Molecular
FluorescentBiosensorsforLiveCellDiscovery
cADDiscAMPAssayforGi
Overview
2 RelevantProducts
2 MaterialsintheKit
2 Storage
3 Additionalmaterialsnotsupplied.
3 BioSafetyConsiderations
3 Warranty
3 AbouttheseAssays
3 SampleReactionSetupforOptimizingGi-assaywithGαs
4 ProtocolforUse
4 SuggestionsforAssaysinAdherentCells
6 FluorescenceDetection
7 AssayPerformanceConsiderations
8 TroubleShooting 10 ContactUs 10 RelatedProducts 11 References 11 !
1 7/15/16 Overview ThefollowingprotocolisoptimizedformeasuringcAMPinhibition,viaaGi-coupledreceptor,inrapidlydividing,immortalizedcelllinesona96-wellplate.TheprotocolhasbeenvalidatedinliveCHO,NIH3T3andHEK293cells[1].Thisassayisveryrobustandcanbeusedforlive-cellimagingorforscreeningonautomatedfluorescenceplatereaders.ForuseiniPSC-derivedoradherentcells,seeSuggestionsforAssaysinAdherentCellssection. TheBacMamvectorcarryingthesesensorsisamodifiedbaculovirus,whichcanbeusedfordeliveryto,andexpressionin,awidevarietyofmammaliancelltypesincludingprimarycultures. RelevantProducts ProductDescription PromotermendedUse X0200GGreencADDisGiAssay CMV FluorescenceimagingandPlatereaderassay(Z’>0.7) MaterialsintheKit -cADDiscAMPsensorBacMam⩰2x1010VG/mLinTNM-FHInsectCultureMedium (AlleleBiotechproduct#ABP-MED-10001). GreenfluorescentsensorthatincreasesinfluorescenceintensityinresponsetodecreasesincAMP.VG/mListhenumberofviralgenespermilliliter,asdistinctfromplaqueformingunits(PFU),thatforbaculovirus,wouldbemeasuredininsectcells. -SodiumButyrate(SigmaAldrichproduct#B5887)500mMinH2O. SodiumButyrateisaddedtotheculturetomaintainBacMamexpression.OtherHDACinhibitorsmayworkaswell. -hD2ReceptorBacMaminTNM-FHInsectCultureMedium(AlleleBiotechproduct #ABP-MED-10001). TheGi-coupledhD2Receptorprovidedasapositivecontrolforthepurposeofassayoptimization.YourownGi-coupledreceptorofinterestmayeitherbepresentinyourcellline,ordeliveredviatransduction/viralvector,orviaplasmid/transfection. -Quinpirolehydrochloride,2mMinSterileWater ActivatesGisignalingthroughthehD2positivecontrolreceptor. -Gαs ConstituitivelyactiveGαs,increasessteady-statelevelsofcAMPandeliminatestheneedforforskolin. !
2 7/15/16 Storage BacMamstocksshouldbestoredat4°Candprotectedfromlight.Avoidrepeatedfreeze/thawcycles. Additionalmaterialsnotsupplied.-GreinerCellCoat(#655946)isourpreferred96-wellplateavailablefromVWR.-o’sPhosphateBufferedSaline(DPBS)availablefromVWR[2]. BioSafetyConsiderations BacMamisamodifiedbaculovirus,Autographacalifornica,AcMNPV.Thevirusinthiskitispseudotypedtoinfectmammaliancells.Inmammaliancells,thebaculovirusgenomeissilent,anditcannotreplicatetoproducenewvirusinmammaliancells.Whileitshouldbehandledcarefully,inasterileenvironment,itisclassifiedasaBiosafetyLevel1(BSL-1)reagent. Thisproductisforresearchuseonlyandisnotmendedforuseorsaleinhumanoranimaldiagnosticortherapeuticproducts. Warranty Materialsareprovidedwithoutwarranty,expressorimplied.Enduserisresponsibleformakingsureproductplieswithapplicableregulations.Norighttoresellproductsorponentsoftheseproductsisconveyed. AbouttheseAssays CyclicAMPisanessentialsecondmessengerusedinmanyimportantcellularprocesses.ThemessagescarriedbycAMParetightlyregulatedinspaceandtime.ThecADDisassaycontainsaically-encodedbiosensorwithacAMPbindingdomainfusedtoafluorescentproteinsuchthatcAMPactivityisconvertedintorobustchangesinfluorescence.cAMPchangesaremeasuredinthelivingcell,incontrasttoumulationassaysmeasuredincelllysates.cADDiscanbinedwithdifferentcoloredsensorstomeasuremultiplesignalssimultaneously.Forexample,greencADDiscanbinedwiththeredGECOCa2+assay(#U0600R) ThecADDisassaydecreasesfluorescenceintensitywhencAMPisincreasinginthecellandincreasesinfluorescenceinresponsetoactivationofGαi.TodetecttheactivationofGαi,thesteadystatelevelsofcAMPshouldfirstbeincreasedwiththeconstitutivelyactiveGαsprovidedinyourkit. TheoptimalratioofcADDisBacMamtotheconstitutivelyactiveGαsBacMammustbedeterminedforagivencellline.InHEK293cells,muchlessGαsisneeded,relativetocADDis.ToolittleortoomuchoftheGαsmutantwilllimittheabilitytodetectthedecreaseincAMP.TooptimizetheratioofcADDistotheGαsmutantforyourparticularcelllineand/orreceptortype,agoodstartingpointistokeeptheamountofsensorconstant,whilegraduallyincreasingtheamountofGαsmutant. Tousetheprovidedpositivecontrol,beginbykeepingthevolumeofthereceptorcontrolconstant.AtypicalpreliminaryexperimentistoselecttwoamountsofcADDis,binedwiththesameamountofhD2receptorcontrol,andvarytheamountsofGsmutant(seeSampleReactionSetUponthenextpage). !
3 7/15/16 SampleReactionSetupforOptimizingGi-assaywithGαs Forasingle50μlrxn(i.e.1wellina96-wellplate): 20μlsensor5μlhD2receptorcontrol2μl,4μl,or8μlGαsmutant0.6μl500mMSodiumButyrate(2mMfinalconcentrationinwell)Xμlsurroundingsolutiontobringfinalvolumeto50μl(DPBS,EMEM,orMediaofchoice) 25μlsensor5μlhD2receptorcontrol2μl,4μl,or8μlGαsmutant0.6μl500mMSodiumButyrate(2mMfinalconcentration)Xμlsurroundingsolutiontobringfinalvolumeto50μl(DPBS,EMEM,orMediaofchoice) Inourexperience,whenusingtheassayinHEK93Tcells,theoptimalamountsofthesensor,Gαsmutant,andhD2receptorare25μl,4μl,and5μl,respectively. AnalternativetousingtheconstitutivelyactiveGαsprovidedinyourkit,istofirstactivateaGscoupledreceptortoincreasebasalcAMP,andthenactivatetheGαi-coupledreceptorandmeasuretheincreaseinfluorescence. Usingforskolintostimulateadenylcyclaseisanoption,howeveritisnotmended,asforskolinbindsadenylcyclaseandcandisrupttheGipathway. ProtocolforUse Day1:TransduceandPlateCells Step1PrepareCells Step2PrepareViralTransductionReaction Step3MixCellsandTransductionMix Media + BacMamStock SodiumButyrate Day2:LiveCellAssay Step1ReplaceculturemediawithDPBS TransductionMix Letrest20-30minutesprotectedfromlightatroom temperature SeedcellsStep2MeasureFluorescence Greenfluorescentsensorwithredfluorescentnucleus,BacMamdeliverytoHEK293cells; 10xobjective !
4 7/15/16 DAY1TRANSDUCEANDPLATECELLSStep1)Preparecells(TubeA) -Detachcellsfromflaskusingnormaltrypsinizationprotocol.Resuspendcellsinculturemedia(EMEM)anddeterminecellcount.Prepareadilutionofcellsat500,000cells/ml.100μlofthiscellresuspensionwillberequiredforasinglewellina96-wellplate,soprepareenoughofthedilutiontoseedthedesirednumberofwellsintheplate.Letcellssitinhoodandmoveontopreparationoftheviraltransductionreaction. Step2)PrepareViralTransductionReaction(TubeB) -Preparea500mMstocksolutionofsodiumbutyrateinsterilewater(providedinkit). -Foreachtransductionreaction(i.e.onewellina96-wellplate),preparethetransductionsolutionbymixing25μloftheBacMamsensorstockwith5μlofthehD2ReceptorControl,optimizedamountofGαsmutant,(inHEK293cells,with5μlhD2receptorcontrol,4μlGαsmutantworkswell)0.6μlofthe500mMstocksolutionofsodiumbutyrate,andenoughCompleteMedia(EMEM,10%FBS)foratotalvolumeof50μl.Mixgently. Step3)MixCellsandTransductionMixfromabove. -MixTubeAandTubeB(100μltubeA+50μltubeB).Mixgentlyandthenseed150μlofthemixperwellonthe96-wellplate. -Placeplateindark(e.g.coverwithaluminumfoil)andincubateatroomtemperaturefor30minutes.Afterthisincubationperiod,placeplatebackincellincubator.Incubatethecellsundernormalgrowthconditions,protectedfromlight,for≈24hrs(5%CO2and37°C). Step4(Optional)* After4-8hrsincubationtimewithvirus(6hrsisoptimal),removetransductionsolutionandadd100μpletegrowthmediumcontaining2mMsodiumbutyrate**. Returncellstonormalgrowthconditionsforapproximately18hrs. *Thisprotocoldoesnotrequireamediaexchangestep.Mediaexchangecanbedonetoreducewell-wellvariability,whichmaybenecessaryforsomeautomatedplatereaders. **1mMsodiumbutyratemayimprovecellhealth,butresultsindimmerfluorescence. DAY2FLUORESCENCEMEASUREMENT-Cellsarenowreadyforassay.Priortoimaging,replaceculturemediawithDPBS.Washgently soasnottodislodgecells.CoverthecellsandallowthemtorestatroomtemperatureinDPBSfor30-35minutesbeforemeasuringfluorescence.Experimentsareperformedat25°CusingstandardGFPexcitationandemissionwavelengths. -Add1μMquinpirole(finalconcentrationinwell)toactivateD2receptorcontrolwellsand measureanincreaseinfluorescenceintensitywhencAMPlevelsdecrease.Theoptimaldoseofquinpirolemayneedtobedeterminedforagivencellline.Addquinpiroleinavolumeof50μlofDPBS,towellscontaining100-150μlDPBS. !
5 7/15/16 SuggestionsforAssaysinAdherentCells Theprotocolaboveisoptimizedforrapidlydividingimmortalizedcells.However,theseassayspatiblewithscreeningprimaryculturesandiPSC-derivedlines,wherethecellsareplatedbeforetransduction.Specificdetailsoftheprotocolwillvarybycelltype,soitisimportanttotakethetimetotitrateBacMamforoptimalresults.Forexpressioninrarecelltypes,orspecificcellsinmixedcultures,Cre-dependentandspecificpromotersystemsareavailableformanyofoursensors. -Preparea500mMstocksolutionofsodiumbutyrateinsterilewater. -Foreachtransductionreaction(i.e.onewellina96-wellplate,containing100μLculturemedia perwell),prepareatransductionsolutionbymixing25μLoftheSensorBacMamstockwith5μLofReceptorcontrol,4μLofGαsmutant,0.6μLofthe500mMstocksolutionofsodiumbutyrate,and15.4μLofDPBS,foratotalvolumeof50μ
L.Mixthesolutiongently. -Sensorexpressionandcellhealthcanbecontrolledbytitratingthevirus,soitisworthtakingthe timetooptimizetheassayforyourparticularcelltype.CellCulturemediamaybeusedinplaceofDPBS. -PrepareadilutionseriesoftransductionreactionsbyvaryingtheamountofBacMam.For example,arangeof10μLto80μL,adjustingtheamountofDPBSordingly. -Addthetransductionreactiondirectlytotheplatedcells(noaspirationofcellmedium necessary).Gentlyrocktheplate4-5timesineachdirectiontomixthroughoutthewell.Incubatethecellsundernormalgrowthconditions(5%CO2and37°C),protectedfromlight,for4-8hours(6hrsisoptimal). -Aspiratetransductionsolutionandadd100μpletegrowthmediumwithsodiumbutyrateat aconcentrationof1-2mM.Returncellstonormalgrowthconditionsforapproximately18-22hrsbeforemeasuringfluorescenceasdescribedabove.Ifcellswillnottolerateafullmediaexchange,partialmediaexchangescanbedone !
6 7/15/16 FluorescenceDetection Ourassayspatiblewithautomatedfluorescentplatereaders.Ourcustomershavereportedgoodresultson: •HamamatsuFDSS•MolecularDevicesFLIPR•MolecularDevicesFlexstation•PerkinElmerEnspireWehavevalidatedon:•BiotekSynergyMX•BiotekCytation•BMGCLARIOstar•Epifluorescencemicroscopes FluorescenceProperties cADDisisconstructedwiththeverybright,mNeongreenfluorescentprotein[6].WemendChroma’sCatalogset#49003foroptimalresults.ThefluorescencepropertiespatiblewithmanyoftheFITCfiltermonlyavailableonmostmicroscopesandplatereaders. excitationmax506 emissionmax517 1.0 0.8 0.6 0.4 0.2 0.0 400 420 440 460 480 500 520 540 560 580 600 620 640 wavelength
(nm) Figure1.AbsorptionandemissionpropertiesofthemNeongreenfluorescentproteinplottedasafunctionofwavelength. !
7 7/15/16 AssayPerformanceConsiderations Timing UnlikemanyassaysthatmeasureumulationofcAMPincelllysates,thecADDisassay measurescAMPinlivingcells,inrealtime.Forbestresults,besuretocapturechangesincAMP duringthepeakresponse.InFigure2,fluorescencewascapturedfromcellsbeforetheadditionof thedrugandthensampledatregularintervals.Themaximalresponseisreachedat~10minutes aftertheadditionofthedrug. Quinpirole Figure2.DownwardcADDiscanbe 1.2 co-expressedwithaconstitutively activeGsAlphasubunittodetectGi- 5μ
M 1.2 25nM DeltaF/F0DeltaF/F0 coupledreceptoractivation.The 0.8 fluorescenceofthesensorincreases 5nM 0.8 inresponsetodecreasingcAMP levels.TheD2receptorwasactivated 0.4 withquinpirole.
0 2.5nM 0.4 0.5nM 5pM
0 EC50=3.4nMEC90~45nM 05101520Minutes 10-310-1101103105Quinpirole(nM) Howwemeasuretheinfectivityoftheviralstock. Typically,virusesarequantifiedintermsofplaqueformingunits(PFU).Inthecaseofbaculoviruses,thiswouldbeameasurementofthevirusesthatarecapableoftransducinganinsectcell,thenaturalhost.Sincemammaliancellexpressionisthegoalforthisassay,wequantifyinfectivitybymeasuringviralgenes(VG)permilliliter(mL)oftheBacMamstock.WeuseprimersspecifictotheVSVGgenepresentintheBacMamgenome.ViralsamplesarepreparedtoreleaseviralgenomicDNA,thenmultipledilutionsofthepreparationareruninqPCRagainstastandardcurvetogenerateanaveragetiterforeachviralstock.CheckthelabelonthetubetofindVG/mLforyourcAMPsensorstock. Expressionlevelsofthesensor. Tooptimizetheassayinyourparticularcelltype,itisimportanttooptimizetheamountofvirususedinthetransduction.Toolittleviruswillproducevariableresults,particularlyifthesensorexpressionlevelsarelowanddifficulttodetectonyourinstrument.InthecaseofHEK293cells,thebaselinefluorescencegoesupasyouaddmorevirus,andwhenaparticularthresholdisreached,theabsolutechangeinsensorfluorescence,aswellastheZ’fortheassay,esconstant.Figure3illustratesthisrelationship,wherethechangeinfluorescenceisinresponsetoactivationofGs.[TewsonPHet.al,2015]. Receptorexpression ThemagnitudeofthesensorresponsecanbeaffectedbythelevelofGPCRexpressioninyourcells.Wehavefoundthatlowlevelsofreceptorexpressionproducethelargestsignals,whilehighlevelsofreceptorexpressionoftenproducesmallerresponses.ThisisconsistentwiththeobservationthatoverexpressionofsomeGPCRscanchangethelevelsofsecondmessengersduetolowlevelsofspontaneousactivity. !
8 7/15/16 Figure3.AstheamountofcADDissensorBacMamaddedtothewells increases,sodoesthebaselinefluorescence,plottedingreen.ThechangeinfluorescenceinresponsetoGssignalingalsoincreaseswith morevirus,butreachesthemaximumpossiblechangeandremainsconstantoverabroadrangeofvirusconcentration(plottedinblue).Oncethemaximumchangeinsensorresponseisobtained,Z’is0.75to0.8overabroadrangeofvirusconcentration(redstars). Z’>0.75 ΔF/Ffluorescenceintensity BacMam(μL) OurgreenfluorescentsensorsunderCMVpromotercontrolhavebeenvalidatedonmanyautomatedfluorescenceplatereaders.Inthissection,wesummarizesomeoftheconsiderationsintheuseofvarioustypesoffluorescencedetectioninstrumentsandthetradeoffsbetweenthesesystems. Thesimplestformat:onedrug,onesensor,onetimepoint. WehavevalidatedoursensorsonaBioTekSynergyMXfluorescenceplatereaderusingthesampleprotocoldescribedonthepreviouspages.Baselinefluorescenceismeasuredfirst,controlreceptoragonistsareaddedbyhand,andthentheplateisinsertedbackintothefluorescenceplatereadertorecordthefluorescencefromeachwellsequentially.Thiscanallbedonebyhandbecausethesesensorsrespondoverseveralminutestoagonistsinthewell(seetheplottedresponseinFigure2).Whilethisprotocolissimple,thedrawbackisthatitdoesnotcapturetheicsoftheresponse,simplythesensorfluorescencebeforeandaftertheadditionofdrug. Twochannelformat:onedrug,twosensors,onetimepoint. bininggreenandredsensors,onecansimultaneouslyrecordtwolimbsofsecondmessengersignaling.Inthiscase,standardfluorescenceplatereaderscanbeused,buttheyneedtobefittedwiththeopticsnecessarytocollecttwodifferentchannelsoffluorescence,whichwillinvolvespecificfiltersetsandeithertwodetectorsorveryfastfilterswitching.Mosttwochannelplatereaderscanscanquickly,buttheicsoftheresponsesarelost.Thiscanbequitelimitingiftheicsofthetwosecondmessengersystemsaredifferent.Forexample,theGqstimulatedreleaseofCa2+storescanbequiterapid,whiletheDAGsignalingursoveralongertimeframe.Ifthetimingisoffwithasinglepointmeasurement,theDAGsignalingmightbedetected,butwithoutcatchingtheCa2+transient. Capturingtheics,onewellatatime. Highcontentimagingsystemsofferexcellentopportunitiesforcapturingicresponses.Inthemodelsthathaveonboardliquidhandling,fluorescencecanberecordedbeforeandaftertheadditionofvehicle,andthencontinuouslyrecordedafteradditionofthedrug.Thisprovidesicdata,buteachwellintheplateisrecordedoverrelativelylongtimeframes.Confocalsystemsarenotmended,asthesignaloutsideofthefocalplaneiseliminatedinthesesystems.. Capturingtheicsinparallel,multi-wellrecording. !
9 7/15/16 Thereareinstrumentsthatcancollectfluorescencedatafromallofthewellsofaplatesimultaneouslyandhaveautomatedliquidhandling.Theseareperfectlysuitedforrecordingtheicsoftheresponsefromeverywell.Becausetherecordingsarealldoneinparallel,thespeedoftheassayisconsiderablyfasterthanasinglechannelinstrument.AnexampleofsuchaninstrumentistheMolecularDevices“FlexStation”and“FLIPR”seriesofplatereaders,andourassayshavebeenvalidatedontheseinstruments. TroubleShooting Hereareafewsimplestepsthatmayhelpyoutroubleshootifneeded. AreYourcellsfluorescent?
Differenttypesofpromotersdriveexpressioninmammaliancells.TheCMVpromoterinourBacMamvectorsisaneffectivepromoterinmanycelllines,butnotall.Twentyfourhoursaftertransduction,youshouldseebrightgreenfluorescentcellsinatypicalepifluorescencemicroscope,orthetransducedwellsina96wellplateshouldbesignificantlymorefluorescentthanuntransducedcellsinwellsonthesameplate. HDACinhibitorsareimportanttoexpressionofthesensors.WhileBacMamtransductionalonewillinitiallygeneratelowlevelsofsensorexpression,sodiumbutyrateoranotherHDACinhibitorsuchasVPAortrichostatinA(TSA)willgenerateoptimallevelsofsensorexpressionandmaintainthislevelofexpression[Kost,T.et.al.2007].Ifcellslookunhealthy,lowerconcentrationsofHDACinhibitormaybeused.Thismayimprovecellhealth,butitwillalsoreducesensorexpression. Finally,thetypeofcellculturemediausedinyourexperimentcanaffectthetransductionefficiencyofBacMam.OurassayshavebeenvalidatedinEMEM,DMEM,andF12Kculturemedia. Isthepositivecontrolworking?
Ifthecellsareexpressingthesensor,andfluorescenceisdetectableonyourinstrument,thenthenextstepistotesttheassayinyourcells.Apositivecontrolreceptorisincludedinthiskit.Add5μlofthehD2receptorcontroltoasetofpositivecontrolwellsasdescribedintheprotocols. Additionofquinpirolewillcauseachangeinfluorescence,asshowninFigure2.Ifitdoesnot,thenitisimportanttousethispositivecontroltooptimizethreeaspectsofyourassay.First,aserialdilutionseriesofthesensorwithaconstantamountofreceptorviruscanbeusedtofindtheoptimalsensorexpressionforyourinstrument(SeeFigure3).Second,itisimportantthatyoufindtheamountofvirussufficienttotransduceallofthecellsinthewell.Third,itisimportanttodeterminewhattheicsoftheresponseisandwhetheryourinstrumentcanmeasureintheappropriatetimeframe. ContactUs Ifyouhaveanyquestionsabouttheprotocolsdescribedhere,orifyouhaveideasabouthowwecanimprovethesetools,thenwewanttohearfromyou.Yourfeedbackisextremelyvaluable.Pleasesendanemailtoinfo@,andwe’llrespondasquicklyaswecan. 1!
0 7/15/16 RelatedProducts ProductSensorDescriptionU0300RRedUpwardDAGD0300RRedDownwardDAGU0600RRedGECOCa2+ PromotermendedUse CMV FluorescenceimagingandPlatereaderassay(Z’>0.5) CMV FluorescenceimagingandPlatereaderassay(Z’>0.7) CMV FluorescenceimagingandPlatereaderassay(Z’>0.5) References
1.GrahamFL,SmileyJ,RussellWC,NairnR:CharacteristicsofahumancelllinetransformedbyDNAfromhumanadenovirustype5.JGenVirol1977,36
(1):59-74. 2.oRandVogtM:Plaqueformationandisolationofpurelineswithpoliomyelitisviruses.TheJournalofexperimentalmedicine1954.
3.ChalfieM,TuY,EuskirchenG,WardWW,PrasherDC:Greenfluorescentproteinasamarkerforgeneexpression.Science1994.
4.KostT,CondreayJ,AmesR,ReesS,RomanosM:ImplementationofBacMamvirusgenedeliverytechnologyinadrugdiscoverysetting.DrugDiscoveryToday2007,12(9-10):396-403.
5.TewsonPH,MartinkaS,ShanerN,HughesTE,QuinnAM:NewDAGandcAMPsensorsoptimizedforlivecellassaysinautomatedlaboratories.JournalofBiomolecularScreening2015. PCT/US2014/063916PatentPending 1!
1
2 RelevantProducts
2 MaterialsintheKit
2 Storage
3 Additionalmaterialsnotsupplied.
3 BioSafetyConsiderations
3 Warranty
3 AbouttheseAssays
3 SampleReactionSetupforOptimizingGi-assaywithGαs
4 ProtocolforUse
4 SuggestionsforAssaysinAdherentCells
6 FluorescenceDetection
7 AssayPerformanceConsiderations
8 TroubleShooting 10 ContactUs 10 RelatedProducts 11 References 11 !
1 7/15/16 Overview ThefollowingprotocolisoptimizedformeasuringcAMPinhibition,viaaGi-coupledreceptor,inrapidlydividing,immortalizedcelllinesona96-wellplate.TheprotocolhasbeenvalidatedinliveCHO,NIH3T3andHEK293cells[1].Thisassayisveryrobustandcanbeusedforlive-cellimagingorforscreeningonautomatedfluorescenceplatereaders.ForuseiniPSC-derivedoradherentcells,seeSuggestionsforAssaysinAdherentCellssection. TheBacMamvectorcarryingthesesensorsisamodifiedbaculovirus,whichcanbeusedfordeliveryto,andexpressionin,awidevarietyofmammaliancelltypesincludingprimarycultures. RelevantProducts ProductDescription PromotermendedUse X0200GGreencADDisGiAssay CMV FluorescenceimagingandPlatereaderassay(Z’>0.7) MaterialsintheKit -cADDiscAMPsensorBacMam⩰2x1010VG/mLinTNM-FHInsectCultureMedium (AlleleBiotechproduct#ABP-MED-10001). GreenfluorescentsensorthatincreasesinfluorescenceintensityinresponsetodecreasesincAMP.VG/mListhenumberofviralgenespermilliliter,asdistinctfromplaqueformingunits(PFU),thatforbaculovirus,wouldbemeasuredininsectcells. -SodiumButyrate(SigmaAldrichproduct#B5887)500mMinH2O. SodiumButyrateisaddedtotheculturetomaintainBacMamexpression.OtherHDACinhibitorsmayworkaswell. -hD2ReceptorBacMaminTNM-FHInsectCultureMedium(AlleleBiotechproduct #ABP-MED-10001). TheGi-coupledhD2Receptorprovidedasapositivecontrolforthepurposeofassayoptimization.YourownGi-coupledreceptorofinterestmayeitherbepresentinyourcellline,ordeliveredviatransduction/viralvector,orviaplasmid/transfection. -Quinpirolehydrochloride,2mMinSterileWater ActivatesGisignalingthroughthehD2positivecontrolreceptor. -Gαs ConstituitivelyactiveGαs,increasessteady-statelevelsofcAMPandeliminatestheneedforforskolin. !
2 7/15/16 Storage BacMamstocksshouldbestoredat4°Candprotectedfromlight.Avoidrepeatedfreeze/thawcycles. Additionalmaterialsnotsupplied.-GreinerCellCoat(#655946)isourpreferred96-wellplateavailablefromVWR.-o’sPhosphateBufferedSaline(DPBS)availablefromVWR[2]. BioSafetyConsiderations BacMamisamodifiedbaculovirus,Autographacalifornica,AcMNPV.Thevirusinthiskitispseudotypedtoinfectmammaliancells.Inmammaliancells,thebaculovirusgenomeissilent,anditcannotreplicatetoproducenewvirusinmammaliancells.Whileitshouldbehandledcarefully,inasterileenvironment,itisclassifiedasaBiosafetyLevel1(BSL-1)reagent. Thisproductisforresearchuseonlyandisnotmendedforuseorsaleinhumanoranimaldiagnosticortherapeuticproducts. Warranty Materialsareprovidedwithoutwarranty,expressorimplied.Enduserisresponsibleformakingsureproductplieswithapplicableregulations.Norighttoresellproductsorponentsoftheseproductsisconveyed. AbouttheseAssays CyclicAMPisanessentialsecondmessengerusedinmanyimportantcellularprocesses.ThemessagescarriedbycAMParetightlyregulatedinspaceandtime.ThecADDisassaycontainsaically-encodedbiosensorwithacAMPbindingdomainfusedtoafluorescentproteinsuchthatcAMPactivityisconvertedintorobustchangesinfluorescence.cAMPchangesaremeasuredinthelivingcell,incontrasttoumulationassaysmeasuredincelllysates.cADDiscanbinedwithdifferentcoloredsensorstomeasuremultiplesignalssimultaneously.Forexample,greencADDiscanbinedwiththeredGECOCa2+assay(#U0600R) ThecADDisassaydecreasesfluorescenceintensitywhencAMPisincreasinginthecellandincreasesinfluorescenceinresponsetoactivationofGαi.TodetecttheactivationofGαi,thesteadystatelevelsofcAMPshouldfirstbeincreasedwiththeconstitutivelyactiveGαsprovidedinyourkit. TheoptimalratioofcADDisBacMamtotheconstitutivelyactiveGαsBacMammustbedeterminedforagivencellline.InHEK293cells,muchlessGαsisneeded,relativetocADDis.ToolittleortoomuchoftheGαsmutantwilllimittheabilitytodetectthedecreaseincAMP.TooptimizetheratioofcADDistotheGαsmutantforyourparticularcelllineand/orreceptortype,agoodstartingpointistokeeptheamountofsensorconstant,whilegraduallyincreasingtheamountofGαsmutant. Tousetheprovidedpositivecontrol,beginbykeepingthevolumeofthereceptorcontrolconstant.AtypicalpreliminaryexperimentistoselecttwoamountsofcADDis,binedwiththesameamountofhD2receptorcontrol,andvarytheamountsofGsmutant(seeSampleReactionSetUponthenextpage). !
3 7/15/16 SampleReactionSetupforOptimizingGi-assaywithGαs Forasingle50μlrxn(i.e.1wellina96-wellplate): 20μlsensor5μlhD2receptorcontrol2μl,4μl,or8μlGαsmutant0.6μl500mMSodiumButyrate(2mMfinalconcentrationinwell)Xμlsurroundingsolutiontobringfinalvolumeto50μl(DPBS,EMEM,orMediaofchoice) 25μlsensor5μlhD2receptorcontrol2μl,4μl,or8μlGαsmutant0.6μl500mMSodiumButyrate(2mMfinalconcentration)Xμlsurroundingsolutiontobringfinalvolumeto50μl(DPBS,EMEM,orMediaofchoice) Inourexperience,whenusingtheassayinHEK93Tcells,theoptimalamountsofthesensor,Gαsmutant,andhD2receptorare25μl,4μl,and5μl,respectively. AnalternativetousingtheconstitutivelyactiveGαsprovidedinyourkit,istofirstactivateaGscoupledreceptortoincreasebasalcAMP,andthenactivatetheGαi-coupledreceptorandmeasuretheincreaseinfluorescence. Usingforskolintostimulateadenylcyclaseisanoption,howeveritisnotmended,asforskolinbindsadenylcyclaseandcandisrupttheGipathway. ProtocolforUse Day1:TransduceandPlateCells Step1PrepareCells Step2PrepareViralTransductionReaction Step3MixCellsandTransductionMix Media + BacMamStock SodiumButyrate Day2:LiveCellAssay Step1ReplaceculturemediawithDPBS TransductionMix Letrest20-30minutesprotectedfromlightatroom temperature SeedcellsStep2MeasureFluorescence Greenfluorescentsensorwithredfluorescentnucleus,BacMamdeliverytoHEK293cells; 10xobjective !
4 7/15/16 DAY1TRANSDUCEANDPLATECELLSStep1)Preparecells(TubeA) -Detachcellsfromflaskusingnormaltrypsinizationprotocol.Resuspendcellsinculturemedia(EMEM)anddeterminecellcount.Prepareadilutionofcellsat500,000cells/ml.100μlofthiscellresuspensionwillberequiredforasinglewellina96-wellplate,soprepareenoughofthedilutiontoseedthedesirednumberofwellsintheplate.Letcellssitinhoodandmoveontopreparationoftheviraltransductionreaction. Step2)PrepareViralTransductionReaction(TubeB) -Preparea500mMstocksolutionofsodiumbutyrateinsterilewater(providedinkit). -Foreachtransductionreaction(i.e.onewellina96-wellplate),preparethetransductionsolutionbymixing25μloftheBacMamsensorstockwith5μlofthehD2ReceptorControl,optimizedamountofGαsmutant,(inHEK293cells,with5μlhD2receptorcontrol,4μlGαsmutantworkswell)0.6μlofthe500mMstocksolutionofsodiumbutyrate,andenoughCompleteMedia(EMEM,10%FBS)foratotalvolumeof50μl.Mixgently. Step3)MixCellsandTransductionMixfromabove. -MixTubeAandTubeB(100μltubeA+50μltubeB).Mixgentlyandthenseed150μlofthemixperwellonthe96-wellplate. -Placeplateindark(e.g.coverwithaluminumfoil)andincubateatroomtemperaturefor30minutes.Afterthisincubationperiod,placeplatebackincellincubator.Incubatethecellsundernormalgrowthconditions,protectedfromlight,for≈24hrs(5%CO2and37°C). Step4(Optional)* After4-8hrsincubationtimewithvirus(6hrsisoptimal),removetransductionsolutionandadd100μpletegrowthmediumcontaining2mMsodiumbutyrate**. Returncellstonormalgrowthconditionsforapproximately18hrs. *Thisprotocoldoesnotrequireamediaexchangestep.Mediaexchangecanbedonetoreducewell-wellvariability,whichmaybenecessaryforsomeautomatedplatereaders. **1mMsodiumbutyratemayimprovecellhealth,butresultsindimmerfluorescence. DAY2FLUORESCENCEMEASUREMENT-Cellsarenowreadyforassay.Priortoimaging,replaceculturemediawithDPBS.Washgently soasnottodislodgecells.CoverthecellsandallowthemtorestatroomtemperatureinDPBSfor30-35minutesbeforemeasuringfluorescence.Experimentsareperformedat25°CusingstandardGFPexcitationandemissionwavelengths. -Add1μMquinpirole(finalconcentrationinwell)toactivateD2receptorcontrolwellsand measureanincreaseinfluorescenceintensitywhencAMPlevelsdecrease.Theoptimaldoseofquinpirolemayneedtobedeterminedforagivencellline.Addquinpiroleinavolumeof50μlofDPBS,towellscontaining100-150μlDPBS. !
5 7/15/16 SuggestionsforAssaysinAdherentCells Theprotocolaboveisoptimizedforrapidlydividingimmortalizedcells.However,theseassayspatiblewithscreeningprimaryculturesandiPSC-derivedlines,wherethecellsareplatedbeforetransduction.Specificdetailsoftheprotocolwillvarybycelltype,soitisimportanttotakethetimetotitrateBacMamforoptimalresults.Forexpressioninrarecelltypes,orspecificcellsinmixedcultures,Cre-dependentandspecificpromotersystemsareavailableformanyofoursensors. -Preparea500mMstocksolutionofsodiumbutyrateinsterilewater. -Foreachtransductionreaction(i.e.onewellina96-wellplate,containing100μLculturemedia perwell),prepareatransductionsolutionbymixing25μLoftheSensorBacMamstockwith5μLofReceptorcontrol,4μLofGαsmutant,0.6μLofthe500mMstocksolutionofsodiumbutyrate,and15.4μLofDPBS,foratotalvolumeof50μ
L.Mixthesolutiongently. -Sensorexpressionandcellhealthcanbecontrolledbytitratingthevirus,soitisworthtakingthe timetooptimizetheassayforyourparticularcelltype.CellCulturemediamaybeusedinplaceofDPBS. -PrepareadilutionseriesoftransductionreactionsbyvaryingtheamountofBacMam.For example,arangeof10μLto80μL,adjustingtheamountofDPBSordingly. -Addthetransductionreactiondirectlytotheplatedcells(noaspirationofcellmedium necessary).Gentlyrocktheplate4-5timesineachdirectiontomixthroughoutthewell.Incubatethecellsundernormalgrowthconditions(5%CO2and37°C),protectedfromlight,for4-8hours(6hrsisoptimal). -Aspiratetransductionsolutionandadd100μpletegrowthmediumwithsodiumbutyrateat aconcentrationof1-2mM.Returncellstonormalgrowthconditionsforapproximately18-22hrsbeforemeasuringfluorescenceasdescribedabove.Ifcellswillnottolerateafullmediaexchange,partialmediaexchangescanbedone !
6 7/15/16 FluorescenceDetection Ourassayspatiblewithautomatedfluorescentplatereaders.Ourcustomershavereportedgoodresultson: •HamamatsuFDSS•MolecularDevicesFLIPR•MolecularDevicesFlexstation•PerkinElmerEnspireWehavevalidatedon:•BiotekSynergyMX•BiotekCytation•BMGCLARIOstar•Epifluorescencemicroscopes FluorescenceProperties cADDisisconstructedwiththeverybright,mNeongreenfluorescentprotein[6].WemendChroma’sCatalogset#49003foroptimalresults.ThefluorescencepropertiespatiblewithmanyoftheFITCfiltermonlyavailableonmostmicroscopesandplatereaders. excitationmax506 emissionmax517 1.0 0.8 0.6 0.4 0.2 0.0 400 420 440 460 480 500 520 540 560 580 600 620 640 wavelength
(nm) Figure1.AbsorptionandemissionpropertiesofthemNeongreenfluorescentproteinplottedasafunctionofwavelength. !
7 7/15/16 AssayPerformanceConsiderations Timing UnlikemanyassaysthatmeasureumulationofcAMPincelllysates,thecADDisassay measurescAMPinlivingcells,inrealtime.Forbestresults,besuretocapturechangesincAMP duringthepeakresponse.InFigure2,fluorescencewascapturedfromcellsbeforetheadditionof thedrugandthensampledatregularintervals.Themaximalresponseisreachedat~10minutes aftertheadditionofthedrug. Quinpirole Figure2.DownwardcADDiscanbe 1.2 co-expressedwithaconstitutively activeGsAlphasubunittodetectGi- 5μ
M 1.2 25nM DeltaF/F0DeltaF/F0 coupledreceptoractivation.The 0.8 fluorescenceofthesensorincreases 5nM 0.8 inresponsetodecreasingcAMP levels.TheD2receptorwasactivated 0.4 withquinpirole.
0 2.5nM 0.4 0.5nM 5pM
0 EC50=3.4nMEC90~45nM 05101520Minutes 10-310-1101103105Quinpirole(nM) Howwemeasuretheinfectivityoftheviralstock. Typically,virusesarequantifiedintermsofplaqueformingunits(PFU).Inthecaseofbaculoviruses,thiswouldbeameasurementofthevirusesthatarecapableoftransducinganinsectcell,thenaturalhost.Sincemammaliancellexpressionisthegoalforthisassay,wequantifyinfectivitybymeasuringviralgenes(VG)permilliliter(mL)oftheBacMamstock.WeuseprimersspecifictotheVSVGgenepresentintheBacMamgenome.ViralsamplesarepreparedtoreleaseviralgenomicDNA,thenmultipledilutionsofthepreparationareruninqPCRagainstastandardcurvetogenerateanaveragetiterforeachviralstock.CheckthelabelonthetubetofindVG/mLforyourcAMPsensorstock. Expressionlevelsofthesensor. Tooptimizetheassayinyourparticularcelltype,itisimportanttooptimizetheamountofvirususedinthetransduction.Toolittleviruswillproducevariableresults,particularlyifthesensorexpressionlevelsarelowanddifficulttodetectonyourinstrument.InthecaseofHEK293cells,thebaselinefluorescencegoesupasyouaddmorevirus,andwhenaparticularthresholdisreached,theabsolutechangeinsensorfluorescence,aswellastheZ’fortheassay,esconstant.Figure3illustratesthisrelationship,wherethechangeinfluorescenceisinresponsetoactivationofGs.[TewsonPHet.al,2015]. Receptorexpression ThemagnitudeofthesensorresponsecanbeaffectedbythelevelofGPCRexpressioninyourcells.Wehavefoundthatlowlevelsofreceptorexpressionproducethelargestsignals,whilehighlevelsofreceptorexpressionoftenproducesmallerresponses.ThisisconsistentwiththeobservationthatoverexpressionofsomeGPCRscanchangethelevelsofsecondmessengersduetolowlevelsofspontaneousactivity. !
8 7/15/16 Figure3.AstheamountofcADDissensorBacMamaddedtothewells increases,sodoesthebaselinefluorescence,plottedingreen.ThechangeinfluorescenceinresponsetoGssignalingalsoincreaseswith morevirus,butreachesthemaximumpossiblechangeandremainsconstantoverabroadrangeofvirusconcentration(plottedinblue).Oncethemaximumchangeinsensorresponseisobtained,Z’is0.75to0.8overabroadrangeofvirusconcentration(redstars). Z’>0.75 ΔF/Ffluorescenceintensity BacMam(μL) OurgreenfluorescentsensorsunderCMVpromotercontrolhavebeenvalidatedonmanyautomatedfluorescenceplatereaders.Inthissection,wesummarizesomeoftheconsiderationsintheuseofvarioustypesoffluorescencedetectioninstrumentsandthetradeoffsbetweenthesesystems. Thesimplestformat:onedrug,onesensor,onetimepoint. WehavevalidatedoursensorsonaBioTekSynergyMXfluorescenceplatereaderusingthesampleprotocoldescribedonthepreviouspages.Baselinefluorescenceismeasuredfirst,controlreceptoragonistsareaddedbyhand,andthentheplateisinsertedbackintothefluorescenceplatereadertorecordthefluorescencefromeachwellsequentially.Thiscanallbedonebyhandbecausethesesensorsrespondoverseveralminutestoagonistsinthewell(seetheplottedresponseinFigure2).Whilethisprotocolissimple,thedrawbackisthatitdoesnotcapturetheicsoftheresponse,simplythesensorfluorescencebeforeandaftertheadditionofdrug. Twochannelformat:onedrug,twosensors,onetimepoint. bininggreenandredsensors,onecansimultaneouslyrecordtwolimbsofsecondmessengersignaling.Inthiscase,standardfluorescenceplatereaderscanbeused,buttheyneedtobefittedwiththeopticsnecessarytocollecttwodifferentchannelsoffluorescence,whichwillinvolvespecificfiltersetsandeithertwodetectorsorveryfastfilterswitching.Mosttwochannelplatereaderscanscanquickly,buttheicsoftheresponsesarelost.Thiscanbequitelimitingiftheicsofthetwosecondmessengersystemsaredifferent.Forexample,theGqstimulatedreleaseofCa2+storescanbequiterapid,whiletheDAGsignalingursoveralongertimeframe.Ifthetimingisoffwithasinglepointmeasurement,theDAGsignalingmightbedetected,butwithoutcatchingtheCa2+transient. Capturingtheics,onewellatatime. Highcontentimagingsystemsofferexcellentopportunitiesforcapturingicresponses.Inthemodelsthathaveonboardliquidhandling,fluorescencecanberecordedbeforeandaftertheadditionofvehicle,andthencontinuouslyrecordedafteradditionofthedrug.Thisprovidesicdata,buteachwellintheplateisrecordedoverrelativelylongtimeframes.Confocalsystemsarenotmended,asthesignaloutsideofthefocalplaneiseliminatedinthesesystems.. Capturingtheicsinparallel,multi-wellrecording. !
9 7/15/16 Thereareinstrumentsthatcancollectfluorescencedatafromallofthewellsofaplatesimultaneouslyandhaveautomatedliquidhandling.Theseareperfectlysuitedforrecordingtheicsoftheresponsefromeverywell.Becausetherecordingsarealldoneinparallel,thespeedoftheassayisconsiderablyfasterthanasinglechannelinstrument.AnexampleofsuchaninstrumentistheMolecularDevices“FlexStation”and“FLIPR”seriesofplatereaders,andourassayshavebeenvalidatedontheseinstruments. TroubleShooting Hereareafewsimplestepsthatmayhelpyoutroubleshootifneeded. AreYourcellsfluorescent?
Differenttypesofpromotersdriveexpressioninmammaliancells.TheCMVpromoterinourBacMamvectorsisaneffectivepromoterinmanycelllines,butnotall.Twentyfourhoursaftertransduction,youshouldseebrightgreenfluorescentcellsinatypicalepifluorescencemicroscope,orthetransducedwellsina96wellplateshouldbesignificantlymorefluorescentthanuntransducedcellsinwellsonthesameplate. HDACinhibitorsareimportanttoexpressionofthesensors.WhileBacMamtransductionalonewillinitiallygeneratelowlevelsofsensorexpression,sodiumbutyrateoranotherHDACinhibitorsuchasVPAortrichostatinA(TSA)willgenerateoptimallevelsofsensorexpressionandmaintainthislevelofexpression[Kost,T.et.al.2007].Ifcellslookunhealthy,lowerconcentrationsofHDACinhibitormaybeused.Thismayimprovecellhealth,butitwillalsoreducesensorexpression. Finally,thetypeofcellculturemediausedinyourexperimentcanaffectthetransductionefficiencyofBacMam.OurassayshavebeenvalidatedinEMEM,DMEM,andF12Kculturemedia. Isthepositivecontrolworking?
Ifthecellsareexpressingthesensor,andfluorescenceisdetectableonyourinstrument,thenthenextstepistotesttheassayinyourcells.Apositivecontrolreceptorisincludedinthiskit.Add5μlofthehD2receptorcontroltoasetofpositivecontrolwellsasdescribedintheprotocols. Additionofquinpirolewillcauseachangeinfluorescence,asshowninFigure2.Ifitdoesnot,thenitisimportanttousethispositivecontroltooptimizethreeaspectsofyourassay.First,aserialdilutionseriesofthesensorwithaconstantamountofreceptorviruscanbeusedtofindtheoptimalsensorexpressionforyourinstrument(SeeFigure3).Second,itisimportantthatyoufindtheamountofvirussufficienttotransduceallofthecellsinthewell.Third,itisimportanttodeterminewhattheicsoftheresponseisandwhetheryourinstrumentcanmeasureintheappropriatetimeframe. ContactUs Ifyouhaveanyquestionsabouttheprotocolsdescribedhere,orifyouhaveideasabouthowwecanimprovethesetools,thenwewanttohearfromyou.Yourfeedbackisextremelyvaluable.Pleasesendanemailtoinfo@,andwe’llrespondasquicklyaswecan. 1!
0 7/15/16 RelatedProducts ProductSensorDescriptionU0300RRedUpwardDAGD0300RRedDownwardDAGU0600RRedGECOCa2+ PromotermendedUse CMV FluorescenceimagingandPlatereaderassay(Z’>0.5) CMV FluorescenceimagingandPlatereaderassay(Z’>0.7) CMV FluorescenceimagingandPlatereaderassay(Z’>0.5) References
1.GrahamFL,SmileyJ,RussellWC,NairnR:CharacteristicsofahumancelllinetransformedbyDNAfromhumanadenovirustype5.JGenVirol1977,36
(1):59-74. 2.oRandVogtM:Plaqueformationandisolationofpurelineswithpoliomyelitisviruses.TheJournalofexperimentalmedicine1954.
3.ChalfieM,TuY,EuskirchenG,WardWW,PrasherDC:Greenfluorescentproteinasamarkerforgeneexpression.Science1994.
4.KostT,CondreayJ,AmesR,ReesS,RomanosM:ImplementationofBacMamvirusgenedeliverytechnologyinadrugdiscoverysetting.DrugDiscoveryToday2007,12(9-10):396-403.
5.TewsonPH,MartinkaS,ShanerN,HughesTE,QuinnAM:NewDAGandcAMPsensorsoptimizedforlivecellassaysinautomatedlaboratories.JournalofBiomolecularScreening2015. PCT/US2014/063916PatentPending 1!
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