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Thawing Cells

Thawing Cells (Schreiber's protocol)

  1. Thaw vial quickly in 37C water. Caution - vial can explode.
  2. Transfer cells to sterile, 15 mL centrifuge tube.
  3. Add 50 l warm FBS (fetal bovine serum, heat inactivated), wait 1 minute.
  4. Add 100 l FBS, wait 1 minute.
  5. Add 200 l FBS, wait 1 minute.
  6. Add 400 l FBS, wait 1 minute.
  7. Add 800 l 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 37C 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)