Overview

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Ex/Em(nm)	575/None
MW	N/A
CAS#	N/A
Solvent	N/A
Storage	F/D/L
Category	Transporters GlucoseTransporters
Related	BiochemicalAssays

GlucosetransportsystemsareresponsIBLefortransportingglucoseacrosscellmembranes.Measuringuptakeof2-deoxyglucose(2-DG),aglucoseanalog,intissuesandcellsiswidelyacceptedasareliablemethodtoestimatetheamountofglucoseuptakeandtoinvestigatetheregulationofglucosemetabolismandmechanismofinsulinresistance.The2-DGuptakeiscommonlydeterminedbyusingnon-metabolized2-DGlabeledwithtritiumorC14.However,routineuseofarADIolabelledprobeiscostlyandrequiresatediousspecialhandlingprocedure.AATBioquest"sScreenQuest™ColorimetricGlucoseUptakeAssayKitprovidesasensitiveandnon-radioactiveassayintissuesorculturedcells.Inthisassay2-DGistakenupbyglucosetransporters,andmetabolizedto2-DG-6-phosphate(2-DG6P).Thenon-metabolizable2-DG6Paccumulatesinthecells,andisproportionaltoglucoseuptakebycells.Theaccumulated2-DG6PisenzymaticallyoxidizedandgeneratesNADPH,whichisspecificallymonitoredbyachromogenicNADPHsensor.ThesignalcanbereadbyaabsorptionmicroplatereaderbyreadingtheODratioatwavelength570nmto610nm.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareCells:

Thefollowingprotocolsareguidelinestoculture3T3-L1adipocytesfor2-DGuptake.

1.1   Preparedifferentiated3T3-L1adipocytes: 3T3-L1fibroblastsweregrown2dayspost-confluenceina75cmflaskwithDMEMsupplementedwith10%FBS.Forinductionofdifferentiationof3T3-L1preadipocytesintomatureadipocytes,thecellswereincubated2dayswithDMEMsupplementedwith10%FBS,0.83µMinsulin,0.25µMdexamethasone,and0.25mMisobutylmethylxanthine. Thecellsweremaintainedfor2dayswithDMEMsupplementedwith10%FBSand0.83µMinsulinalone.ThemediumwaschangedtoDMEMsupplementedwith10%FBSforanother3-5days. Differentiatedcells(atleast95%ofwhichshowedanadipocytephenotypebyaccumulationoflipiddroplets)wereusedonday8to12afterinductionofdifferentiation.

1.2   Plate3T3-L1adipocytesingrowthmediumat50,000-80,000cells/well/100µL/96-wellor12,500-20,000cells/well/25µL/384-wellblackwall/clearbottomcellculturePoly-Dlysineplatefor4-6hoursbeforeexperiment.

1.3   Removethecellplatefromtheincubator,aspiratethemediumfromthewells,anddeprivethecellswith100µl/well/96well-plateor25µl/well/384well-plateserumfreemedium.Incubatethecellsat37ºC,5%CO₂incubatorfor6hourstoovernight.

2.      TreatCells:

2.1   Prepare1×KRPHbuffer:Add20mLof5×KRPHBuffer(ComponentH)to80mLofdeionizedwater.

Note:50mLvolumeof1×KRPHBufferisenoughforapproximatelyone96-wellplate.Preparetheneededvolumeproportionally.Storetheunused1×KRPHat4ºCor-20ºC.

2.2   Removethecellplatefromtheincubator,aspiratethemediumfromthewells,andgentlywashthecellstwicewith100µL/well1×KRPHbuffer.

2.3   Add90µL/wellGlucoseUptakeBuffer(ComponentB)andincubatethecellsat37ºC,5%CO₂incubatorfor1hour.

2.4   Stimulatewithorwithoutinsulinorcompoundoftestfor20min.Add10µL/wellofthe10×insulinsolutiontoafinalconcentrationof1µMor10×compoundsolutionoftest.Andalsoadd10µLinsulinvehiclebufferorcompoundvehiclebuffertotheuntreatedwellsascontrol,andincubateat37ºC,5%CO₂incubatorfor20min.

2.5   Forglucoseuptakeinhibitionstudy,add10×Phloretintoafinalconcentrationof200uMorinhibitorsoftest,andincubateat37ºC,5%CO₂for2-5min.

Note:10µLinhibitorvehiclebufferissuggestedtobeaddedtoboththeinsulintreatedanduntreatedwellsascontrol.

2.6   Add10µL/well2-DGsolution(ComponentA)toeachwell,andincubateat37ºC,5%CO₂incubatorfor20-40min.Fornegativecontrols,leavesomewellsuntreatedwithinsulin,inhibitorand2-DG.

 

Table1.Anexamplelayoutofexperimentina96-well-plate

1	3	5
1	3	5
1	3	5
1	3	5
2	4	6
2	4	6
2	4	6
2	4	6

Table2.Treatmentconditionexamples

1	-insulin+2-DG	4	+insulin-Phloretin+2DG
2	+Insulin+2DG	5	+Insulin+Glucose(5mM)+2DG
3	+Insulin+Phloretin+2DG	6	-Insulin-2DG

-Insulin:onlyaddinsulinvehiclebuffer;-Phloretin:onlyaddPhloretinvehiclebuffer;-2DG:onlyaddH₂O

 

3.      LyseCells:

3.1   Aftertreatment,removesolutionineachwellandgentlywashcells3times,100µL/wellwithKRPHtoremovetheextra2-DGfromthesolution.

3.2   Add25µL/wellAcidicLysisBuffer(ComponentC)toeachwellandincubateat37ºCfor20mintolysethecells.Andthe2DGuptakeassaymixturecouldbepreparedinthemeantime(seeStep4).

3.3   Add25µL/wellNeutralizationBuffer(ComponentD)toeachwell,mixthoroughly,leaveatroomtemperaturefor5-10minutestoneutralizethecelllysate.

4.      Runglucoseuptakeassay:

4.1   Add100µLofH₂OintothevialofNADP(ComponentG)toreconstituteNADP.

4.2   Add5mLofAssayBuffer(ComponentF)intothebottleofEnzymeProbe(ComponentE).

4.3   Add100µLreconstitutedNADPsolution(fromStep4.1)intothebottleofComponentE(fromStep4.2)tomakethe2DGuptakeassaymixture.

4.4   Add50μLof2DGuptakeassaymixture(fromStep4.3)toeachwellof2DG6Pstandardorcelllysate.

4.5   Incubatethereactionatroomtemperaturefor30minutesto2hours,protectedfromlight.

4.6   Monitortheabsorbanceratioincreaseat570/610nmwithanabsorbanceplatereader.

References&Citations

CitationExplorer

Corticotropinreleasinghormonecanselectivelystimulateglucoseuptakeincorticotropinomaviaglucosetransporter1
Authors:JieLu,BlakeKMontgomery,GrégoirePChatain,AlejandroBugarini,QiZhang,XiangWang,NancyAEdwards,AbhikRay-Chaudhury,MarshaJMerrill,RussellRLonser
Journal:MolecularandCellularEndocrinology(2017)

ANon-RadioactiveEnzymaticPhotometricAssayforGlucoseUptakeinInsulin-Responsive3T3-L1Adipocytes
Authors:QinZhao,JinfangLiao,ZhenjunDiwu
Journal:BiophysicalJournal(2014):369a

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