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Ex/Em(nm)	485/540
MW	N/A
CAS#	186689-07-6
Solvent	DMSO
Storage	F/D/L
Category	Transporters GlucoseTransporters
Related	CellFunctionalAnalysis

Glucosemetabolism,aprocesswhichconvertsglucoseintoenergy,isaprimarysourceofenergysupplyinmostorganisms.2-NBDG[2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose],afluorescentlytaggedglucosetracer,hasbeenproventoeffectivelymonitorglucosetransportationincells,as2-NBDGtransportsintocellsbythesameglucosetransporters(GLUTs)asglucose.Once2-NBDGisuptakenincells,itundergoesphosphorylationatC-6positiontogive2-NBDG-6-phosphate,whichiswellretainedwithinthecells.Comparedtootherglucosetracers,suchas2-DGorFDG,2-NBDGallowsinsitumeasurementsof2-NBDGwithhightemporalandspatialresolutionatsinglecelllevel.AATBioquest"sCellMeter™2-NBDGGlucoseUptakeAssayKitprovidesasensitiveandnon-rADIoactiveassayformeasuringglucoseuptakeinculturedcells.Inthiskit,AssayBufferIisusedtoenhancetheuptakeandretentionof2-NBDGincells,whileAssayBufferIIcanimprovethesignal-to-backgroundratioof2-NBDGinthecells.ThefluorescencesignalcanbemonitoredbyfluorescencemicroscopeorflowcytometerwithaFITCfilterset.CellMeter™2-NBDGGlucoseUptakeAssayKitisthemostrobusttoolformonitoringglucosetransporters.

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1. Preparecells:
   1. Foradherentcells:Platecellsovernightingrowthmediumat10,000to40,000cells/well/100μLfora96-wellplateor2,500to10,000cells/well/20μLfora384-wellplatewithblackwallandclearbottom.
   2. Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandsUSPendthecellpelletsinculturemediumat50,000-100,000cells/well/100µLfora96-wellpoly-Dlysineplateor10,000-25,000cells/well/20µLfora384-wellpoly-Dlysineplate.
   3. Addtestcompoundsintothecellsandincubateforadesiredperiodoftime(suchas24,48or96hours)ina37°C,5%CO2incubator.Forblankwells(mediumwithoutthecells),addthesameamountofcompoundbuffer.
      Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityandincubationtime.WeincubatedCHO-K1cellswith20mMGlucoseforglucosecompetitionassay,and100µMPhloretinforGLUTsinhibitionassay.SeeFigure2fordetails.


2. Staincells:
   1. Preparestainingsolutionbyadding5μLof2-NBDG(10mg/mL)(ComponentA)to1.5mLofAssayBufferI(ComponentB).Thedye-loadingsolutionisstablefor1houratroomtemperature,protectedfromlight.
      Note:Astheoptimalstainingconditionsmayvarydependingondifferentcelltypes,it’srecommendedtodeterminetheoptimalconcentrationofComponentAforeachspecificexperiment.
   2. Attheendofthetreatment,centrifugetheplatefor5minutesat800rpmwithbrakeoffpriortoyourexperiment.Aspiratethesupernatantwithoutdisturbingcells.
   3. Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofstainingsolution(fromStep2.1).
      Note:Opticalincubationtimewillneedtobedeterminedforeachcelllineandforeachspecificexperiment.WeincubatedCHO-K1cellswith100µM2-NBDG(~34µg/mL)for20minutestoshowsufficientglucoseuptake.Seefigure2fordetails.
   4. Attheendoftheincubation,centrifugetheplatefor5minutesat800rpm.Removestainingsolutionwithoutdisturbingcells.
   5. Forfluorescencemicroscope:WashcellswithAssayBufferI(ComponentB)once.Keepcellsin100µL/well(96-wellplate)or25µL/well(384-wellplate)ofAssayBufferII(ComponentC).MonitorthefluorescencesignalusingafluorescencemicroscopewithFITCfilter.
   6. Forflowcytometer:DetachcellsifrequiredusingEDTAandresuspendcellsin100µL/sampleofAssayBufferI(ComponentB).MonitorthefluorescencesignalusingaflowcytometeratFITCchannel.

References&Citations

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NT1014,anovelbiguanide,inhibitsovariancancergrowthinvitroandinvivo
Authors:LuZhang,JianjunHan,AmandaLJackson,LeslieNClark,JoshuaKilgore,HuiGuo,NickLivingston,KennethBatchelor,YajieYin,TimothyPGilliam
Journal:JournalofHematology&Oncology(2016):91

GlutaminepromotesovariancancercellproliferationthroughthemTOR/S6pathway
Authors:LingqinYuan,XiuguiSheng,AdamKWillson,DarioRRoque,JessicaEStine,HuiGuo,HannahMJones,ChunxiaoZhou,VictoriaLBae-Jump
Journal:Endocrine-relatedcancer(2015):577--591

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