| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | R/D/L |
| Category | GPCR cAMPGPCRAssays |
| Related | DiagnosticMolecules CellSignaling BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
Note1.Allowallthekitcomponentstowarmtoroomtemperaturebeforeusingthem;Note2:Somematerialmightbesticktothevialcapduringtheshipment.Brieflycentrifugethevialtocollectallthecontent.
1.Preparesamples:
1.1 CellSamples:
Foradherentcells:Platecellsovernightingrowthmediumat30,000-100,000cells/wellfora96-wellplate.
Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsinculturemediumat100,000-300,000cells/wellfora96-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiment.
Treatcellsasdesired:ThefollowingisanexampleforHelacellstreatedwithFoskolintoinducecAMPina96-wellplateformat.
a).Aspirateoffcellgrowthmedium,add100µL/well100µMForskolininHanksand20mMHepesbuffer(HHBS),incubateina5%CO2,37oCincubatorfor15minutes;b).Aspirateoffcellsolutionaftertheincubation,add100µL/wellofCellLysisBuffer(ComponentE),andincubateatroomtemperatureforanother10minutes.ThiscelllysatecanbeassayeddirectlyordilutedinAssayBuffer(ComponentB).
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.Cellsmaybeseededthedaybeforeoronthedayoftheexperimentdependinguponthecelltypeand/ortheeffectofthetestcompounds.
1.2 TissueSamples:Itisimportanttorapidlyfreezetissuesaftercollection(e.g.,usingliquidnitrogen)duetoquickmetabolismofcyclicnucleotidesintissue.Weighthefrozentissueandadd10-20μL/mgofcelllysisbuffer.Homogenizethesampleonice.Spinattopspeedfor5minutesandcollectthesupernatant.Thesupernatantmaybeassayeddirectly.
1.3 Urine,PlasmaandCultureMediumSamples:Urineandplasmamaybetesteddirectlywith1:200to1:1000dilutionsin1XLysisBuffer.Culturemediumcanalsobetestedwith1:10to1:200dilutionsinLysisBuffer.
Note:RPMImediummaycontain>350fmol/μLcAMP.
2.PreparecAMPassaysolutions:
2.1 Prepare100µMcAMPstocksolutionbyadding1mLofAssayBuffer(ComponentB)tothevialofcAMPStandard(ComponentA).Make1:10,1:100and1:3serialdilutionsinAssayBuffer(ComponentB)tohave10,000,100,30,10,3,1,0.3,and0nMcAMPdilutedsolutions.Storeoniceor4oC.
Note:Theunusedreconstituted100µMcAMPstocksolutionshouldbealiquotedandstoredat-20oC.
2.2 Prepare50XHRP-cAMPconjugatestocksolutionbyadding55µL(forKit36373)or550µL(forKit36374)ofAssayBuffer(ComponentB)intothevialofHRP-cAMPConjugate(ComponentC).Make1:50dilutionwithassaybuffertohave1XHRP-cAMPconjugateworkingsolutionbeforeuse.Storeitoniceor4oC.
Note1:25µLof1XHRP-cAMPconjugateworkingsolutionisenoughforoneassaypoint;prepareappropriatelyvolumeforsingleuseonly;
Note2:Theunused50XHRP-cAMPconjugatestocksolutionshouldbedividedintosingleusealiquotsandstoredthemat-20oC.
2.3 Prepare1Xwashingsolutionbyadding1mLof10XWashSolution(ComponentD)to9mLdistilledwater.
2.4 Prepare200XAmplite™Redstocksolutionbyadding50µL(forKit36373)or500µL(forKit36374)ofDMSOintothewellofAmplite™Red(ComponentG).
Note:0.5µLof200XAmplite™Redstocksolutionisenoughforoneassaypoint.Theunusedreconstitutedstocksolutionshouldbealiquotedandstoredat-20oC.
3.RuncAMPassay:
3.1 Alltheassaywellswillbepreparedinthefollowingorders:A)cAMPstandards,control,ortestssamples;B)HRP-cAMPconjugate.
3.2 Add75µL/wellofthecAMPdilutedsolution(fromStep2.1)andtestsamplesintoeachwelloftheanti-cAMPAbcoated96-wellplate(ComponentH).Itisrecommendedtoduplicatetheassaysforeachstandardandtestsample.Incubateatroomtemperaturefor5to10minutes.
3.3 Add25µL/wellof1XHRP-cAMPconjugateworkingsolution(fromStep2.2).Incubateatroomtemperaturefor2hoursbyplacingtheplateonshaker.
3.4 Aspirateplatecontents,andwash4timeswith200µL/wellof1Xwashsolution(fromStep2.3).
3.5 PrepareAmplite™Redworkingsolutionbyadding50µLof200XAmplite™Redstocksolution(fromStep2.4)and11.5µLof3%H2O2(ComponentF)into10mLofSubstrateBuffer(ComponentI).
Note:TheAmplite™Redworkingsolutionisnotstable,useitpromptly.
3.6 Add100µL/wellofAmplite™Redworkingsolution(fromStep3.5)intoeachwell,andincubateatroomtemperaturefor10minutesto2hours,orupto24hours,protectedfromlight.
3.7 MonitorthefluorescenceincreaseatEx/Em=540/590nm(cutoff570nm)byusingafluorescenceplatereader(topreadmode).
| References&Citations |  CitationExplorer |
ActivationofP2X7andP2Y11purinergicreceptorsinhibitsmigrationandnormalizestumor-derivedendothelialcellsviacAMPsignaling
Authors:DAvanzato,TGenova,AFiorioPla,MBernardini,SBianco,BBussolati,DMancardi,EGiraudo,FMaione,PCassoni
Journal:ScientificReports(2016)
TheM2muscarinicreceptorsareessentialforsignalingintheheartleftventricleduringrestraintstressinmice
Authors:HanaTomankova,PaulinaValuskova,EvaVarejkova,JanaRotkova,JanBenes,JaromirMyslivecek
Journal:Stress(2015)
