FreezingCells:

• Cellsshouldbegrowingwellorknowntobeinlogphase
• Count,collectandpelletcellsina15mLtesttube
• ResUSPendinfreezingmediasothattheconcentrationisnomorethan5x10^6cells/mLofcoldfreezingmedia
• Transfer1mLofcellstoappropriatelylabelledcryovialsandmaintainoniceforapproximately30minutes
• Transfervialsto-80Cfreezerfor24hrs
• Transfertoliquidnitrogendewaror-140Cfreezerforlong-termstorage.
• Freezingmedia
  • 10%DMSO
  • 90%FCS
  • you"llneed1mLper5x10^6cells

ThawingCells:

• RemovevialfromLiquidNitrogenor-140Cfreezerandimmediatelytransferto37Cwaterbath
• Whileholdingthetipofthevial,gentlyagitatethevial,beingcarefulhnottoallowwatertopenetratethecaporseal
• Whencompletelythawed,transfercontentsofvialto15mLtesttube
• Slowlyadd10mLwarmcompletemediaandspinat1000gfor5min
• Decantmediaandresuspendpelletinavolumeofcompletemediaappropriateforflaskormacrowell
• Transfercellstoflaskor24wellplateandincubateat37Cand5%CO2
• Cellscanbecheckedvisuallyorcounted,beginningatapproximately1hr,foranestimateofviABIlity.ImmediatecellcountscanbemisleADIng

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