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