Defrosting Eukaryotic Cells Frozen In Liquid N2
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<center> <h1> <font><font>Defrosting Eukaryotic Cells Frozen In Liquid N<sub>2</sub> </font> </font></h1> </center>
Reagents / Solutions
- Frozen cell line.
- Growth media (e.g. RPMI1640)
- Foetal calf serum (FCS).
Protocol
- Make 20ml growth media/10% FCS for each vial of cells to be thawed.
- Place a small sandwich box containing warm (~ 37�) water in the CO2 incubator.
- Remove vial(s) of cells and thaw rapidly by placing in an expanded polystyrene 'floater' in the water bath. Transfer cells to a universal.
- Add 10ml media/10% FCS very slowly . First ml should take ~ 1 min, second ml should take ~ 45 seconds etc.
- When cells are resuspended in 10ml spin down in bench-top centrifuge and resuspend in 10 ml media/10% FCS (this does not need to be added slowly).
- Place cells in large flask and allow to settle for >= 2 hours.
- Take a sample, assess % viability and cell number.
- Allow to grow overnight.
- Assess cell number and viability, if the cell density is too high for the normal grwoth of the cell line add further normal growth media (e.g. RPMI1640/10% FCS.
