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ESandTScellfreezing/thawing
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FreezingandThawingcells Itisbesttofreezecellsthataregrowingrapidly.Withadherentcells,itiseasiesttosetup100mmdishes,givethemfreshmediumthedaybeforeyoufreezethem,andfreezethemjustastheybecomeconfluent.IfyouareworkingwithsUSPensioncells,makesurethattheyaregrowingvigorouslybeforeyoufreezethem.1.Trypsinizethecellsifnecessary,andthenspinthemdown.Resuspendtheminonehalfthevolumethatyouwanttoendupwith. ThecellsshouldberesuspendedinDMEMcontaining20%fetalcalfserum.Putthetubeinanicebucketandletthecellscool. 2.Now,addtheDMSOtothecellsslowly.ThisstepisaccomplishedbyaddingtotheresuspendedcellsanequalvolumeofDMEMcontaining20%fetalcalfserumand20%DMSO. ThereasontodothisslowlyistoavoidosmoticshockandtominimizetheheatingthatoccurswhenDMSOisaddedtowater.Whenyouarefinished,youshouldhavethecellssuspendedinDMEMcontaining20%fetalcalfserumand10%DMSOwithallthecellsfromasingle100mmdishin1ml. 3.Chillthecellsonice,inthecentrifugetube,forexactly30min.Thentransferthemtofreezingvialswhichhavebeenpre-chilledinthefreezerintheplasticrack,whichhasalsobeenpre-chilledinthefreezer.Use1mlpervial.4.Putthevialsinawellinsulatedboxthathasalsobeenpre-chilledinthefreezer.Insulationisbestaccomplishedwithcotton.Thenputtheboxinthe-70°Cfreezerovernight,forslowcooling,andthenputthemintoliquidnitrogen.Cellswillstoreforeverinliquidnitrogen.Storageat-70°Cisriskier. Cellsshouldbethawedrapidlyandthendilutedslowlyintowarmgrowthmedium. Thenputthecellsona100mldish.Thiswaythecellsdon"tsufferosmoticshock.
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Freezing
Inotherwords,use0.5mlper100mmdishyouareusing.Thisshouldbedonedropwiseandthecellsuspensionshouldbemixedbyswirlingaftertheadditionofeachdrop.Thebestwaytodothisistotransferthecontentsofthethawedvialtoa50mlcentrifugetubeandthenadd--dropwise,withcontinualswirling--10mlofwarmmedium.
Somecellsseemtobenefitfrombeingdilutedintomedium,spundown,andthenresuspendedinwarmmedium.ThisgetsridoftheDMSO,butmaydisturbfragilecells.Ifthecellsareseededwithoutspinning,themediumshouldbechangedassoonasthecellshaveattached.
