Sean Lauber:Thawing Mammalian Cells

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1. Remove a vial from liquid nitrogen and transport (quickly) on ice (best is dry ice but ice is okay)

2. Thaw the vial in a 37°C water bath by constantly swirling the vial in the bath (don't just leave it in there) - you want it to thaw as quickly as possible

3. Once about 90% thawed (very small piece of ice left), remove from the water bath and enter the hood

4. Add 1 ml of PBS to the vial and allow the cells time to equilibrate to the new osmolarity (10 seconds)

5. Dispense this 2 ml into 8 ml of PBS (in a 15 ml falcon tube)

3. Spin at 1000 rpm for 3 min to pellet

4. Discard the supernatant and resuspend the pellet in the residual liquid by tapping

5. Add 10 ml of warmed media and resuspend by pipetting

6. Take a volume of this and add to a T75 or T25 (depends on how many cells you have)

7. After some time the cells need to be split (depends on the cell line)

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