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AAT Bioquest/Screen Quest™ Fluorimetric Fatty Acid Uptake Assay Kit/36385/100 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 490/515 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | Transporters FattyAcidTransporters |
| Related | BiochemicalAssays |
1.PrepareCells:
Preparecellsasdesired.Thefollowingprotocolsareguidelinestoprepare3T3-L1ADIpocytes.
1.1 Preparedifferentiated3T3-L1adipocytes(seeRef1):3T3-L1fibroblastsweregrown2daysina75cmflaskpost-confluenceinDMEM/FBS,andthenfor2daysinDMEM/FBSsupplementedwith0.83µMinsulin,0.25µMdexamethasone,and0.25mMisobutylmethylxanthine.ThemediumischangedtomaintaintheinsulinconcentrationwithdexamethasoneandIBMXabsentforanother2days.ThemediumwasthenchangedtoDMEM/FBSaloneforanother3-5days. 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.Centrifugetheplateat800rpmfor2minuteswithbrakeoff.
Note1:Itisrecommendedtoplate3wellswithgrowthmediumonly(withoutcells)asblankwellsfordatanormalization;Note2:Wefindthatadipocytesplatedatthesameday(4-6hours,andthenserumdeprivedfor1hour)givebetterresultsthanthatplatedforovernight.
1.3 Removethecellplatefromtheincubator,aspiratethemediumfromthewells,anddeprivethecellswith90µl/well/96well-plateor20µl/well/384well-plateserumfreemedium.Incubatethecellsat37ºC,5%CO2incubatorfor1hr.
1.4 Treatthecellsbyadding10µl/well/96-wellplate(5µl/well/384-wellplate)ofthetestcompoundsor1XHanksand20mMHepesbuffer(1XHBSS,pH7.4)orbufferofyourchoiceasthecompounddiluent. Forblankwells,addthecompounddiluents.Incubatethecellsat37ºC,5%CO2incubatorforadesiredperiodoftime(30minutesfor3T3-L1cellstreatedwithInsulin).
2.Preparefattyaciddye-loadingsolution:
2.1 Thawallthekitcomponentsatroomtemperaturebeforeuse.
2.2 MakeTF2-C12FattyAcidstocksolution:Add20µLDMSO(ComponentC)intothevialoftheTF2-C12FattyAcid(ComponentA),andmixthemwell.
Note:20µLofthefluorescentfattyacidsubstratestocksolutionisenoughforoneplate.Theunusedfluorescentfattyacidsubstratestocksolutioncanbealiquotedandstoredat<-20oCforuptotwomonthsifthetubesaresealedtightlyandkeptfromlight.Avoidrepeatedfreeze-thawcycles.
2.3 Makefattyaciddye-loadingsolution:Add20µLoftheTF2-C12FattyAcidstocksolution(fromStep2.2)to10mLofAssayBuffer(ComponentB),andmixthemwell.
Note1:10mLoffattyaciddye-loadingsolutionisenoughforoneplate,preparefreshforeachexperiment.
3.RunFattyAcidUptakeAssay:
3.1 Removecompound-treatedcellplatesfromtheincubator(fromStep1.4),add100µl/well/96-wellplateor25µl/well/384-wellplate(includingblankwells)ofthefattyaciddye-loadingsolution(fromStep2.3).
3.2 MeasurethefluorescencesignalwithafluorescencemicroplatereaderatEx/Em=485/515nm(cutoffat495nm)usingabottomreadmode.
Forkineticreading: Readthefluorescenceintensityimmediatelyat20secondsintervalfor30-60minutes.
Forendpointreading:Readthefluorescenceintensityattheendofthe30-60minutesincubation.
| References&Citations |  PrinterFriendlyVersion |
1. ShapiroGD,FraserWD,SeguinJR.(2012)Emergingriskfactorsforpostpartumdepression:SEROtonintransportergenotypeandomega-3fattyacidstatus.CanJPsychiatry,57,704.
2. NarendranR,FrankleWG,MasonNS,MuldoonMF,MoghaddamB.(2012)Improvedworkingmemorybutnoeffectonstriatalvesicularmonoaminetransportertype2afteromega-3polyunsaturatedfattyacidsupplementation.PLoSOne,7,e46832.
3. KoricanacG,TepavcevicS,RomicS,ZivkovicM,StojiljkovicM,MilosavljevicT,StankovicA,PetkovicM,KamcevaT,ZakulaZ.(2012)EstradiolenhanceseffectsoffructoserichdietoncardiacfattyacidtransporterCD36andtriglyceridesaccumulation.EurJPharmacol,694,127.
4. YuanJ,ZhangB,GuoY.(2012)PoultryfatdecreasedfattyacidtransporterproteinmRNAexpressionandaffectedfattyacidcompositioninchickens.JAnimSciBiotechnol,3,17.
5. MoschenI,BroerA,GalicS,LangF,BroerS.(2012)Significanceofshortchainfattyacidtransportbymembersofthemonocarboxylatetransporterfamily(MCT).NeuRochemRes,37,2562.
6. JainSS,SnookLA,GlatzJF,LuikenJJ,HollowayGP,ThurmondDC,BonenA.(2012)Munc18cprovidesstimulus-selectiveregulationofGLUT4butnotfattyacidtransportertraffickinginskeletalmuscle.FEBSLett,586,2428.
7. SteinbuschLK,LuikenJJ,VlasblomR,ChabowskiA,HoebersNT,CoumansWA,VroegrijkIO,VosholPJ,OuwensDM,GlatzJF,DiamantM.(2011)AbsenceoffattyacidtransporterCD36protectsagainstWestern-typediet-relatedcardiacdysfunctionfollowingpressureoverloadinmice.AmJPhysiolEndocrinolMetab,301,E618.
8. SchipperP,KiliaanAJ,HombergJR.(2011)Amixedpolyunsaturatedfattyaciddietnormalizeshippocampalneurogenesisandreducesanxietyinserotonintransporterknockoutrats.BehavPharmacol,22,324.
9. BabuM,GreenblattJF,EmiliA,StrynadkaNC,ReithmeierRA,MoraesTF.(2010)StructureofaSLC26aniontransporterSTASdomainincomplexwithacylcarrierprotein:implicationsforE.coliYchMinfattyacidmetabolism.Structure,18,1450.
10. KeithDJ,EshlemanAJ,JanowskyA.(2011)Melittinstimulatesfattyacidreleasethroughnon-phospholipase-mediatedmechanismsandinteractswiththedopaminetransporterandothermembrane-spanningproteins.EurJPharmacol,650,501.
