ES and TS cell freezing/thawing
Needed:
EScellfreezingmedium(2x)
2xEScellfreezingmediumshouldbemadeupfresheachtimeitistobeused,andshouldcomprisefreshlyprepared60%DMEM+,20%FCS,and20%DMSO(Sigma,Cat.No.D-5879).
Freezingincryovials
Thegeneralprotocolforfreezingcellsgrowninastandard10cmdishat70%confluencyisgivenbelow:
1.Changemedia2-3hoursbeforefreezingthecells.
2.Freshlyprepare2xfreezingmedia.
3.Harvestthecellsina15ml.tubecontainingDMEM+mediumaftertrypsinization.
4.Spindownat1000rpmfor5minatroomtemperature.
5.Removethesupernatantthenoneortwodrops(200microliters)ofDMEM+mediumtothetube.Shakegentlybutthoroughly,todispersethecells.
6.AddanadditionalDMEM+mediumtoatotalvolumeof1.5mlanddispersethecellscarefullysothattheycompriseasinglecellsUSPension.
7.Addanequalvolume(1.5ml)of2xfreezingmediumandmixbypipettingseveraltimes.
8.QuicklyaliquotthecellsuspensionintothreevialsandimmediatelyplacetheminaStyrofoambox(thiswillallowthemtocooldowngradually).Alternatively
specialboxesdedicatedtothistaskcanbepurchasedfromanumberofmanufacturers(forexampleStratagene).
9.Placetheboxina-700Cfreezerfor1-2days,thentransfertheindividualcryovialsintoaliquidnitrogencontainerforlongtermstorage.
Freezingin96wellplates
1.Workingonerowatatimeusingamultichannelpipettorchangethemedium2-3hourspriortofreezing.
2.Freshlyprepare2xcellfreezingmedia.
3.AspiratethemediumfromeachwellandwashthecellswithPBS(approximately200ml).
4.Add50microlitertrypsintoeachwell,thenplaceplateinanincubatorfor5-10min.
5.Workingonice,preferablyinawideflatcontainer,aliquot50microliterofDMEM+mediumintoeachwell.Pipettethecellsseveraltimessoastogetthem
intoahomogenoussuspension.
6.Thenadd100microliter2xcellfreezingmediatothewells,andagainpipettetomix.
7.Finallyadd80-100microlitersterilemineraloil(Sigma,Cat.No.M-8410)tocoverthecell/freezingmediummixture.
8.WraptheplatesinParafilm,placeinastyrofoambox,andstoreina-700Cfreezeruntilsuchtimeasthedesiredcloneshavebeenidentifiedandneedtobe
recovered.
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