| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 492/516 |
| MW | 994.99 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | EnzymeDetection PeptidasesandProteases |
| Related | CellApoptosis ApoptosisandCytotoxicity BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1. GeneralSolutionCaspaseAssaysUsingAMC,AFC,pNA,R110andProRedSubstrates
1.1. Preparea10mMstocksolutioninDMSO.
1.2. Preparea2Xcaspase substrate(50µM)assaysolutionasthefollowing:
50µLsubstratestocksolution
100 µLDTT(1M)
400 µLEDTA(100mM)
10mLTrisBuffer(20mM),pH=7.4
1.3. Mixequalvolumeofthecaspasestandardsorsampleswith2Xcaspasesubstrateassaysolution(fromStep1.2),andincubatethesolutionsatroomtemperatureforatleast1hour.
1.4. Monitorthefluorescenceusingafluorescencemicroplatereader,orabsorbanceusinganabsorbancemicroplatereader.
2. CellCaspaseAssaysUsingCell-PermeableFMKCaspaseProbes
2.1. Preparea2-5mMstocksolutioninDMSO.
2.2. Treatcellsasdesired.
2.3. Preparea2Xpermeablecaspasesubstrate(20µM)assaysolutionbydilutingtheDMSOstocksolution(fromStep2.1)inHankswith20mMHepesbuffer(HHBS).
2.4. Mixequalvolumeofthetreatedcellswith2Xcaspasesubstrateassaysolution(fromStep2.3),andincubatethecellsina37°C,5%CO2incubatorforatleast1hour.
2.5. WashthecellswithHHBSforatleastonce.
2.6. Monitorthefluorescenceintensitybyaflowcytometer,afluorescencemicroscopeorafluorescencemicroplatereader.
3. CellCaspaseAssaysUsingCell-PermeableFMKCaspaseProbes(For#13470-13476only)
3.1. Preparea250Xstocksolutionbyadding50µLDMSOintothevial.
3.2. Treatcellsasdesired.
3.3. Add250XDMSOstocksolution(fromStep3.1)intothecellsolutionata1:250ratio(suchas2uLto500uLcells),andincubatethecellsina37°C,5%CO2incubatorfor1hour.
3.4. WashthecellswithHHBSforatleastonce.
3.5. Monitorthefluorescenceintensitybyflowcytometer,fluorescencemicroscopyorfluorescentmicroplatereader.
| References&Citations |  PrinterFriendlyVersion |
1. LiJN,SongDQ,JiangJD.(2004)[Antitumormechanismof3-bromopropionylaminobenzoylureaonleukemiaandlymphoma].YaoXueXueBao,39,491.
2. ThraneC,KaufmannU,StummannBM,OlssonS.(2004)Activationofcaspase-likeactivityandpoly(ADP-ribose)polymerasedegradationduringsporulationinAspergillusnidulans.FungalGenetBiol,41,361.
3. PandeyS,SmithB,WalkerPR,SikorskaM.(2000)Caspase-dependentandindependentcelldeathinrathepatoma5123tccells.Apoptosis,5,265.
4. HashiramotoA,SanoH,MaekawaT,KawahitoY,KimuraS,KusakaY,WilderRL,KatoH,KondoM,NakajimaH.(1999)C-mycantisenseoligodeoxynucleotidescaninduceapoptosisanddown-regulateFasexpressioninrheumatoidsynoviocytes.ArthritisRheum,42,954.
