| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 555/575 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy LabelingCells |
| Related | FluorescenceImaging BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparecells:
1.1. Foradherentcells:Platecellsovernightingrowthmediumat10,000to40,000cells/well/90μLfora96-wellplateor2,500to10,000cells/well/20μLfora384-wellplate.
1.2. Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandsUSPendthecellpelletsinculturemediumat50,000-100,000cells/well/90µLfora96-wellpoly-Dlysineplateor10,000-25,000cells/well/20µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortoyourexperiment.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.Prepareworkingsolution:
2.1 Thawoneofeachkitcomponentatroomtemperaturebeforeuse.
2.2 MakeCellpaint™Orange500Xstocksolution:Add100µLofDMSO(ComponentC)intothevialofCellpaint™Orange(ComponentA)tomake500Xstocksolution.
2.3 MakeCellpaint™Orangeworkingsolution:Add20µLof500Xstocksolution(ComponentA)into10mLofAssayBuffer(ComponentB),andmixwell.Theworkingsolutionisstableforatleast8hoursatroomtemperature.
Note:20µLof500XCellpaint™Orange500Xstocksolution(ComponentA)isenoughforone96-wellplate.UnusedCellpaint™Orange500Xstocksolutioncanbealiquotedandstoredat≤-20ºCforonemonthifthetubesaresealedtightly.Protectfromlightandavoidrepeatedfreeze-thawcycles.
3.Staincells:
3.1 Add100µL/well(96-wellplate)or50µL/well(384-wellplate)ofCellpaint™Orangeworkingsolution(fromStep2.3)inthecellplate.Incubatethecellsat37ºCfor10-20minutes,protectedfromlight.
Note:Theoptimalconcentrationofthecellmembraneprobevariesdependingonthespecificapplication.Thestainingconditionsmaybemodifiedaccordingtotheparticularcelltypeandthepermeabilityofthecellsortissuestotheprobe.
3.2 Removeworkingsolutionineachwell.Washcellswithphysiologicalbuffer(suchasHHBS,PBSorbufferofyourchoice)forthreetimes.
3.3 Fixcellsafterstaining(Optional).Fixthecellswith4%formaldehydefor15-30minutes.Washcellswithphysiologicalbufferforthreetimes.
3.4 ObservethefluorescencesignalincellsusingfluorescencemicroscopewithaTRITCfilterset.
| References&Citations |  PrinterFriendlyVersion |
1. DanielsenEM.(2015)Probingendocytosisfromtheenterocytebrushborderusingfluorescentlipophilicdyes:lipidsortingattheapicalcellsurface.HistochemCellBiol,143,545.
2. FlynnFW,Kinney-LangE,HoekstraC,PrattDL,ThakarA.(2015)Activationoftheneurokinin3receptorpromotesfilopodiagrowthandsproutinginratembryonichypothalamiccells.DevNeurobiol,75,12.
3. MonkemollerV,SchuttpelzM,McCourtP,SorensenK,SmedsrodB,HuserT.(2014)Imagingfenestrationsinliversinusoidalendothelialcellsbyopticallocalizationmicroscopy.PhysChemChemPhys,16,12576.
4. ChenY,AllarsM,PanX,MaitiK,AngeliG,SmithR,NicholsonRC.(2013)Effectsofcorticotrophinreleasinghormone(CRH)oncellviabilityanddifferentiationinthehumanBeWochoriocarcinomacellline:apotentialsyncytialisationinducerdistinctfromcyclicadenosinemonophosphate(cAMP).ReprodBiolEndocrinol,11,30.
5. Shibai-OgataA,KakinumaC,HiokiT,KasaharaT.(2011)Evaluationofhigh-throughputscreeningforinvitromicronucleustestusingfluorescence-basedcellimaging.Mutagenesis,26,709.
