Background:TheCD45moleculeistypicallyexpressedathighlevelsonallhematopoieticcells.CD45isamajorcomponentoftheglycocalixofthesecellsandcanbeexpressedindifferentisoforms.AntibodyVIT200recognizesapanCD45epitope,whichisexpressedonallhematopoieticcells.CD14isaGPI-anchoredmoleculeexpressedbyvirtuallyallhumanmonocytesandmacrophagesand–toalesserdegree-granulocytes.CD14togetherwithToll-likereceptor4andMD-2formstheLPS-receptorcomplexthatrecognizesandsignalsthepresenceofLPS.WhileCD14hasnosignalingstructureitsmainroleseemstobethebindingofLPS.TheVIT200/MEM18COMBI-REAGENT-GateControlpermitstheidentificationandenumerationofhumanleukocytesusingflowcytometry.Resultsmustbeputwithinthecontextofotherdiagnostictestsaswellastheclinicalhistoryofthepatientbyacertifiedprofessionalbeforefinalinterpretation.Analysesperformedwiththisantibodyshouldbeparalleledbypositiveandnegativecontrols.Ifunexpectedresultsareobtained,whichcannotbeattributedtodifferencesinlaboratoryprocedures,pleasecontactusProduct:PBSpH7.2,1%BSA,0.05%NaN3Applications:StainingProcedureDirectImmunofluorescence(StainingProcedure)Nordic-MubioFluorochromelabeledantibodiesaredesignedforusewitheitherwholebloodorisolatedmononuclearcell(MNC)preparations.Proposedstainingprocedureforwholebloodinshort:-Foreachsampleadd50µlofEDTAanti-coagulatedbloodtoa3-5mltube-Add20µloftheappropriateNordic-MUbiomonoclonalantibodyconjugate-Incubatethetubefor15minutesat4°Coratroomtemperatureinthedark-Add100µlNM-LYSE(Cat.No.GAS-003)toeachtubeandincubatefor10minutesatroomtemperature-Add3-4mlofdestilledwaterandvortex,incubatefor5-10minutesatroomtemperature-Centrifugetubefor5minutesat300g-AspiratesupernatantandresUSPendpelletin0.3mlofsheathfluid-Analyzeimmediatelyorstoresamplesat2-8°Cinthedarkandanalyzewithin24hoursFor“No-Wash”protocolpleaserefertowww.Nordicmubio.comProposedstainingprocedureforMNCinshort:-Carefullyadd20µlantibodyconjugateand50-100µlMNCtothebottomofatube-Vortexatlowspeedfor1-2seconds-Incubatefor15-30minutesat2-8°Coratroomtemperature-Centrifugetubesfor5minutesat300g-Removesupernatant,resuspendcellsin2-5mlofphosphatebufferedsaline(PBS)andcentrifugecellsagainfor5minutesat300g-Removesupernatantandresuspendcellsinsheathfluidforimmediateanalysisorresuspendcellsin0.5ml1%formaldehydeandstorethemat2-8°Cinthedark.Analyzefixedcellswithin24hoursSpecificity:TheCD45mAb(cloneVIT200)recognizesapanCD45epitope.TheCD14mAb(cloneMEM18)recognizessurfaceCD14onhumanmonocytesandmacrophagesaswellasneutrophils.ThesensitivityofCD45/CD14mAbisdeterminedbystainingwell-definedbloodsamplesfromrepresentativedonorswithserial-foldmAbdilutionstoobtainatitrationcurvethatallowsrelatingthemAbconcentrationtothepercentageofstainedcellsandgeometricMFI(meanfluorescenceintensity).Forthispurpose,amAb-concentrationrangeisselectedtoincludeboththesaturationpoint(i.e.themAbdilutionexpectedtobindallepitopesonthetargetcell)andthedetectionthreshold(i.e.themAbdilutionexpectedtorepresenttheleastamountofmAbneededtodetectanidenticalpercentageofcells).Inpractice,50µlofleukocytescontaining10^7cells/mlarestainedwith20µlmAbofvariousdilutionstoobtainatitrationcurveandtoidentifythesaturationpointanddetectionthreshold.Thefinalconcentrationoftheproductisthenadjustedtobeatleast3-foldabovethedetectionthreshold.Inadditionandtocontrollot-to-lotvariation,thegivenlotiscomparedandadjustedtofluorescencestandardswithdefinedintensity.Storage:Nordic-MUbiomonoclonalantibodyreagentscontainoptimalconcentrationsofaffinity-purifiedantibody.ForstABIlityreasonsthismonoclonalantibodysolutioncontainssodiumazide.Thesereagentsshouldbestoredat2-8°C(DONOTFREEZE!)andprotectedfromprolongedexposuretolight.Stabilityofthereagent:Pleaserefertotheexpirydateprintedontothevial.Theuseofthereagentaftertheexpirationdateisnotrecommended.References:1.Battifora,H.&Trowbridge,I.S.(1983)Cancer51,816-21.2.Beutler,B.(2002)CurrTopMicrobiolImmunol270,109-20.3.Brocklebank,A.M.&Sparrow,R.L.(2001)Cytometry46,254-61.4.Cobbold,S.,Hale,G.&Waldmann,H.(1987)LeucocyteTypingIII.p788-803(OxfordUniversityPress,Oxford-NewYork-Tokyo).5.Dalchau,R.,Kirkley,J.&Fabre,J.W.(1980)EurJImmunol10,737-44.6.Goyert,S.M.(1989)LeucocyteTypingIV.p789-793(OxfordUniversityPress,Oxford-NewYork-Tokyo).7.Goyert,S.M.,Ferrero,E.,Rettig,W.J.,Yenamandra,A.K.,Obata,F.&LeBeau,M.M.(1988)Science239,497-500.8.Goyert,S.M.,Ferrero,E.M.,Seremetis,S.V.,Winchester,R.J.,Silver,J.&Mattison,A.C.(1986)JImmunol137,3909-14.9.Jing,S.,Ralph,S.,Head,M.T.A.,Chain,A.&Trowbridge,I.(1987)StructuralstudiesofthetransferrinreceptorandT200glycoprotein(CD45).LeucocyteTypingIII.p899-905(OxfordUniversityPress,Oxford-NewYork-Tokyo).10.Knapp,W.(1982)Blut45,301-8.11.Knapp,W.(1989)LeucocyteTypingIV.p747-780(OxfordUniversityPress,Oxford-NewYork-Tokyo).12.Means,T.K.,Lien,E.,Yoshimura,A.,Wang,S.,Golenbock,D.T.&Fenton,M.J.(1999)JImmunol163,6748-55.13.Nicholson,J.K.,Hubbard,M.&Jones,B.M.(1996)Cytometry26,16-21.14.Sugita,K.,Majdic,O.,Stockinger,H.,Holter,W.,Burger,R.&Knapp,W.(1987)Transplantation43,570-4.15.Sun,T.,Sangaline,R.,Ryder,J.,Gibbens,K.,Rollo,C.,Stewart,S.&Rajagopalan,C.(1997)AmJClinPathol108,152-7.16.Tapping,R.I.,Akashi,S.,Miyake,K.,Godowski,P.J.&Tobias,P.S.(2000)JImmunol165,5780-7.17.Thomas,M.L.(1989)AnnuRevImmunol7,339-69.18.Ugolini,V.,Nunez,G.,Smith,R.G.,Stastny,P.&Capra,J.D.(1980)ProcNatlAcadSciUSA77,6764-8.19.Yoshimura,A.,Lien,E.,Ingalls,R.R.,Tuomanen,E.,Dziarski,R.&Golenbock,D.(1999)JImmunol163,1-5.Caution:Forprofessionalusersonly.Thisreagentcontainssodiumazide.Toavoidthedevelopmentofhazardousconditions,reagentscontainingazideshouldbedilutedinrunningwaterpriortobediscarded.SimilartotheworkwithotherBIOLOGicalproducts,properhandlingproceduresarerecommended.