Overview | PrinterFriendlyVersion |
Ex/Em(nm) | 554/578 |
MW | N/A |
CAS# | N/A |
Solvent | N/A |
Storage | F/D/L |
Category | CellAnalysis CellCytotoxicity |
Related | ApoptosisandCytotoxicity CellApoptosis BiochemicalAssays |
1.Preparecells:
1.1 Foradherentcells:Platecellsovernightingrowthmediumat20,000cells/well/90µLfora96-wellplateor5,000cells/well/20µLfora384-wellplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletinculturemediumat80,000to200,000cells/well/90µLfora96-wellpoly-Dlysineplate,or20,000to50,000cells/well/20µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforapoptosisinduction.
2.PrepareApopxin™Orangeassaysolution:
2.1 WarmKitComponentBatroomtemperaturebeforeuse.
2.2 Add10µLofApopxin™Orange(ComponentA)into1mLofAssayBuffer(ComponentB),andmixthemwell.
Note:100µLofApopxin™Orangeassaysolutionisenoughforonewell.Preparefreshbeforeuse.
3.Runapoptosisassay:
3.1 Treatcellswithtestcompoundsbyadding10µL/well(96-wellplate)or2.5µL/well(384-wellplate)of10XtestcompoundstocksolutionintoPBSorthedesiredbuffer.Forblankwells(mediumwithoutthecells),addthesameamountofcompoundbuffer.
3.2 Incubatethecellplateina5%CO2,37°Cincubatorforadesiredperiodoftime(4-6hoursforJurkatcellstreatedwithcamptothecin)toinduceapoptosis.
3.3 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofApopxin™Orangeassaysolution(fromStep2.2)intoeachwell.
3.4 Incubatethecellplateatroomtemperatureforatleast1hour,protectedfromlight.
3.5 Centrifugecellplate(especiallyforthenon-adherentcells)at800rpmfor2minutes(brakeoff).
3.6 MonitorthefluorescenceintensityatEx/Em=540/590nm(cutoffat570nm)byusingafluorescentmicroplatereader(bottomreadmode)orusingafluorescentmicroscope(Cy3®channel).
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