| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 443/505 |
| MW | ~110K |
| CAS# | N/A |
| Solvent | Water |
| Storage | F/D/L |
| Category | CellBIOLOGy pHandIonIndicators |
| Related | CellMetabolism BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparecellsasdesired.Forexample,plateadherentcellsovernightingrowthmediumat40,000to80,000cells/well/100µLfor
96-wellor10,000to20,000cells/well/25µLfor384-wellplates.
Note: Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.PrepareRatioWorks™Protonex™GreenDextranloadingsolution:
2.1 Preparea1mg/mLstocksolutionofProtonex™GreenDextranin1mLofsterilewaterorHanksand20mMHepesbuffer(HHBS).Thestocksolutionshouldbeusedpromptly.Anyunusedsolutionneedtobealiquotedandrefrozenat<-20oC.
Note:Avoidrepeatedfreeze-thawcycles,andprotectfromlight.
2.2 Preparea20-100ug/mLProtonex™GreenDextranloadingsolutioninHHBS.
3. RunEndocytosisAssay
3.1 Removethemedium,andadd100µL/well(96-wellplate)or25µL/well(384-wellplate)Protonex™GreenDextranloadingsolutionintothecellplate(fromStep2.2).
Note1: ItisimportanttoreplacethegrowthmediumwithHHBSbuffer(100µL/wellfor96-wellplateor25µL/wellfor384-wellplatebeforedye-loading)ifyourcompoundsinterferewiththeserum.
Note2:RapidtraffickingofProtonex™Greendextranfromearlyendosomestolateendosomesandsubsequentfusionwithlysosomescanoccur.ToaidthevisualizationofProtonex™Greendextranwithintheendosomes,werecommendincreasingthelabelingconcentrationanddecreasingtheloadingtime,andimagingimmediately.
3.2 Incubatethedye-loadingplateatcellincubatorfor5to20minutes.
3.3 Washandreplacethedye-loadingsolutionwithHHBSorgrowthmedium.
3.4 RuntheendocytosisassaybymonitoringthefluorescenceatEx/Em=443/505nm.
| References&Citations |  CitationExplorer |
PHD2isaregulatorforglycolyticreprogramminginmacrophages
Authors:AnnemarieGuentsch,AngelikaBeneke,LijaSwain,KatjaFarhat,ShunmugamNagarajan,BenWielockx,KaaminiRaithatha,JanDudek,PeterRehling,AnkeZieseniss
Journal:MolecularandCellularBiology(2016):MCB--00236
