Tissue Culture Thawing Cells from Liquid Nitrogen
来自 : 蚂蚁淘
TissueCulture-ThawingCellsfromLiquidNitrogenMETHOD:- Preparebeforethawing:Fillatesttubeof10-15mlsofcoldmedia(appropriateforyourcellline)with10-20%fetalcalfserum(use2xthe%youuseforgrowthsupplement).PlaceinabeakerofcrushedicetoCHILL.(DMSOistoxicatroomtemperature.)Haveeverythinginthehoodthatyouwillneed:70%alcohol,papertowels,pipets,etc.
- GetvialfromLiquidNitrogen-useglovesandfaceshield.*Makesurethecoverisreplacedproperly!!
- QUICKTHAW"in37ºCH2Obathbyshakingvialrapidlyinwatertillapproximately3/4thawed,withasmallpeasizedportionstillfrozen.
- RemovefromH2Obath,butcontinuetoshakevialuntilithasthawedcompletely.
- RinsevialwithEthanol.
- Openvialcarefully,pipetupcontentsand"layer"itontocoldmedia(inthetesttubeyouhavechilling)
- Spinatapprox.1000rpmfor5min.
- Wash1xwith10-15mlsofmedia,1000rpm5min(towashoutallDMSO).GentlyresUSPendthepellet.
- SeedintoasmallflaskT25orT75withappropriatevolumeofmediacontaining2xtheregularamountofFCS,andtheregularamountofglutamineandantibiotic.
- NEXTDAY:Changemediaifgrowingadherentcellstogetridofexcessdeadcells
- Checknon-adherentcellstoseehowsoontheywillneedfreshmediaand/orneedtobesplit.
- 1-4DAYS:Passagecellswhenrequiredinusualfashion.
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