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.