DissociationofCellsfromPrimaryTissueTrypLE™ProductsTrypLE™productsareformulatedtoallowdirectsubstitutionintoyourexistingprotocols.Thefollowinggeneralprocedurecanbeusedtoremovevariouscelllinesfromculturewarewhilemaintainingcellularintegrity.Optimalconditionsandconcentrationsemployedforindividualsystemsshouldbedeterminedempirically.1.Decantmediafromflask.Rinseflaskwith5mlofDulbecco’sPhosphateBufferedSaline(D-PBS)withoutcalciumandwithoutmagnesium(GIBCO®cat.number14190).DecantD-PBS.2.Addanappropriatevolume(i.e.2mlina75cm2flask)ofprewarmedTrypLE™toflask.Rockvesseltocoatcellsheetcompletely.3.Incubateat37°Cuntilcellshavedetached(observeat5minuteintervals).Gentlyrapvesseltodislodgecells.4.Dilutein2to5mlofcellculturegrowthmediaandtransfercellsuspensiontoa15mlcentrifugetube.5.Centrifugefor5to10minutesat100×g.Discardsupernatantandsuspendcellpelletwith2to5mloffreshgrowthmedium.Trypsin1.Afterdissectingoffunusabletissue,mincetheremainingtissueinto3to4mmpieceswithasterilescalpelorscissors.Washthetissuepiecesbyresuspendinginabalancedsaltsolutionwithoutcalciumandmagnesium.Allowthetissuepiecestosettle,andremovethesupernate.Repeatthewash2or3times.2.Placethecontainerwiththetissuepiecesonice,andremoveanyremainingsupernate.Add0.25%trypsininabalancedsaltsolutionwithoutcalciumormagnesium(1mloftrypsinforevery100mgoftissue).3.Incubateat4°Cfor6to18htomaximizepenetrationoftheenzymewithlittletrypsinactivity.4.Decantanddiscardthetrypsinfromthetissuepieces.Incubatethetissuepieceswithresidualtrypsinat37°Cfor20to30min.5.Addwarm,completemediatothetissuepiecesandgentlydispersethetissuebypipetting.Ifusingaserum-freemedium,alsoaddsoybeantrypsininhibitor.6.Filterthecellsuspensionthroughsterile,stainlesssteelmesh(100to200µM)tocompletelydisperseanyremainingtissue.Countandseedthecellsforculture.Collagenase1.Mincetissueinto3to4mmpieceswithasterilescalpelorscissors.WashthetissuepiecesseveraltimeswithHanks'BalancedSaltSolution(HBSS).2.Addcollagenase(50to200U/mlinHBSS).3.Incubateat37°Cfor4to18h.Additionof3mMCaCl2increasestheefficiencyofdissociation.4.Filterthecellsuspensionthroughasterilestainlesssteelornylonmeshtoseparatethedispersedcellsandtissuefragmentsfromthelargerpieces.Freshcollagenasecanbeaddedtothefragmentsiffurtherdisaggregationisrequired.5.WashsuspensionseveraltimesbycentrifugationinHBSS.6.Resuspendthepelletinculturemedium.Countandseedthecellsforculture.Dispase1.Mincetissueinto3to4mmpieceswithasterilescalpelorscissors.Washthetissuepiecesseveraltimesinacalciumandmagnesium-freebalancedsaltsolution.2.Adddispase(0.6to2.4U/mlincalciumandmagnesium-freebalancedsaltsolution).3.Incubateat37°Cfor20mintoseveralhours.4.Filterthecellsuspensionthroughasterile,stainlesssteelornylonmeshtoseparatethedispersedcellsandtissuefragmentsfromthelargerpieces.Freshdispasecanbeaddedtothefragmentsiffurtherdisaggregationisrequired.5.Washsuspensionseveraltimesbycentrifugationinthebalancedsaltsolution.
