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Fluorescent_dyes >
AAT Bioquest/Cell Navigator™ Lysosome Staining Kit *Deep Red Fluorescence*/22659/500 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 596/619 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy LabelingCells |
| Related | FluorescenceImaging BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparelysosomal-stainingsolution:
1.1 WarmLysolite™DeepRed(ComponentA)toroomtemperature.
1.2 Preparedyeworkingsolutionbydiluting10µLofLysolite™DeepRed(ComponentA)to5mLofLiveCellStainingBufferA(ComponentB).
Note1:10µLofLysolite™DeepRed(ComponentA)isenoughforone96-wellplate.AliquotandstoreunusedLysolite™DeepRed(ComponentA)at<‑20oC.Protectfromlightandavoidrepeatedfreeze-thawcycles.
Note2:Theoptimalconcentrationofthefluorescentlysosomeindicatorvariesdependingonthespecificapplication.Thestainingconditionsmaybemodifiedaccordingtotheparticularcelltypeandthepermeabilityofthecellsortissuestotheprobe.
2.Prepareandstaincells:
2.1 Foradherentcells:
2.1.1 Growcellseitherina96-wellblackwall/clearbottomplate(100µL/well/96-wellplate)oroncover-slipsinsideapetridishfilledwiththeappropriateculturemedium.
2.1.2 Whencellsreachthedesiredconfluence,add1/2volume(suchas50µL/well/96-wellplate)ofthedye-
workingsolution(fromStep1.2).Incubatethecellsina37°C,5%CO2incubatorfor30minutesto1hour.
2.1.3 Add50µL/well(96-wellplate)ofAssayBufferB(ComponentC)intothedye-loadingplate,incubateat
roomtemperaturefor15-30minutes.
2.1.4 ObservethecellsusingafluorescencemicroscopefittedwithaTexasredfilterset(Ex/Em=590/620nm).
Note1:TheLysoBrite™DeepRedhasminimumornocelltoxicity;itcanbeusedforcelltracking. Forcelltrackingpurpose,skiptheStep2.1.3,simplyreplacethedye-workingsolution(fromStep2.1.2)togrowthmedium,andthenobserveunderafluorescencemicroscope(Step2.1.4).
Note2:Itisrecommendedtoincreaseeitherthelabelingconcentrationortheincubationtimetoallowthedyetoaccumulateifthecellsdonotappeartobesufficientlystained.
2.2 Forsuspensioncells:
2.2.1 Centrifugethecellsat1000rpmfor5minutestoobtainacellpelletandaspiratethesupernatant.
2.2.2 Resuspendthecellpelletgentlyinpre-warmedgrowthmedium(suchas100µL/tube),andthenadd1/2volume(50µL/tube)ofthedye-workingsolution(fromStep1.2).Incubatethecellsina37°C,5%CO2incubatorfor30minutesto1hour.
2.2.3 Add1/3volume(50µL/tube)ofAssayBufferB(ComponentC)intothedye-loadingcells(fromStep2.2.2),incubateatroomtemperaturefor15-30minutes.
2.2.4 ObservethecellsusingafluorescencemicroscopefittedwithaTexasredfilterset(Ex/Em=590/620nm).
Note1:TheLysoBrite™DeepRedhasminimumornocelltoxicity;itcanbeusedforcelltracking. Forcelltrackingpurpose,skiptheStep2.1.3,simplyreplacethedye-workingsolution(fromStep2.1.2togrowthmedium,andthenobserveunderafluorescencemicroscope(Step2.1.4).
Note2:Itisrecommendedtoincreaseeitherthelabelingconcentrationortheincubationtimetoallowthedyetoaccumulateifthecellsdonotappeartobesufficientlystained.
Note3:Suspensioncellsmaybeattachedtocover-slipsthathavebeentreatedwithBDCell-Tak®(BDBiosciences)andstainedasadherentcells(seeStep2.1).
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