| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 640/659 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy LabelingCells |
| Related | FluorescenceImaging |
| Spectrum | AdvancedSpectrumViewer |
1.Preparemitochondrialstainingsolution:
1.1 Warmallthecomponentstoroomtemperature.
1.2 Preparedyeworkingsolutionbydiluting20µLofMitolite™DeepRedFX660(ComponentA)into10mLofLiveCellStainingBuffer(ComponentB).
Note1:20µLof500XMitolite™DeepRedFX660(ComponentA)isenoughforone96-wellplate.Aliquotandstoreunused500XMitolite™DeepRedFX660at≤-20oC.Protectfromlightandavoidrepeatedfreeze-thawcycles.
Note2:Theoptimalconcentrationofthefluorescentmitochondrialindicatorvariesdependingonthespecificapplication.Thestainingconditionsmaybemodifiedaccordingtotheparticularcelltypeandthepermeabilityofthecellsortissuestotheprobe.
2.Prepareandstaincells:
2.1 Foradherentcells:Growcellseitherina96-wellblackwall/clearbottomplateoroncover-slipsinsideapetridishfilledwiththeappropriateculturemedium.Whencellsreachthedesiredconfluence,addequalvolume(e.g.100µLfora96-wellplateand25µLfora384-wellplate)ofthedye-workingsolution(fromStep1.2).Incubatethecellsina37°C,5%CO2incubatorfor30minutesto2hours.ReplacethedyeloadingsolutionwithHanksand20mMHepesbuffer(HHbuffer)orbufferofyourchoice(e.g.thebufferwithgrowthmediumat1:1concentration).ObservethecellsusingafluorescencemicroscopefittedwithaCy5filterset.
Note:Itisrecommendedtoincreaseeitherthelabelingconcentrationortheincubationtimetoallowthedyetoaccumulateifthecellsdonotappeartobesufficientlystained.
2.2 Forsuspensioncells:Centrifugethecellsat1000rpmfor5minutestoobtainacellpelletandaspiratethesupernatant.Resuspendthecellpelletsgentlyinpre-warmed(37°C)growthmedium,andaddequalvolumeofthedye-workingsolution(fromStep1.2).Incubatethecellsina37°C,5%CO2incubatorfor30minutesto2hours.Replacethedye-loadingsolutionwithHanksand20mMHepesbuffer(HHbuffer)orbufferofyourchoice(e.g.thebufferwithgrowthmediumat1:1concentration).ObservethecellsusingafluorescencemicroscopefittedwithaTexasRedfilterset.
Note1:Itisrecommendedtoincreaseeitherthelabelingconcentrationortheincubationtimetoallowthedyetoaccumulateifthecellsdonotappeartobesufficientlystained.
Note2:Suspensioncellsmaybeattachedtocover-slipsthathavebeentreatedwithBDCell-Tak®(BDBiosciences)andstainedasadherentcells(seeStep2.1).
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