Updated 01/21/09 MDR

CELL THAWING1)

MATERIALS

1. Cell-appropriate culture medium
2. 50mL tube, sterile
3. Tissue culture flask
4. Cryogloves
5. Frozen cells (liquid nitrogen tank is in the cold room)
6. BSC P1000 and tips

PROTOCOL

• *ALWAYS FOLLOW SUPPLIER’S RECOMMENDATIONS**

1. Prewarm culture medium to 37C in water bath. Place 10mL warm medium in 50mL tube
2. Set P1000 to 1ml in BSC
3. Retrieve frozen cells. Be sure to use cryogloves to prevent personal injury.
4. Thaw cells rapidly, within 1-3 minutes of removal from tank, by swirling vigorously in 37C water bath. Stop when a sliver of ice remains, wipe down with EtOH and work in BSC.
5. Transfer cells to 50mL tube of warm medium. Mix thoroughly by gently pipetting solution up and down or closing and inverting.
6. Pellet the cells by centrifugation at 700 rpm for 5 minutes
7. Aspirate off medium to remove DMSO-containing freezing medium.
8. Resuspend cells in warm culture medium (5ml for T25 or 15ml for T75) by gently pipetting up and down.
9. Transfer to tissue culture flask. Write cell line, passage number, date, and initials on flask.
10. Examine cells under microscope. Place in 37C incubator.
11. Update frozen cell log online if taken from frozen stock.