//Updated 01/21/09 MDR//


**CELL THAWING**((Adapted from Stratagene AAV Helper-Free System))

**MATERIALS**
  - Cell-appropriate culture medium
  - 50mL tube, sterile
  - Tissue culture flask 
  - Cryogloves
  - Frozen cells (liquid nitrogen tank is in the cold room)
  - BSC P1000 and tips

**PROTOCOL**
    **ALWAYS FOLLOW SUPPLIER'S RECOMMENDATIONS**

    - Prewarm culture medium to 37C in water bath. Place 10mL warm medium in 50mL tube
    - Set P1000 to 1ml in BSC
    - Retrieve frozen cells. Be sure to use cryogloves to prevent personal injury.
    - 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.
    - Transfer cells to 50mL tube of warm medium. Mix thoroughly by gently pipetting solution up and down or closing and inverting.
    - Pellet the cells by centrifugation at 700 rpm for 5 minutes  
    - Aspirate off medium to remove DMSO-containing freezing medium. 
    - Resuspend cells in warm culture medium (5ml for T25 or 15ml for T75) by gently pipetting up and down.
    - Transfer to tissue culture flask. Write cell line, passage number, date, and initials on flask. 
    - Examine cells under microscope. Place in 37C incubator. 
    - Update frozen cell log online if taken from frozen stock.