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AAT Bioquest/PhosphoWorks™ Luminometric ATP Assay Kit *Steady Glow*/21609/1 Plate
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | None/560 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | SmallMoleculeDetection Anions |
| Related | CellCytotoxicity ProteinKinases BiochemicalAssays |
1.Preparecells(orsamples):
1.1 Foradherentcells:Platecellsovernightingrowthmediumat1,000-10,000cells/90µL/well(fora96-wellplateor250-2,000cells/20µL/wellfora384-wellplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsinculturemediumat2,000-20,000cells/90µL/wellfora96-wellpoly-Dlysineplateor500-5,000cells/20µL/wellfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments.
Note1:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforproliferationorcytotoxicityinduction.Fortoxicityassays,startwithmorecells.
Note2:Forallluminescentexperiments,itisrecommendtousewhiteplatestoachievethebestresults.
2.PrepareATPassaysolution:
2.1 Thawallthecomponentstoroomtemperaturebeforeuse.
2.2 Transferthewholevialof10mLReactionBuffer(ComponentC)intoATPSensor(ComponentB),andmixwell.
2.3 Add20µLofATPMonitoringEnzyme(ComponentA)intothesolutionpreparedatStep2.2.
Note:AliquotandstoretheunusedComponentsAandCat-20oC.Avoidrepeatedfreeze/thawcyclesandpotentialATPcontaminationfromexogenousbiologicalsources.
3.RunATPassay:
3.1 Treatcells(orsamples)withtestcompoundsbyadding10µLof10Xcompoundsfora96-wellplateor5 µLof5Xcompoundsfora384-wellplateindesiredcompoundbuffer.Forblankwells(mediumwithoutthecells),addthecorrespondingamountofcompoundbuffer.
3.2 Incubatethecellplateina37oC,5%CO2incubatorforadesiredperiodoftime,suchas24,48or96hours.
3.3 Add100µL(96-wellplate)or25µL(384-wellplate)ofATPassaysolution(fromStep2.3)intoeachwell,andincubateatroomtemperaturefor10-20minutes.
3.4 Monitorluminescenceintensitywithastandardluminometer.
4.GenerateastandardATPcalibrationcurve:
Note:AnATPstandardcurveshouldbegeneratedtogetherwiththeaboveassayiftheabsoluteamountofATPinsamplesneedstobecalculated.
4.1 MakeaseriesofdilutionsofATPinPBSbufferwith0.1%BSAbyincludingasamplewithoutATP(asacontrol)formeasuringbackgroundluminescence.
Note:TypicallyATPconcentrationsfrom1nMto10µMareappropriate.
4.2 AddthesameamountofthedilutedATPsolutionintoanemptyplate(100µLfora96-wellplate,and25 µLfora384-wellplate).
4.3 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofATPassaysolution(fromStep2.3).
4.4 Incubatethereactionmixtureatroomtemperaturefor10to20minutes.
4.5 Monitortheluminescenceintensitywithastandardluminometer.
4.6 GeneratetheATPstandardcurve.
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