TissueCulture-ThawingCellsfromLiquidNitrogenMETHOD:

1. Preparebeforethawing:Fillatesttubeof10-15mlsofcoldmedia(appropriateforyourcellline)with10-20%fetalcalfserum(use2xthe%youuseforgrowthsupplement).PlaceinabeakerofcrushedicetoCHILL.(DMSOistoxicatroomtemperature.)Haveeverythinginthehoodthatyouwillneed:70%alcohol,papertowels,pipets,etc.
2. GetvialfromLiquidNitrogen-useglovesandfaceshield.*Makesurethecoverisreplacedproperly!!
3. QUICKTHAW"in37ºCH₂Obathbyshakingvialrapidlyinwatertillapproximately3/4thawed,withasmallpeasizedportionstillfrozen.
4. RemovefromH₂Obath,butcontinuetoshakevialuntilithasthawedcompletely.
5. RinsevialwithEthanol.
6. Openvialcarefully,pipetupcontentsand"layer"itontocoldmedia(inthetesttubeyouhavechilling)
7. Spinatapprox.1000rpmfor5min.
8. Wash1xwith10-15mlsofmedia,1000rpm5min(towashoutallDMSO).GentlyresUSPendthepellet.
9. SeedintoasmallflaskT25orT75withappropriatevolumeofmediacontaining2xtheregularamountofFCS,andtheregularamountofglutamineandantibiotic.
10. NEXTDAY:Changemediaifgrowingadherentcellstogetridofexcessdeadcells
11. Checknon-adherentcellstoseehowsoontheywillneedfreshmediaand/orneedtobesplit.
12. 1-4DAYS:Passagecellswhenrequiredinusualfashion.

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