| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 575/600 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy LabelingCells |
| Related | FluorescenceImaging BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparemitochondria-stainingsolution:
1.1 WarmMitoLite™dyestoroomtemperature.
1.2 Preparedyeworkingsolutionbydiluting20µLof500XMitolite™dyesto10mLofHanksand20mmHepesbufferorbuffer(HBSS)ofyourchoice.
Note1:20µLofMitolite™dyeisenoughforone96-wellplate.AliquotandstoreunusedMitoLite™dyestocksolutionsat<-15oC.Protectitfromlightandavoidrepeatedfreeze-thawcycles.
Note2:Theoptimalconcentrationofthefluorescentmitochondriaindicatorsvariesdependingonthespecificapplication.ThestainingconditionsmaybemodifiedaccordingtotheparticularcelltypeandthepermeABIlityofthecellsortissuestotheprobe.
2.Prepareandstaincells:
2.1 Foradherentcells:a).Growcellseitherina96-wellblackwall/clearbottomplate(100µL/well/96-wellplate)oroncover-slipsinsideapetridishfilledwiththeappropriateculturemedium.Whencellsreachthedesiredconfluence,addequalvolumeofthedye-workingsolution(fromStep1.2).b).Incubatethecellsina37°C,5%CO2incubatorfor30minutesto2hours.c).Replacethedye-loadingsolutionorwash(especiallyforcat#22679)thecellswithprewarmed(37°C)Hanksand20mMHepesbuffer(HBSS)orbufferofyourchoice(e.g.thebufferwithgrowthmediumat1:1concentration).FillthecellwellswithHBSSorgrowthmedium.d).Observethecellsusingafluorescencemicroscopefittedwithadesiredfilterset.
Note:Itisrecommendedtoincreaseeitherthelabelingconcentrationortheincubationtimetoallowthedyetoaccumulateifthecellsdonotappeartobesufficientlystained.
2.2 Forsuspensioncells:Centrifugethecellsat1000rpmfor5minutestoobtainacellpelletandaspiratethesupernatant.Resuspendthecellpelletsgentlyinpre-warmed(37°C)growthmedium,andaddequalvolumeofthedye-workingsolution(fromStep1.2).Incubatethecellsina37°C,5%CO2incubatorfor30minutesto2hours.Replacethedye-loadingsolutionorwash(especiallyforcat#22679)thecellswithHanksand20mMHepesbuffer(HHbuffer)orbufferofyourchoice(e.g.thebufferwithgrowthmediumat1:1concentration).FillthecellwellswithHBSSorgrowthmedium.Observethecellsusingafluorescencemicroscopefittedwithadesiredfilterset.
Note1:Itisrecommendedtoincreaseeitherthelabelingconcentrationortheincubationtimetoallowthedyetoaccumulateifthecellsdonotappeartobesufficientlystained.
Note2:Suspensioncellsmaybeattachedtocover-slipsthathavebeentreatedwithBDCell-Tak®(BDBiosciences)andstainedasadherentcells(seeStep2.1).
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