Thawing Cells (Schreiber protocol)

Thawing Cells (Schreiber's protocol) Thaw vial quickly in 37°C water. Caution - vial can explode. Transfer cells to sterile, 15 mL centrifuge tube. Add 50 µl warm FBS (fetal bovine serum, heat inactivated), wait 1 minute . Add 100 µl FBS , wait 1 minute. Add 200 µl FBS , wait 1 minute. Add 400 µl FBS , wait 1 minute. Add 800 µl 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°C 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)