oC freezer.