Description:TLR4/MD-2,Mouse,mAbMTS510,functionalstudyantibody,lowendotoxinThemonoclonalantibodyMTS510reactswiththeToll-likereceptor4(TLR4,CD284)thatisassociatedwithMD2.TLRsareexpressedbyvariouscellsoftheimmunesystem,suchasmacrophagesanddendriticcells.TLRsareclassIreceptors,withasingleα-helixthatspansthecellmembrane.Theyrecognizeandrespondtomoleculesderivedfrombacterial,viralandfungalpathogens,suchaslipopolysaccharide(LPS)fromtheoutermembraneofGramnegativebacteria,peptidoglycanfragmentsfrombacterialcellwallsandsingle-strandedanddouble-strandedRNAfromviruses.Toll-likereceptor4(TLR4;CD284)hasbeenidentified,nexttoMD-2andCD14,asareceptorthatiscentraltotheinnateimmuneresponsetoLPSofGram-negativebacteria.TLR4isuniqueamongTLRsinitsABIlitytoactivatetwodistinctsignalingpathways;onepathwayisactivatedbytheadaptorsTIRAP(Toll/interleukin-1-receptor(TIR)-domain-containingadaptorprotein)andMyD88,whichleadstotheinductionofpro‑inflammatorycytokines.ThesecondpathwayisactivatedbytheadaptorsTRIF(TIR-domaincontainingadaptorproteininducinginterferon‑β)andTRAM(TRIFrelatedadaptormolecule),whichleadstotheinductionoftypeIinterferons.MD-2existsasacellsurfaceproteininassociationwithTLR4.ItalsoexistsassecretedformsconsistingofMD-2monomerandmultimers.CirculatingsMD-2ismainlypresentasadoubletof~20and25kD,representingdifferentiallyglycosylatedforms.UnlikeTLR4,sMD-2bindsdirectlyLPSwithouttheneedofsolubleCD14(sCD14).However,LPS-MD-2interactionsareincreasedwhenLPSispretreatedwithCD14.OnlymonomericsMD-2isBIOLOGicallyactiveandabletoassociatewithTLR4andLPS.sMD-2circulatesinplasmaofhealthyindividualsasanon-active,polymericprotein.Insepticplasma,thetotalamountofsMD-2wasstronglyelevatedandcontainedbothsMD-2polymersandmonomers.SolubleMD-2isproposedtobeanimportantmediatoroforganinflammationduringsepsis.Duringexperimentalhumanendotoxemia,themonomericandtotalsMD-2contentinplasmaincreasedwiththekineticsofanacutephaseprotein.ThisparallelsenhancedTLR4costimulatoryactivity.InvitrostudiesrevealedthatsMD-2releaseappearstoberestrictedtoendothelialanddendriticcells.ThemonoclonalantibodyMTS510reactspreferentially,especiallyinflowcytometry,withmouseTLR4thatisassociatedwithMD-2.MTS510isaTLR4function-blockingantibodythatisusefulforstudiesontheroleofTLR4asareceptorforLPSinducedcytokineproductionbyTLR4bearingcells.TheantibodywasshowntocoprecipitateMD-2(30kDa)withTLR4(100kDa).Specifications:CatalognumberHM1029-FSProducttypeMonoclonalantibodiesQuantity0.5mgFormulation0.5mg(100µg/ml)0.2µmfilteredantibodysolutioninPBS(exactconcentrationisindicatedonthelabel)ApplicationFlowcytometry,Frozensections,Functionalstudies,Immunofluorescence,ImmunoprecipitationApplicationNotesF:6µmacetonefixedsectionsblockedwith0.3%hydrogenperoxidaseinmethanolandsubsequentlywithnormalserum.Sectionswereincubatedfor2hatRTat1/100dilutionofmAbFc:Cellswereincubatedwith0.1 µmmAbfor30minutesat4 °CFS:Cellswerepre-incubatedwith10 µg/mlofantagoNISTicmAbMTS510RAW264.7cells(105)werestainedwith4 µg/mlmAbfor1hat4 °C(black-isotypecontrol,red-irrelevantmAb,Blue-HM1029)UseForimmunohistologyandflowcytometrydilutionstobeuseddependondetectionsystemapplied.Itisrecommendedthatuserstestthereagentanddeterminetheirownoptimaldilutions.Thetypicalstartingworkingdilutionis1:50.ImmunogenBa/F3cellsexpressingmouseTLR4andMD-22IsotypeRatIgG2aSpeciesMouseAliasToll-likereceptor4:-TLR4,CD284,ARMD10MD2:ESOP-1;myeloiddifferentiationprotein-2;lymphocyteantigen96(Ly-96)PositiveControlRAW264.7cellsReferences1.Nomura,Fetal;Cuttingedge:Endotoxintoleranceinmouseperitonealmacrophagescorrelatewithdown-regulationofsurfacetoll-likereceptor4expression.JImmunol2000,164:34762.Akashi,Setal;Cuttingedge:Cellsurfaceexpressionandlipopolysaccharidesignallingviathetoll-likereceptor4-MD-2complexonmouseperitonealmacrophages.JImmunol2000,164:34713.Sato,Setal;Synergyandcross-tolerancebetweentoll-likereceptor(TLR)2-andTLR4-mediatedsignalingpathways.JImmunol2000,165:70964.Ortega-Cava,Cetal;Strategiccompartmentalizationoftoll-likereceptor4inthemousegut.JImmunol2003,170:39775.Tsujimoto,H.AcriticalroleofCpGmotifsinamurineperitonitismodelbytheirbindingtohighlyexpressedtoll-likereceptor-9onliverNTKcells.Jhepatology2006,45:8366.Cook,Cetal.Lipopolysaccharide,TumorNecrosisFactoralpha,orInterleukin-1βtriggersreactivationoflatentCMVinimmunocompetentmice.Jvirology2006,80:91517.Tsukumo_Detal.Loss-of-functionmutationinToll-likereceptor4preventsdiet-inducedobesityandinsulinresistance.Diabetes2007,56:19868.Burton,Oetal.ImportanceofTLR2inthedirectresponseofTlymphocytestoSchistosomamansoniantigens.EurJImmunol2010,40:2221StorageandstabilityProductshouldbestoredat4 °C.Underrecommendedstorageconditions,productisstableforoneyear.PrecautionsForresearchuseonly.Notforuseinoronhumansoranimalsorfordiagnostics.Itistheresponsibilityoftheusertocomplywithalllocal/stateandFederalrulesintheuseofthisproduct.HycultBiotechisnotresponsIBLeforanypatentinfringementsthatmightresultwiththeuseoforderivationofthisproduct.DiseaseInfectiousdiseases