| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 556/579 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellAnalysis CellCytotoxicity |
| Related | ApoptosisandCytotoxicity CellApoptosis |
| Spectrum | AdvancedSpectrumViewer |
Note:ThawComponentsCatroomtemperature,keepComponentsAandBonicebeforeuse.
1. Culturecellstoanoptimaldensityforapoptosisinductionaccordingtoyourspecificprotocol.Werecommendabout30,000to50,000cells/wellforadherentcellsgrownina96-wellmicroplateculture,orabout1to2x106cells/mLfornon-adherentcells. Atthesametime,cultureanon-inducednegativecontrolcellpopulationatthesamedensityastheinducedpopulationforeverylabelingcondition.
Note:WetreatedHeLacellswith100nM-1µMstaurosporinefor4hourstoinducecellapoptosis.SeeFigure1fordetails.
2. 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)
3. TUNELreaction
3.1 Preparereactionmixturejustbeforeusebasedonthenumberofsamplestobeassayed:
ReactionComponents | Volume PerWell |
100XTunnelyte™Red(ComponentA) | 0.5µL |
ReactionBuffer(ComponentB) | 50µL |
Totalvolume | 50.5µL |
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
3.2 Add50µLofthereactionmixture(fromStep3.1)toeachwellortubeandincubateat37°Cfor60minutes.
3.3 Removethereactionmixture,andwashthecells3-5timeswith200µL/wellofPBS.
4. Monitorthefluorescenceintensitybyfluorescencemicroscope,flowcytometer,orfluorescencemicroplatereaderatEx/Em=550/590-650nm.
5. Optional:Stainthenucleuswith1XHoechst(ComponentC,Ex/Em=350/460nm)forimageanalysis.
| References&Citations |  CitationExplorer |
Vaccarinalleviateshypertensionandnephropathyinrenovascularhypertensiverats
Authors:WeiweiCai,ZhenpengZhang,YiqiHuang,HaijianSun,LiyingQiu
Journal:ExperimentalandTherapeuticMedicine(2018):924--932
CO-releasingmolecules-2attenuatesox-LDL-inducedinjuryinHUVECsbyamelioratingmitochondrialfunctionandinhibitingWnt/β-cateninpathway
Authors:Hai-JianSun,Dong-YanXu,Yi-XinSun,TongXue,Chen-XingZhang,Zhi-XuanZhang,WeiLin,Ke-XueLi
Journal:BiochemicalandBiophysicalResearchCommunications(2017)
Salusin-βmediateshighglucose-inducedendothelialinjuryviadisruptionofAMPKsignalingpathway
Authors:XuexueZhu,YuetaoZhou,WeiweiCai,HaijianSun,LiyingQiu
Journal:BiochemicalandBiophysicalResearchCommunications(2017)
VaccarinprotectshumanmicrovascularendothelialcellsfromapoptosisviaattenuationofHDAC1andoxidativestress
Authors:XuexueZhu,YueyueLei,FanggenTan,LeileiGong,HaifengGong,WeiYang,TingChen,ZhixuanZhang,WeiweiCai,BaoHou
Journal:EuropeanJournalofPharmacology(2017)
AxlisrequiredforTGF-β2-induceddormancyofprostatecancercellsinthebonemarrow
Authors:KenjiYumoto,MatthewREber,JingchengWang,FrankCCackowski,AnnMDecker,EunsohlLee,AnaRitaNobre,JulioAAguirre-Ghiso,YounghunJung,RussellSTaichman
Journal:ScientificReports(2016)
GrowthArrest-Specific6(GAS6)PromotesProstateCancerSurvivalbyG1Arrest/SPhaseDelayandInhibitionofApoptoticPathwayDuringChemotherapyinBoneMarrow
Authors:EunsohlLee,AnnMDecker,FrankCCackowski,LuliaAKana,KenjiYumoto,YounghunJung,JingchengWang,LauraButtitta,ToddMMorgan,RussellSTaichman
Journal:Journalofcellularbiochemistry(2016)
RFX1--dependentactivationofSHP-1inducesautophagybyanovelobatoclaxderivativeinhepatocellularcarcinomacells
Authors:Jung-ChenSu,Ping-HuiTseng,Cheng-YiHsu,Wei-TienTai,Jui-WenHuang,Ching-HuaiKo,Mai-WeiLin,Chun-YuLiu,Kuen-FengChen,Chung-WaiShiau
Journal:Oncotarget(2014):4909
