| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 497/520 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellAnalysis CellCytotoxicity |
| Related | ApoptosisandCytotoxicity CellApoptosis |
| Spectrum | AdvancedSpectrumViewer |
- Culturecellstoanoptimaldensityforapoptosisinductionaccordingtoyourspecificprotocol.Werecommendabout30,000to50,000cells/wellforadherentcellsgrownina96-wellmicroplateculture,orabout1to2x106cells/mLfornon-adherentcells.Atthesametime,cultureanon-inducednegativecontrolcellpopulationatthesamedensityastheinducedpopulationforeverylabelingcondition.
Note:WetreatedHeLacellswith100nM-1µMstaurosporinefor4hourstoinducecellapoptosis.SeeFigure1fordetails. - FixationandPermeABIlization
a.Removecellmedia.
b.Add100μL/well/96-wellplateof4%formaldehydefixativebuffer(notsupplied)toeachwell.
Note:Fornon-adherentcells,adddesiredamount(suchas2x106cells/mL)of4%formaldehydefixativebuffer.
c.Incubateplatesfor20to30minutesatroomtemperature.
d.Removefixative.
Optional:add100μL/well/96-wellplateofthepermeabilizationreagent(0.2%TritonX-100inPBS,notsupplied)afterthefixationifneeded,andincubatetheplatefor10minutesatroomtemperature.
e.WashthecellswithPBS2-3times.
Optional:YoumayalsoprepareapositivecontrolforTUNELreactionusingDNAaseIbydigestingcellswithDNAaseIfor30minatroomtemperaturebeforeproceedtoTUNELreaction(Step3) - TUNELreaction
a.Preparereactionmixturejustbeforeusebasedonthenumberofsamplestobeassayed:ReactionComponents VolumePerWell 100XTunnelyte™Green(ComponentA) 0.5µL ReactionBuffer(ComponentB) 50µL Totalvolume 50.5µL
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
b.Add50µLofthereactionmixture(fromStep3.1)toeachsampleandincubateat37ºCfor60minutes.
c.Removethereactionmixture,andwashthecells3-5timeswith200µL/wellofPBS - Monitorthefluorescenceintensitybyfluorescencemicroscope,flowcytometer,orfluorescencemicroplatereaderatEx/Em=490/520nm.
- Optional:Stainthenucleuswith1XHoechst(ComponentC,Ex/Em=350/460nm)forimageanalysis.
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