Itisrecommendedtoquicklythawthefrozencellsfromliquidnitrogenina37°Cwater-bath,transfertoatubecontaining10mlofgrowthmediumwithoutBlasticidin,spindowncells,resUSPendcellsinpre-warmedgrowthmediumwithoutBlasticidin,transferresuspendedcellstoT25flaskandcultureat37°CinCO2incubator.Thenextday,replacethemediumwithfreshgrowthmediumwithoutBlasticidin,andcontinuegrowingcultureinaCO2incubatorat37°Cuntilthecellsarereadytobesplit.Cellsshouldbesplitbeforetheyreachcompleteconfluence.AtfirstpassageswitchtogrowthmediumcontainingBlasticidin.Cellsshouldbesplitbeforetheyreachcompleteconfluence.
Topassagethecells,rinsecellswithphosphatebufferedsaline(PBS),detachcellsfromculturevesselwithTrypsin/EDTA,addcompletegrowthmediumandtransfertoatube,spindowncells,resuspendcellsandseedappropriatealiquotsofcellsuspensionintonewculturevessels.
Justafterthawingandatlowdensity,thecellsmaygrowataslowerrate.Itisrecommendedtosplitthecellswith~1:4ratioatthebeginningofculturing.Afterseveralpassages,thecellgrowthrateincreasesandthecellscanbesplitwith1:8-1:20ratioweekly.
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