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