| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 549/565 |
| MW | 765.55 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | CellBIOLOGy LabelingCells |
| Related | Lipoproteins VitalStains BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.PrepareDiO,DiIDiD,DiSorDiRmembranestainsolutions:
1.1 PrepareDMSOorEtOHstocksolutions:ThestocksolutionsshouldbepreparedinDMSOorEtOHat1-5mM.
Note:Theunusedportionofthestocksolutionshouldbestoredat-20oC.Avoidrepeatedfreeze/thawcycles.
1.2 Prepareworkingsolutions:Dilutethestocksolutions(fromStep1.1)intoasuitablebuffersuchasserum-freeculturemedium,HBSSorPBStomake1to5µMworkingsolutions.
Note:Thefinalconcentrationoftheworkingsolutionshouldbeempiricallydeterminedfordifferentcelltypesand/orexperimentalconditions.Itisrecommendedtotestattheconcentrationsthatareatleastoveratenfoldrange.
2.StainthecellsinsUSPension:
2.1 Suspendcellsatadensityof1×106/mLindyeworkingsolution(fromStep1.2).
2.2 Incubateat37°Cfor2–20minutes.Theoptimalincubationtimevariesdependingonthecelltype.Startbyincubatingfor20minutesandsubsequentlyoptimizeasnecessarytoobtainuniformlabeling.
2.3 Centrifugethelabeledsuspensiontubesat1000to1500rpmfor5minutes.
2.4 Removethesupernatantandgentlyresuspendthecellsinpre-warmed(37°C)growthmedium.
2.5 WashtwomoretimesasSteps2.3and2.4.
3.Stainadherentcells:
3.1 Growadherentcellsonsterileglasscoverslips.
3.2 Removecoverslipsfromgrowthmediumandgentlydrainoffexcessmedium.Placecoverslipinahumiditychamber.
3.3 Pipet100μLofthedyeworkingsolution(fromStep1.2)ontothecornerofacoverslipandgentlyagitateuntilallcellsarecovered.
3.4 Incubatethecoverslipat37°Cfor2–20minutes.Theoptimalincubationtimevariesdependingonthecelltype.Startbyincubatingfor20minutesandsubsequentlyoptimizeasnecessarytoobtainuniformlabeling.
3.5 Drainoffthedyeworkingsolutionandwashthecoverslipstwotothreetimeswithgrowthmedium.Foreachwashcycle,coverthecellswithpre-warmedgrowthmedium,incubatefor5-10minutesandthendrainoffthemedium.
4.MicroscopyDetection:
4.1 TheselectionofDiD,DiO,DiI,DiSandDiR’sfiltersetsissummarizedinTable1.
4.2 Forsimultaneousdetectionofmultipledyes,multibandfiltersetsareavailableasfollows:
a) DiIandDiO=OmegaXF52,Chroma51004
b) DiIandDiD=OmegaXF92,Chroma51007
c) DiI,DiOandDiD=OmegaXF93,Chroma61005
5.FlowCytometryDetection:
CellslabeledwithDiO,DiI,DiD,DiSandDiRcanbeanalyzedusingtheconventionalFL1,FL2,FL3andFL4flowcytometerdetectionchannels,respectively.
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