1)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. --------------------------------------------------------------------------------Freezingmedia 60mlRPMI1640orDMEM40mlGammaglobulinfreeHorseSerum(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.