Thaw vial quickly in 37癈 water. Caution - vial can explode.
Transfer cells to sterile, 15 mL centrifuge tube.
Add 50 祃 warm FBS (fetal bovine serum, heat inactivated), wait 1 minute.
Add 100 祃 FBS, wait 1 minute.
Add 200 祃 FBS, wait 1 minute.
Add 400 祃 FBS, wait 1 minute.
Add 800 祃 FBS, wait 1 minute.
Centrifuge for 5 minutes at 1000 rpm.
After aspirating supernatant, resuspend in 5 - 6 mL warm media.
Transfer to T 25 (25 cm2 flask).
Incubate at 37癈 and 5% CO2 with top of flask loose.
Next day, check that cells are viable and attached. Pour off or aspirate media. Add 5 - 6 mL fresh warm media.
Passage into T75 when cells become confluent (some cells, like RCHO-1 never become confluent)