MediaUsed

Toprepare100mlmedium

Dulbecco"sModificationofEaglesMedium(DMEM)

1xwithoutL-glutaminewith4.5g/LGlucose.Storeat4

Cytosystems500mlCat.No.11.016.0500V

Nonessentialaminoacids100x

Cytosystems100mlCatNo.21-145-0100V.Storeat4

Penicillin-streptomycin(5,0001U/ml,5,000ug/ml)

Cytosystems100mlCatNo.21-140-0100V.Storeat-20

FoetalCalfSerum(FCS)

NeedstobetestedforABIlitytosupportgrowthofESCells.Serumshouldbestoredfrozenandheatinactivatedbeforeusebyheatingat56for30mins.Itmaybestoredat4followinginactiavation.Theheatinactivationremovescomplementactivitywhichalongwithnaturalheterophileantibodies,maybetoxicforEScells.

100xNucleosideStock

Toprepare100ml(100x)

Addto100mlTravenolwateranddissolvebywarmingto37.Filtersterilizeandaliquotwhilewarm.Storeat4formonths.Thenucleosideswillcomeoutofsolution.Warmto37beforeusetore-solubilize.Solutionseemstobestableat4C.

Othersolutionsrequired.

-1xDulbecco"sPhosphateBufferedSaltSolution(PBS)withoutcalciumandmagnesium.

-Trypsin2.5%Cytosystems100ml21-159-0100V

-Trypsin-EDTA(1:250)Cytosystems(0.05%Trypsin)100ml21-160-0100V

Solutionstobemadeup

PBS/EGTA(0.5mM)

To100mlsofPBSadd1mlof0.05MstockEGTA(SigmaE4378)togiveafinalconcentrationof0.5mM.

StockEGTAinH200.05Maddconc.NaOHtodissolve

Trypsin0.25%.

To20mlsCytosystemsTrypsin/EDTA0.05%,add1.6mls2.5%Trypsin

0.1%GelatininPBS

To100mlsPBSadd0.1gmGelatinSigmaG-1890andautoclave

0.1M2-mercaptoethanol(E)SigmaM-6250

Add0.1mlE(14.4M)to14.3mlsPBSandfilterthrougha0.2uMACRODISC.Storeat-20foruptoonemonth.

LIF(ifnofeedersareused)

LIFESGROAMRAD(murineLifinPBS/BSAsolution)1mlampoule

107U/ml.Dilute1/100inDMEM10%FCS,aliquotinto10x10mltubes,

storeat-20untiluse.

107U/mldilute1/100=105U/ml.(100xconc)

Dilute1/100foruse=1000U/ml

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