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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