| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 575/597 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy LabelingCells |
| Related | FluorescenceImaging BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.PrepareLysosome-stainingsolution:
1.1 WarmLysoBrite™Red(ComponentA)toroomtemperature.
1.2 Preparedyeworkingsolutionbydiluting20µLofLysoBrite™Red(ComponentA)to10mLofLiveCellStainingBuffer(ComponentB).
Note1:20µLofLysoBrite™Red(ComponentA)isenoughforone96-wellplate.AliquotandstoreunusedLysoBrite™Red(ComponentA)at<-20oC.Protectitfromlightandavoidrepeatedfreeze-thawcycles.
Note2:Theoptimalconcentrationofthefluorescentlysosomeindicatorvariesdependingonthespecificapplication.Thestainingconditionsmaybemodifiedaccordingtotheparticularcelltypeandthepermeabilityofthecellsortissuestotheprobe.
2.Prepareandstaincells:
2.1 Foradherentcells:Growcellseitherina96-wellblackwall/clearbottomplate(100µL/well/96-wellplate)oroncover-slipsinsideapetridishfilledwiththeappropriateculturemedium.Whencellsreachthedesiredconfluence,addequalvolumeofthedye-workingsolution(fromStep1.2).Incubatethecellsina37°C,5%CO2incubatorfor30minutes.Washthecellstwicewithpre-warmed(37°C)Hanksand20mMHepesbuffer(HBSS)orbufferofyourchoice,fillthecellwellswithHBSSorgrowthmedium.ObservethecellsusingafluorescencemicroscopefittedwithaTRITCfilterset.
Note:Itisrecommendedtoincreaseeitherthelabelingconcentrationortheincubationtimetoallowthedyetoaccumulateifthecellsdonotappeartobesufficientlystained.
2.2 Forsuspensioncells:Addequalvolumeofdye-workingsolution(fromStep1.2)intothecells.Incubatethecellsina37°C,5%CO2incubatorfor30minutes.Washthecellstwicewithpre-warmed(37°C)Hanksand20mMHepesbuffer(HBSS)orbufferofyourchoice,fillthecellwellswithHBSSorgrowthmedium.ObservethecellsusingafluorescencemicroscopeequippedwithaTRITCfilterset.
Note1:Itisrecommendedtoincreaseeitherthelabelingconcentrationortheincubationtimetoallowthedyetoaccumulateifthecellsdonotappeartobesufficientlystained.
Note2:Suspensioncellsmaybeattachedtocover-slipsthathavebeentreatedwithBDCell-Tak®(BDBiosciences)andstainedasadherentcells(seeStep2.1).
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