BackgroundFlowcytometricanalyseswithmonoclonalantibodiesweresofarmainlyrestrictedtocellsurfacemolecules.Intracellularstructuressuchascytoplasmicornuclearenzymes,oncoproteins,cytokines,immunoglobulinsetc.werelargelyexcludedfromsuchstudies.Alsoexcludedfromflowcytometricstudieswerecytoplasmiclocalizationsofwell-establishedmembranemoleculeslikeCD3andCD22,which,intheircytoplasmicform,arethemostreliablelineageMarkersinundifferentiatedleukemia.WiththeFIX&PERM®Kitflowcytometricanalysisofintracellularantigenshasbecomeaseasyassurfaceantigenstudies.Theonlyprerequisiteistheavailabilityofsuitableantibodyconjugates.MostoftheavailablemonoclonalantibodyconjugatescanbeusedwiththeFIX&PERM®Kit,somedeterminantsaresensitive,however,tothefixationstepinvolved.Thisandtheoptimalfixationtimehavetobetestedforeachreagent.

ProductThisFIX&PERM®CellFixationandPermeabilizationKitcontains2reagents:FixationMedium(ReagentA)andPermeabilizationMedium(ReagentB).ItisintendedforfirstfixingcellsinsUSPensionwithReagentAandthenpermeabilizingthecellmembraneswithReagentB.Thisproceduregivesantibodiesaccesstointracellularstructuresandleavesthemorphologicalscattercharacteristicsofcellsintact.SpecificformulationsreducebackgroundstainingandallowsimultaneousadditionofpermeabilizationmediumandFluorochromelabeledantibodies.FIX&PERM®issuitablefortheanalysisofnormalandmalignantleukocytepopulationsderivedfromvarioushumanBIOLOGicalsamples(blood,bonemarrowandothers)usingflowcytometry.Resultsmustbeputwithinthecontextofotherdiagnostictestsaswellastheclinicalhistoryofthepatientbyacertifiedprofessionalbeforefinalinterpretation.Format:4x5mlvialofFixationMedium(ReagentA)and4x5mlvialPermeabilizationMedium(ReagentB).FIX&PERM®Reagentsaredesignedforusewithallcommerciallyavailableflowcytometers.Alignmentandcompensationshouldbeperformedaccordingtomanufacturer´sinstructions.ThequalityofeachFIX&PERM®Lotisdeterminedbyfixationandpermeabilizationofwelldefinedbloodsamplesfromrepresentativedonorsandsubsequentcomparisonofforwardandsidescattercharacteristicsofobtainedleukocytes,aswellasimmunostainingefficiencyforseveralmembranousandcytoplasmicantigens

Formulation:4x5mlvialofFixationMedium(ReagentA)and4x5mlvialPermeabilizationMedium(ReagentB).FIX&PERMReagentsaredesignedforusewithallcommerciallyavailableflowcytometers.Alignmentandcompensationshouldbeperformedaccordingtomanufact

ApplicationsBiologicalfluids(blood,bonemarrow,andothers)mustbecollectedundersterileconditions.AnticoagulationwithEDTAorheparinisrecommended.Thesamplesshouldbestoredatroomtemperatureuntilused.Foroptimalresults,samplesshouldbeprocessedandanalyzedwithin24hours.Sampleswithhighnumbersofnon-viablecellsmightcausefalseresults,suchcasesrequiredeterminationofcellviabilitywithe.g.propidiumiodide.Allbiologicalsampleshavetobehandledwithcaution.Alwaysconsiderthemaspotentiallyinfective.Useappropriateprecautionssuchasgloves,lab-coat,etc.Fixation,permeabilizationandstainingprocedure:•Foreachsampletobeanalyzedadd50µlofwholeblood,bonemarrowormononuclearcellsuspensionina5mltube•Add100µlofReagentA(FixationMedium,storedandusedatroomtemperature)•Incubatefor15minutesatroomtemperature•Add5mlphosphatebufferedsalineandcentrifugecellsfor5minutesat300g•Removesupernatantandaddtocellpellet100µlReagentB(PermeabilizationMedium)and20µloftheappropriateNordic-Mubiomonoclonalantibodyconjugate•Vortexatlowspeedfor1-2seconds•Incubatefor15minutesatroomtemperature•Washcellswithphosphatebufferedsalineasdescribedabove•Removesupernatantandresuspendcellsinsheathfluidforimmediateanalysisorresuspendcellsin0.5ml1.0%formaldehydeandstorethemat2-8°Cinthedark.Analyzefixedcellswithin24hours.Comments:Specialcases(dilutedbonemarrowsamples,othersamplescontaininglowsolubleprotein)mightbenefitfromreplenishmentwithplasmacomponentsbeforetheFIX&PERM®treatmentinordertocreateamilieu,whichmorecloselyresemblesthestuationinanti-coagulatedblood.ForthatpuposeadditionofIgGpreparations(e.g.BeriglobulinP,ZLBBehring,finalconcentration10mg/ml)andhumanserumalbumin(e.g.humanalbumin“Behring”20%-infusionsolution,finalconcentration40mg/ml)isrecommended.

Limitationsofthetechnique:Flowcytometryshouldbeperformedbyprofessionalusersonly.Improperalignmentoftheflowcytometer,inaccuratecompensationoffluorescenceleakingintootherchannelsaswellasincorrectpositioningofregionsmayleadtofalseresults.LysisofredcellsmightbeimpossIBLeforvariousreasons.Insuchinstancesitisrecommendedtoisolatemononuclearcells(MNC)viadensitygrADIentcentrifugationpriortostaining.Resultswillbecorrectandreproducibleaslongastheproceduresusedrespectthetechnicalrecommendationsandobeygoodlaboratorypractice.TheFIX&PERM®solutionsareprovidedinaconcentrationthatwillallowtofixandpermeabilizehumanhematopoieticcells.Itisthereforestronglyrecommendedtosticktotheworkingprotocolintermsofconcentrationandvolumeregardingcellsandantibody.ThepropertiesofFIX&PERM®havebeendeterminedusingEDTAanti-coagulatedperipheralblood.

StorageFIX&PERM®CellFixationandPermeabilizationKitreagentsshouldbestoredandusedatroomtemperature.Donotfreeze.Stabilityofthereagent:Pleaserefertotheexpirydateprintedontothevial.Theuseofthereagentaftertheexpirationdateisnotrecommended.Ifreagentsarestoredunderanyconditionsotherthanthosespecified,theconditionsmustbeverifiedbytheuser.Donotusereagentsifaprecipitateshouldformordiscolorationoccurs.Ifunexpectedresultsareobtainedwhichcannotbeattributedtodifferencesinlaboratoryprocedures,pleasecontactus.

CautionForprofessionalusersonly.ReagentAofFIX&PERM®CellFixationandPermeabilizationKitcontainsfomaldehydeandislabelled:Harmful.Formaldehydeistoxic,allergenicandasuspectedcarcinogen.NeverPipettebymouthandavoidcontactwitheyes,skinandclothing.Properhandlingproceduresarerecommended.Asamainrule,personsunder18yearsofagearenotallowedtoworkwiththisproduct.Usersmustbecarefullyinstructedintheproperworkingprocedure,thedangerouspropertiesoftheproductandthenecessarysafetyinstructions.PleaserefertotheSafetyDataSheet(SDS)foradditionalinformation.Disposeproductremaindersaccordingtolocalregulations.

ReferencesRecentapplicationpaper:NiesKPH,KraaijvangerR,LindelaufKHK,DrentRJMR,RuttenRMJ,RamaekersFCS,LeersMPG.Determinationoftheproliferativefractionsindifferentiatinghematopoieticcelllineagesofnormalbonemarrow.CytometryA.2018Sep3.doi:10.1002/cyto.a.23564.Furtherreading:-Gerna,G.,Percivalle,E.,Lilleri,D.,Lozza,L.,Fornara,C.,Hahn,G.,Baldanti,F.&Revello,M.G.(2005)JGenVirol86,275-84.-Groeneveld,K.,teMarvelde,J.G.,vandenBeemd,M.W.,Hooijkaas,H.&vanDongen,J.J.(1996)Leukemia10,1383-9.-Haranaga,S.,Yamaguchi,H.,Friedman,H.,Izumi,S.,&Yamamoto,Y.(2001)InfectImmun69,7753-9.-Hegazy,A.N.&Klein,C.(2008)Leukemia22,2070-9.-Kappelmayer,J.,Gratama,J.W.,Karaszi,E.,Menendez,P.,Ciudad,J.,Rivas,R.&Orfao,A.(2000)JImmunolMethods242,53-65.-Kline,M.P.,Rajkumar,S.V.,Timm,M.M.,Kimlinger,T.K.,Haug,J.L.,Lust,J.A.,Greipp,P.R.&Kumar,S.(2007)Leukemia21,1549-60-Knapp,W.,Majdic,O.&Strobl,H.(1993)RecentResultsCancerRes131,31-40.-Knapp,W.,Strobl,H.&Majdic,O.(1994)Cytometry18,187-98.-Knapp,W.,Strobl,H.,Scheinecker,C.,Bello-Fernandez,C.&Majdic,O.(1995)AnnHematol70,281-96.-Konikova,E.,Glasova,M.,Kusenda,J.&Babusikova,O.(1998)Neoplasma45,282-91.-Lanza,F.,Latorraca,A.,Moretti,S.,Castagnari,B.,Ferrari,L.&Castoldi,G.(1997)Cytometry30,134-44.-Millard,I.,Degrave,E.,Philippe,M.&Gala,J.L.(1998)ClinChem44,2320-30.-Nakase,K.,Sartor,M.&Bradstock(1998)Cytometry34,198-202.-Pascale,F.,Contreras,V.,Bonneau,M.,Courbet,A.,Chilmonczyk,S.,Bevilacqua,C.,Epardaud,M.,Niborski,V.,Riffault,S.,Balazuc,A.M.,Foulon,E.,Guzylack-Piriou,L.,Riteau,B.,Hope,J.,Bertho,N.,Charley,B.&Schwartz-Cornil,I.(2008)JImmunol180,5963-72-Pickl,W.F.,Majdic,O.,Kohl,P.,Stockl,J.,Riedl,E.,Scheinecker,C.,Bello-Fernandez,C.&Knapp,W.(1996)JImmunol157,3850-9.-Riera-Sans,L.,&Behrens,A.(2007)JImmunol178,5690-700-Roberts,J.L.,Lengi,A.,Brown,S.M.,Chen,M.,Zhou,Y.J.,O"Shea,J.J.&Buckley,R.H.(2004)Blood103,2009-18-Sargent,R.L.,Craig,F.E.&Swerdlow,S.H.(2009)IntJClinExpPathol2,574-82-Scheinecker,C.,Strobl,H.,Fritsch,G.,Csmarits,B.,Krieger,O.,Majdic,O.&Knapp,W.(1995)Blood86,4115-23.-Sedlmayr,P.,Grosshaupt,B.&Muntean,W.(1996)Cytometry23,284-9.-Strobl,H.&Knapp,W.(2004)JBiolRegulHomeostAgents18,335-9.-Strobl,H.,Scheinecker,C.,Csmarits,B.,Majdic,O.&Knapp,W.(1995)BrJHaematol90,774-82.-Strobl,H.,Scheinecker,C.,Riedl,E.,Csmarits,B.,Bello-Fernandez,C.,Pickl,W.F.,Majdic,O.&Knapp,W.(1998)JImmunol161,740-8.-Strobl,H.,Takimoto,M.,Majdic,O.,Fritsch,G.,Scheinecker,C.,Hocker,P.&Knapp,W.(1993)Blood82,2069-78.-Wang,X.,Chang,X.,Facchinetti,V.,Zhuang,Y.&Su,B.(2009)JImmunol182,3597-608-FanJ,TangX,WangQ,ZhangZ,WuS,LiW,LiuS,YaoG,ChenHandSunL,Mesenchymalstemcellsalleviateexperimentalautoimmunecholangitisthroughimmunosuppressionandcytoprotectivefunctionmediatedbygalectin-9,StemCellResearch&Therapy2018,https://doi.org/10.1186/s13287-018-0979-xBingyuShi,JingjingQi,GenhongYao,RuihaiFeng,ZhuoyaZhang,DandanWang,ChenChen,XiaojunTang,LiweiLu,WanjunChenandLingyunSunMesenchymalstemcelltransplantationamelioratesSjögren’ssyndromeviasuppressingIL-12productionbydendriticcells.StemCellResearch&Therapy,2018https://doi.org/10.1186/s13287-018-1023RossellaZanin,SilviaPegoraro,GloriaRos,YariCiani,SilvanoPiazza,FleurBossi,RobertaBulla,CristinaZennaro,FedericaTonon,DejanLazarevic,EliaStupka,RiccardoSgarra&GuidalbertoManfiolettiHMGA1promotesbreastcancerangiogenesissupportingthestability,nuclearlocalizationandtranscriptionalactivityofFOXM1JournalofExperimental&ClinicalCancerResearchvolume38,Articlenumber:313(2019)https://doi.org/10.1186/s13046-019-1307-8ManabuItoh,YosukeMukae,TakahiroKitsuka,KenichiArai,AnnaNakamura,KazuyoshiUchihashi,ShujiToda,KumikaMatsubayashi,Jun-ichiOyama,KoichiNode,DaisukeKami,SatoshiGojo,ShigekiMorita,TakahiroNishida,KoichiNakayama&EijiKobayashiDevelopmentofanimmunodeficientpigmodelallowinglong-termaccommodationofartificialhumanvasculartubes.NatureCommunicationsvolume10,Articlenumber:2244(2019)https://doi.org/10.1038/s41467-019-10107-1

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