| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 390/650 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | GPCR cAMPGPCRAssays |
| Related | DiagnosticMolecules CellSignaling BiochemicalAssays |
Foradherentcells:Platecellsovernightingrowthmediumat30,000-100,000cells/wellfora96-wellplate.
Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsinculturemediumat100,000-300,000cells/wellfora96-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiment.
Treatcellsasdesired:ThefollowingisanexampleforHelacellstreatedwithForskolintoinducecAMPina96-wellplateformat.25µLcellsingrowthmedium,add25µL/well100µMForskolininHanksand20mMHepesbuffer(HHBS),incubateina5%CO2,37°Cincubatorfor15minutes.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.Cellsmaybeseededthedaybeforeoronthedayoftheexperimentdependinguponthecelltypeand/ortheeffectofthetestcompounds.
Add100µLComponentE(Diluent)toComponentC(cAMPStandard)tomake1mMstocksolution.Dilutioncanbecarriedoutwiththediluent(orwithcellculturemedia).Add2.24µL1mMstocksolutioninto200µLDiluenttomake11200nMstandard,andthenget50µL11200nMcAMPstandardinto150µLDilenttoget2800nMStandard,andrepeatthisproceduretomaketheserialdilutions.ThecAMPstandarddilutionsare:11200,2800,700,175,43.75,10.94,2.73,0.68nM(finalconcentrationof2800~0.17nMcAMPperwell).
3.PrepareAnticAMP-trFluor™Euworkingsolution:Add55µLComponentEintovialAtoreconstituteAnticAMP-trFluor™Eufor#36379,(or550µLComponentEintovialAfor#36380and#36381),andthenprepareworkingsolutionbyadding50µLreconstitutedComponentAto2.5mLCellLysisBuffer(ComponentD),ormaketheneededvolumeproportionally.Preparebeforeuse!
4.PreparecAMP-trFluor™650workingsolution:Add55µLComponentEintovialBtoreconstitutecAMP-trFluor™650for#36379,(or550µLComponentEintovialAfor#36380and#36381),andthenprepareworkingsolutionbyadding50µLreconstitutedComponentBto2.5mLCellLysisBuffer(ComponentD),ormaketheneededvolumeproportionally.Preparebeforeuse!
5.RuncAMPassay:RuncAMPAssay(includingthestandardcurveandcellbasedassay)asinstructioninthefollowingtable:
| cAMPStandard | Cells | ||||
| NegativeControl | PositiveControl | StandardCurve | NegativeControl | Non-stimulated | Stimulated |
| 25µLDiluent | 25µLDiluent | 25µLStandard | 25µLCells | 25µLCells | 25µLCells |
| 25µLCompoundBuffer | 25µLCompoundBuffer | 25µLCompoundBuffer | 25µLCompoundBuffer | 25µLCompoundBuffer | 25µLCompound |
| Incubate30minatRT | |||||
| 25µLLysisBuffer | 25µLcAMP-trFluor™650workingsolution | 25µLcAMP-trFluor™650workingsolution | 25µLLysisBuffer | 25µLcAMP-trFluor™650workingsolution | 25µLcAMP-trFluor™650workingsolution |
| 25µLAnticAMP-trFluor™Euworkingsolution | |||||
| Incubate30minatRT | |||||
ReadonacompatIBLeTR-FRETreader.
| 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)
