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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:ThefollowingisanexampleofHelacellstreatedwithForskolintoinducecAMPina96-wellplateformat.

a).Aspirateoffcellgrowthmedium,add100µL/well100µMForskolininHanksand20mMHepesbuffer(HHBS),incubateina5%CO₂,37^oCincubatorfor15minutes;b).Aspirateoffcellsolutionaftertheincubation,add100µL/wellofCellLysisBuffer(ComponentE),andincubateatroomtemperatureforanother10minutes.ThiscelllysatecanbeassayeddirectlyorafterdilutedinAssayBuffer(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)tohave10,000,100,30,10,3,1,0.3,0.1,0.03,0.01,0.003and0nMcAMPdilutedsolutions.Storeoniceor4^oC.

Note:Theunusedreconstituted100µMcAMPstocksolutionshouldbealiquotedandstoredat-20^oC.

2.2   Prepare50XHRP-cAMPconjugatestocksolutionbyadding55µL(forKit36370)or550µL(forKit36371)ofAssayBuffer(ComponentB)intothevialofHRP-cAMPConjugate(ComponentC).Make1:50dilutionwithAssayBuffer(ComponentB)tohave1XHRP-cAMPconjugateworkingsolutionbeforeuse.Storeitoniceor4^oC.

Note1:25µLof1XHRP-cAMPconjugateworkingsolutionisenoughforoneassaypoint;prepareappropriatelyvolumeforsingleuseonly.

Note2:Theunused50XHRP-cAMPconjugatestocksolutionshouldbedividedintosingleusealiquotsandstoredthemat-20^oC.

2.3   Prepare1Xwashingsolutionbyadding1mLof10XWashSolution(ComponentD)to9mLdistilledwater.

3.RuncAMPassay:

3.1   Alltheassaywellswillbepreparedinthefollowingorders:A)cAMPstandards,control,ortestssamples;B)HRP-cAMPconjugate.

3.2   Add75µL/wellofthecAMPdilutedstandardsolution(fromStep2.1)andtestsamplesintoeachwelloftheanti-cAMPAbcoated96-wellplate(ComponentF).Werecommendedduplicatingtheassaysforeachstandardandtestingsample.Incubateatroomtemperaturefor5to10minutes.

3.3   Add25µL/wellof1XHRP-cAMPconjugateworkingsolution(fromStep2.2).Incubateatroomtemperaturefor3hoursbyplacingtheplateonshaker.

3.4   Aspirateplatecontents,andwash4timeswith200µL/wellof1Xwashsolution(fromStep2.3).

3.5   Add100µL/wellofAmplite™Green(ComponentG)intoeachwell,andincubateatroomtemperaturefor60minto3hours,protectedfromlight.

3.6   Monitortheabsorbanceincreaseat405nm,650nm,or740nmusinganabsorbanceplatereader.

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