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 1.PrepareassaymixforOne96-wellplate:

1.1   Prepareβ-galactosidasesubstratestocksolution(200X):Add50µLofDMSO(ComponentE)intothevialofResorufinβ-galactoside(ComponentA)tomakestocksolution(200X).

Note:25µLofβ-galsubstratesstocksolution(200X)isenoughforone96-wellplate.Unusedstocksolutionshouldbealiquotedandstoredat<-20^oC.Keepfromlightandavoidrepeatedfreeze-and-thawcycles.

1.2   Prepare0.3%β-mercaptoethanolassaybuffer:Add30µLofb-mercaptoethanol(ComponentF)to10mLofReactionBuffer(ComponentB),andmixwell.

Note:Additionalbufferisneededforpreparingserialdilutionofβ-galactosidase,whichisusedtogenerateastandardcurve.

1.3   Prepareassaymixture:Add25µLofβ-galactosidasesubstratestocksolution(fromStep1.1)into5mLofassaybuffer(fromStep1.2)tomakeassaymixture.

Note1:Thisβ-galassaymixtureisenoughforone96-wellplate.Itisnotstable,useitpromptly.

Note2:Onecandivideunusedβ-galassaymixtureintosingleusealiquotsandstoredat-20^oC.

2.Preparelysisbufferworkingsolution:

Makelysisbufferworkingsolutionbyadding5µLofβ-mercaptoethanol(ComponentF)to5mLofLysisBuffer(ComponentD)beforeuse.

Note:Alwaysadd0.1%β-mercaptoethanolintolysisbufferbeforelysingthecells.

3.Preparecellextractsfrommammaliancells:

3.1   TreatcellscontainingLacZgenewithtestcompoundsforadesiredperiodoftime.

3.2   Washthecellstwicewith1XPBS.Donotdislodgethecells.

3.3   Foradherentcells:Addlysisbufferworkingsolution(fromStep2)tothecultureplates.Recommendedvolumesforvariousplatesarelistedinthefollowingtable.

Fornon-adherentcells:Pelletthecellsintocentrifugetube,andadd50-2000µL(dependingonthesizeofthecellpellet)of1Xlysisbuffertothetube.

3.4   Incubatethecellswithcelllysisbuffer(fromStep3.3)atroomtemperaturefor10-15minutes,andgentlyswirltheplatesortubesseveraltimestoensurecompletelysis.

3.5   Proceedtotheβ-galactosidaseassayorfreezethesampleat-80^oCtilluse.

Note1:Agoodlysiscanalsobeobtainedbyaquickfreeze-and-thawcycle(freeze1-2hoursat-20^oCto‑80^oCandthawatroomtemperature).

Note2:Alternatively,centrifugethecelllysisfor2-3minutestopellettheinsolublematerial,andthenassaythesupernatant.

4.Runb-galactosidaseassay:

4.1   Thawthetubeorplateoflysedcellsatroomtemperatureifneeded.Performtheassaydirectlyonthe96-wellplateifthecellswereseededina96-wellplate.

4.2   Add50µL/wellofβ-galactosidasecontainingcellextracts(fromStep3.4)intoa96-wellsolidbackplate.

Note1:Ifnecessary,dilutethelysatein1XLysisBufferwhentransfectionefficiencyisveryhigh.Orreducethevolumeoflysisbufferwhentransfectionefficiencyislow.Ifthetransfectionisperformedina96-wellplate,orastablecelllinewasseededintoa96-wellplate,performtheassaydirectlyontheplate.

Note2:Forendogenousß-galactosidaseactivitycontrol,add50µLofcelllysatefromnon-transfectedcells.Forblankcontrol,add50µLoflysisbuffer.

4.3   Optional(ifastandardcurveisdesired):Prepareβ-galactosidase(E.Coli)standardsbyperforming1:3serialdilutionsin0.3%β-mercaptoethanolassaybuffer(fromStep1.2)togetapproximately100,30,10,3,1,0.3,0.1and0mU/mLβ-galactosidasestandards.Add50µL/wellofseriallydilutedb-galactosidasestandardsintoa96-wellsolidbackplate.

Note1:Adjustthestandardcurve(concentrationrange)tosuitthespecificexperimentalconditions,suchascelltype,number,transfectionefficiency,andsizeofthecultureplates.

Note2:Thedilutionsforthestandardcurvemustbepreparedfreshlyeachtimetheassayisperformed.

4.4   Add50µLofassaymixture(fromStep1.3)toeachwell.Incubatetheplateatroomtemperatureor37^oCforapproximately10minutesto4hoursdependingonthecelltype.

4.5   Add50µLofStopBuffer(ComponentC)toeachwell.Thestopbuffercausesanincreaseinthefluorescenceintensityoftheproduct,inadditiontoterminatethereaction.

4.6   MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540/590nm(cutoff570nm).

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