TLR1StableCellLine

Description（描述）

TLR1stablecelllineexpressesfull-lengthhumanToll-likereceptor1(TLR1)withanN-terminalHAtag.TLR1expressionhasbeenvalidatedbyWesternblotting(Fig.1)andflowcytometry(Fig.2).

CompleteGrowthMedium（完全培养基）

DMEMwith4.5g/Lglucose+10%FBS+4mML-glutamine+1mMsodiumpyruvate+100units/mlpenicillin+100ug/mlstreptomycin+10ug/mlblasticidin.Note:Theselectionagent fortheTLR1stablelineis blasticidin.

Application（应用）

TheTLR1stablecelllinecanbeusedforTLR1flowcytometriccalibrationanddetectioncontrol.

ProductHandlingProtocol（产品处理协议）

Note:Pleasereadtheentiredatasheetbeforethawing.Itisrecommendedthatusersfollowgoodtissueculturepractice.The TLR1stablelineissterileandallworkshouldbeperformedundersterileconditions.1.Prepareasterile15-mltubewith9mlfreshmediumwithoutselectionagentspre-warmedat37oC.2.ThawtheTLR1stable celllinevialquicklyina37oCwaterbath,keepingthecapportionoutofthewatertoavoidanypossiblecontamination.3.Uponthawing,takethevialoutofthewaterandcleanitwith70%ethanoltodecontaminate.4.Transfercontentstothe15-mltube(Step1)andmixwithmediumbygentleinversionoftube.5.Centrifugeat1,000RPMfor5minutes.6.Removesupernatantandresuspendpelletin10mloffreshmediumwithoutselectionagents.Note:Itisimportanttogrowthe stablecellsatthisstagewithoutanyselectionagents.7.TransfertheTLR1stablelineintoa25-cm2tissuecultureflaskandincubateat37oCina95%air-5%CO2mixture.8.Aftercellssettledown(in1-3days),removethemediumandreplacewithfreshcompletegrowthmediumcontainingselectionagents.9.At70-80%confluency,detachthecellsbytrypsinizationandsplitintonewflaskswithfreshcompletegrowthmedium.10.FreezetheTLR1stable celllineat3~4x10^6cells/mlpercryogenicvial.Foroptimalviabilityafterfreezing,freezecellswhentheyhavereachedlogphasegrowth(95-98%confluency).Detachbytrypsinizationat37oCfor5min,andharvestbymixingwith3volumesoffreshmediumfollowedbycentrifugation(Step5).Resuspendthepelletinfreezemedia(FBSwith10%DMSO).Addsuspensiontocryogenicvialsin1mlaliquots.Placecryogenicvials,inatissuecultureapprovedcryogenicvialcontainer,in-80oCfreezerfor24-48hours.After24-48hours,movethevialsintoliquidnitrogenstorage.

SafetyConsiderations（安全注意事项）

Assumeallculturesarehazardoussincetheymayharborlatentvirusesorotherorganismsthatareuncharacterized.Thefollowingsafetyprecautionsshouldbeobserved.•Usepipetteaidstopreventingestionandkeepaerosolsdowntoaminimum.•Noeating,drinkingorsmokingwhilehandlingtheTLR1 stableline.•WashhandsafterhandlingtheTLR1stablelineandbeforeleavingthelab.•Decontaminateworksurfacewithdisinfectantor70%ethanolbeforeandafterworkingwithcells.•Allwasteshouldbeconsideredhazardous.•Disposeofallliquidwasteaftereachexperimentandtreatwithbleach.

Figure1.WesternblotanalysisofTLR1expressionintheTLR1stablecelllineusinganHAantibody(20ugtotalprotein/lane).Legend.Vect:Vector/HEK293(IML-200);TLR1:TLR1stablecellline(IML-201).

Figure2.CellsurfaceexpressionofTLR1intheTLR1stablecelllinewasanalyzedbyflowcytometryusingAlexaFluor®488-conjugatedTLR1antibody(IMG-5012AF488)andcomparedwiththeVector/HEK293cellline(IML-200).IMGENEX’srabbitIgGisotypecontrol(20304AF488)and theSurfaceTLRStainingFlowKit(10099K)wereusedforthistest.*Note:AnendogenouslevelofTLR1ispresentinHEK293cells.

Reference（参考文献）

1.MarkM.Wurfel,AnthonyC.Gordon,TarahD.Holden,FrankRadella,JeannaStrout,OsamuKajikawa,JohnT.Ruzinski,GailRona,R.AnthonyBlack,SethStratton,GailP.Jarvik,AdelineM.Hajjar,DeborahA.Nickerson,MarkRieder,JonathanSevransky,JamesP.Maloney,MarcMoss,GregMartin,CarlShanholtz,JoeG.N.Garcia,LiGao,RoyBrower,KathleenC.Barnes,KeithR.Walley,JamesA.Russell,ThomasR.Martin.Toll-likeReceptor1PolymorphismsAffectInnateImmuneResponsesandOutcomesinSepsis.AmJRespirCritCareMed.2008October1;178(7):710–720.2.SunnyH.Wong,SaileshGochhait,DheerajMalhotra,FredrikH.Pettersson,YikY.Teo,ChieaC.Khor,AnnaRautanen,StephenJ.Chapman,TaraC.Mills,AmitSrivastava,AlekseyRudko,MaximB.Freidin,ValeryP.Puzyrev,ShafatAli,ShwetaAggarwal,RupaliChopra,BelumS.N.Reddy,VijayK.Garg,SuchismitaRoy,SarahMeisner,SunilK.Hazra,BibhutiSaha,SianFloyd,BrendanJ.Keating,CeciliaKim,BenjaminP.Fairfax,JulianC.Knight,PhilipC.Hill,RichardA.Adegbola,HakonHakonarson,PaulE.M.Fine,RamasamyM.Pitchappan,RameshwarN.K.Bamezai,AdrianV.S.Hill,FredrikO.Vannberg.LeprosyandtheAdaptationofHumanToll-LikeReceptor1.PLoSPathog.2010July;6(7):e1000979.3.HoomanIzadi,AmirrezaT.Motameni,TonyaC.Bates,EliasR.Olivera,VegaVillar-Suarez,IlaJoshi,RenuGarg,BarbaraA.Osborne,RogerJ.Davis,MercedesRincón,JuanAnguita.c-JunN-TerminalKinase1IsRequiredforToll-LikeReceptor1GeneExpressioninMacrophages.InfectImmun.2007October;75(10):5027–5034.4.ELISAbethElass,LaëtitiaAubry,MaryseMasson,AgnèsDenys,YannGuérardel,EmmanuelMaes,DominiqueLegrand,JoëlMazurier,LaurentKremer.MycobacterialLipomannanInducesMatrixMetalloproteinase-9ExpressioninHumanMacrophagicCellsthroughaToll-LikeReceptor1(TLR1)/TLR2-andCD14-DependentMechanism.InfectImmun.2005October;73(10):7064–7068.5.HelenKWLaw,ChungYanCheung,SinFunSia,YukOnChan,JSMalikPeiris,YuLungLau.Toll-likereceptors,chemokinereceptorsanddeathreceptorligandsresponsesinSARScoronavirusinfectedhumanmonocytederiveddendriticcells.BMCImmunol.2009;10:35.6.ChristinaL.Lancioni,QingLi,JeremyJ.Thomas,XueDongDing,BonnieThiel,MichaelG.Drage,NicoleD.Pecora,AssemG.Ziady,SamuelShank,CliffordV.Harding,W.HenryBoom,RoxanaE.Rojas.MycobacteriumtuberculosisLipoproteinsDirectlyRegulateHumanMemoryCD4+TCellActivationviaToll-LikeReceptors1and2.InfectImmun.2011February;79(2):663–673.

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