Thawing Cells (Schreiber's protocol)

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