流式测Treg

Description:TheFJK-16santibodyreactswithmouse/ratFoxp3alsoknownasFORKHEADBOXP3,SCURFIN,andJM2;crossreactivityofthisantibodytootherproteinshasnotbeendetermined.Foxp3,a49-55kDaprotein,isamemberoftheforkhead/winged-helixfamilyoftranscriptionalregulators,andwasidentifiedasthegenedefectivein‘scurfy’(sf)mice.ConstitutivehighexpressionofFoxp3mRNAhasbeenshowninCD4+CD25+regulatoryTcells(Tregcells),andectopicexpressionofFoxp3inCD4+CD25-cellsimpartsaTregphenotypeinthesecells.（Foxp3是叉状头转录因子家族中的一个成员，被认为是调节性T细胞（Treg）的标志性分子，Foxp3基因突变能引起严重的自身免疫性疾病，因此Foxp3在调节机体免疫自稳中起关键作用。Foxp3作为一个转录调控因子，通过直接调控多种基因来调节Treg的活性，目前关于Foxp3通过何种方式调控这些基因表达的机制尚未阐明。)ImmunoblottingwithFJK-16santibodyhasmappedtheepitope(抗原表位）toaminoacids75-125ofthemouseFoxp3protein.Inthehuman,thisregionhasbeenshowntobealternativelysplicedatthemRNAlevel.Boththealternatively-splicedandnon-splicedisoforms（亚型）arepresentintheCD4+CD25+subsetoflymphocytes.PreliminaryRT-PCRexperimentshavenotrevealedthisalternatively-splicedisoforminmousesplenocytes,suggestingdifferentgeneregulationinthemouseandhuman.IntracellularstainingofmousesplenocyteswithFJK-16susingthemouseFoxp3stainingsetsandprotocolrevealsapproximately2%oftotalsplenocytesintheC57Bl/6strainandapproximately3-5%intheBALB/cmousestrain.Multicolorflowcytometricanalysisdemonstratesapproximately90%oftheCD4+CD25+cellsand4%oftheCD4+CD25-cellsstainingwithFJK-16s.B220+,CD11b+,CD11c+,andLy6G/Gr-1+cellsdonotshowsignificantco-stainingwithFJK-16s.ThesedataareconsistentwitharecentreportwhichfollowsexpressionofFoxp3,usingaGFPknock-in(Fontenotetal,2005).FJK-16scross-reactswithratFoxp3.ThishasbeendemonstratedbyintracellularstainingofFoxp3andflowcytometryofratsplenocytesusingthesamemethodandreagentsasusedformousetissue.PleasenotethattheCD4andCD25antibodiesincludedinthiskitonlyrecognizethemouseantigens.Forstainingrattissue,pleaseuse(CD4FITCcat.11-0040andCD25PEcat.12-0390)Theanti-mouseFoxp3StainingSethasbeenformulatedandoptimizedforthestainingofmousesplenocyteswiththeFJK-16smonoclonalantibody.Notincluded:Isotypecontrolsforanti-CD4(ratIgG2a,cat.11-4321)andanti-CD25(ratIgG1,cat.12-4301)ReactivityMouseComponentsAnti-MouseCD4FITC(RM4-5):50ug.Useat0.125ug/test.Storeat4℃inthedark.（0.2mg/mL=0.2ug/uL）Anti-MouseCD25PE(PC61.5):50ug.Useat0.06ug/test.Storeat4℃inthedark.（0.2mg/mL=0.2ug/uL）Anti-Mouse/RatFoxp3PE-Cy5(FJK-16s):25ug.Useat0.5ug/test.Storeat4℃inthedark.（0.2mg/mL=0.2ug/uL）RatIgG2aIsotypeControlPE-Cy5:25ug.Useat0.5ug/test.Storeat4℃inthedark.（0.2mg/mL=0.2ug/uL）Anti-MouseCD16/32(FcBlock)Purified:50ug.Useat1-5ug/test（0.2mg/mL=0.2ug/uL）FlowCytometryStainingBuffer:200mlFixation/PermeabilizationConcentrate:30ml.Storea4℃.Avoidagitation.Thisisa4XstocksolutionthatmustbedilutedpriortousewiththeFixation/PermeabilizationDiluent.Dilute1partFixation/PermeabilizationConcentratewith3partsFixation/PermeabilizationDiluent.Usewithin6monthsofreceipt.Caution:ThissolutioncontainsParaformaldehyde,whichistoxicandasuspectedcarcinogen.Contactwitheyes,skinandmucousmembranesshouldbeavoided.Wearproperprotectiveclothingandgloves.Fixation/PermeabilizationDiluent:100ml.Storeat4℃.ThediluentisintendedtobeusedincombinationwiththeFixation/PermeabilizationConcentrate.PermeabilizationBuffer(10X):100ml.Storeat4℃.Diluteto1Xwithdeionized/distilledwaterandstoreat4℃.Note:The10xPermeabilizationBufferhasanaturaltendencytoprecipitate,however,itsfunctionisnotaffectedbythis.Toclarify,thesolutioncanbefilteredafterdilutionto1Xworkingsolution.Caution:Harmfulifswallowedorirritantbycontact.Wearproperprotectiveclothingandgloves.ReportedApplicationsIntracellularStainingFollowedbyFlowCytometricAnalysis过程：在标记前，配制所需要体积的固定/破膜工作液（每个样本1mL），现配现用：即将Fixation/Permeabilizationconcentrate(浓缩液，1part)加入至Fixation/PermeabilizationDiluent(稀释液，3parts)；配制破膜缓冲液（10×Permeabilizationbuffer→1×Permeabilizationbuffer），现配现用：用三蒸水稀释。步骤：1.在每个流式管内加入100uL的细胞（大约1×106个细胞）2.标记表面抗原分子（每个样本100uL）：每管加入100uL的含有0.125ug/testanti-mouseCD4antibodies和0.06ug/testanti-mouseCD25antibodies的FlowStainingBuffer(流式细胞仪染色液）0.125ug/testanti-mouseCD4antibodies(假设有a个样本，则需要加入(a×0.125ug/uL)/0.2ug/uL的抗CD抗体，每管加0.625uL,)0.06ug/testanti-mouseCD25antibodies(假设有a个样本，则需要加入(a×0.06ug/uL)/0.2ug/uL的抗CD25抗体，每管加0.3uL)则吸取a×0.625uL的抗CD4抗体和a×0.3uL的抗CD25抗体，加至a×100uL的FlowStainingBuffer(流式细胞仪染色液）中，混匀。于4℃避光孵育至少1h。3.加入2mL的coldFlowCytometryStainingBuffer（流式细胞仪染色液），涡旋重悬细胞，1500rpm,5min离心后，弃去上清；（离心时可配制固定/破膜工作液，即浓缩液：稀释液=1:3）4.每管加入1mL固定/破膜工作液，涡旋重悬细胞5.于4℃避光孵育2h左右。孵育结束后，离心，1700rpm,5min，倒弃上清。6.然后加入2mL的1×Permeabilizationbuffer（破膜缓冲液），涡旋重悬后，于1700rpm,离心5min，倒弃上清（尽量倒干净些）。（注：由10×Permeabilizationbuffer→1×Permeabilizationbuffer，现配现用：用三蒸水稀释。）7.标记Foxp3分子（Foxp3为膜内的抗原分子，故需之前的破膜过程）每管加入100uL的含有0.5uganti-mouse/ratFoxp3(FJK-16s)antibody的.1×Permeabilizationbuffer（破膜缓冲液），于4℃避光孵育1h。（假设有a个样本，则需配制(a×0.5ug/uL)/0.2ug/uL的抗Foxp3抗体，即每管加入0.25uL的抗体，则吸取约a×0.25uL抗Foxp3抗体至a×100uL的1×Permeabilizationbuffer（破膜缓冲液）中，混匀。）8.孵育结束后，每管加入2mL的1×Permeabilizationbuffer（破膜缓冲液），涡旋重悬后，于1700rpm,离心5min，倒弃上清9.每管加入300uL的FlowCytometryStainingBuffer（流式细胞仪染色液），涡旋重悬细胞后，避光置于4℃保存。然后测流式。