Contributor:SupryaJayadevDate:January10,19911)Keeppreparedsolutionsonice.2)Determinetotalcellcountofcellstobefrozen.(e.g.1X108)3)Determinenumberofvialstobefrozen.(e.g.10vialsat1X107)4)CentrifugecellsUSPension.5)Resuspendcellpelletinhalftherequiredvolumeoffreezingmedia.(egiffreezing10vials,add5mlsfreezingmedia)6)Keepcellsuspensiononice.7)SlowlyadddropwisewithmixinganequalvolumeofDMSOsolution.8)Dispense1mloffinalmixturepersterileNuncfreezingvial.9)FreezeinliquidnitrogenplugorincontrolledratePlanarfreezer.
Freezingmedia60mlRPMI1640orDMEM40mlGammaglobulinfreeHorseSerum(orFCS)1mlPenicillin-Streptomycin-Filterthrough0.2µfilter.-Aliquotin15mlconicaltubesandstorein-200Cfreezerforlongtermperiods.-Aliquotscanbestoredintissuecultureflasksinrefrigeratorforshortperiods.20%DMSOsolutionMAKEFRESHONDAYOFUSE!Ifusing100%DMSO:4mlpHadjustedmediumRPMI(orange-redcolor)1mlsterile100%DMSO0.1mlversene-Filterthrougha0.2µfilter.Ifusing50%DMSO:3mlpHadjustedmedium2ml50%DMSO0.1mlversene-Filterthrougha0.2µfilter.NOTE:Versenecanbeleftout,butaddingitpreventscellsfromclumping.
