免疫磁珠说明书

Contents1.Description1.1PrincipleoftheMACS®Separation1.2Backgroundinformation1.3Applications1.4Reagentandinstrumentrequirements试剂和仪器的要求2.Protocol2.1Samplepreparation样品制备2.2Magneticlabeling磁性标记2.3Magneticseparation磁性分离3.ExampleofaseparationusingtheCD133MicroBeadKit4.References1.DescriptionComponents2mLCD133MicroBeads,human:MicroBeadsconjugatedtomonoclonalantihumanCD133antibodies(isotype:mouseIgG1,cloneAC133).2mLFcRBlockingReagent,humanSpecificityCD133antigen,epitope(CD133/1)1.CapacityFor2亊10⁹totalcells,upto100separations.ProductformatCD133MicroBeadsaresuppliedinbuffercontainingstabilizerand0.05%sodiumazide.StorageStoreprotectedfromlightat2−8°C.Donotfreeze.Theexpirationdateisindicatedontheviallabel.1.1PrincipleoftheMACS®SeparationFirst,theCD133+cellsaremagneticallylabeledwithCD133MicroBeads.Then,thecellsuspensionisloadedontoaMACS®Column,whichisplacedinthemagneticfieldofaMACSSeparator.ThemagneticallylabeledCD133+cellsareretainedwithinthecolumn.Theunlabeledcellsrunthrough;thiscellfractionisthusdepletedofCD133+cells.Afterremovingthecolumnfromthemagneticfield,themagneticallyretainedCD133+cellscanbeelutedasthepositivelyselectedcellfraction.Toincreasethepurity,thepositivelyselectedcellfractioncontainingtheCD133+cellsisseparatedoverasecondcolumn.1.2BackgroundinformationTheCD133MicroBeadKitisamagneticlabelingsystemdesignedforthepositiveselectionofCD133+cells.Itallowsthesingle-stepisolationofnonhematopoieticandearlyhematopoieticprogenitorsandstemcells.TheCD133moleculeisa5-transmembranecellsurfaceantigenwithamolecularweightof117kD.2TheCD133/1(cloneAC133)antibodyrecognizesepitope1oftheCD133antigen.1Inthehematopoieticsystem,CD133expressionisrestrictedtoasubsetofCD34brightstemandprogenitorcellsinhumanfetalliver,bonemarrow,cordblood,andperipheralblood.3Isolatedfromhematopoieticsources,CD133+cellscanbecomeadherentandarereportedtobecomeCD133–duringculture⁴.TheCD34+CD133+cellpopulation,whichincludesCD34+CD38–cells,wasshowntobecapableofrepopulatingNOD/SCIDmice.⁵Recently,CD133hasalsobeenfoundtobeexpressedoncirculatingendothelialprogenitorcells⁶,⁷andfetalneuralstemcells⁸,⁹aswellasonothertissue-specificstemcells,suchasrenal1⁰andprostate11stemcells.Lately,whenisolatedfromtumortissue,theCD133+populationcanbeenrichedfortumor-initiatingcells.11-1⁵CD133MicroBeadshavebeenusedtoisolateadultstemcellsfromcordbloodandasastartingpopulationforreprogramingtowardsiPScells.1⁶CD133expressionhasbeenfoundonundifferentiatedhumanEScells.ThereforeCD133MicroBeadscouldbeusedforenrichmentordepletionofthesecells.1⁷1.3Applications●PositiveselectionordepletionofcellsexpressinghumanCD133antigen.●IsolationordepletionofCD133+cellsfromperipheralbloodmononuclearcells(PBMCs)orsingle-cellsuspensionsfromtissue.●CD133+cellsareusedinbasicstemcellresearch,stemcellevaluation,stemcellexpansion,researchinhematologicalmalignancies,stemcellplasticity,andpotentialcellulartherapiesaswellasintissueregenerationandcancerresearch.1.4Reagentandinstrumentrequirements●Buffer:Prepareasolutioncontainingphosphate-bufferedsaline(PBS),pH7.2,0.5%bovineserumalbumin(BSA),and2mMEDTAbydilutingMACSBSAStockSolution(#130‑091-376)1:20withautoMACS_RinsingSolution(#130-091-222).Keepbuffercold(2−8°C).Degasbufferbeforeuse,asairbubblescouldblockthecolumn.▲Note:EDTAcanbereplacedbyothersupplementssuchasanticoagulantcitratedextroseformula-A(ACD-A)orcitratephosphatedextrose(CPD).BSAcanbereplacedbyotherproteinssuchashumanserumalbumin,humanserum,orfetalbovineserum(FBS).BuffersormediacontainingCa2+orMg2+arenotrecommendedforuse.●(Optional)Fluorochrome-conjugatedantibodiesforflowcytometricanalysis,e.g.,CD133/2(293C3)-PE(#130-090-853),CD133/2(293C3)-APC(#130-090-854),CD133/2(293C3)‑Biotin,CD34-FITC(#130-081-001),CD34‑APC(#130-090-954),orCD34-PE(#130-081-002).Formoreinformationaboutfluorochrome-conjugatedantibodiessee.●MACSColumnsandMACSSeparators:CD133+cellscanbeEnriched浓缩byusingMS,LS,orXSColumnsordepletedwiththeuseofLD,CS,orDColumns.CellswhichstronglyexpresstheCD133antigencanalsobedepletedusingMS,LS,orXSColumns.Positiveselection正向ordepletioncanalsobeperformedbyusingtheautoMACSProortheautoMACSSeparator.▲Note:ColumnadaptersarerequiredtoinsertcertaincolumnsintotheVarioMACS™orSuperMACS™Separators.FordetailsseetherespectiveMACSSeparatordatasheet.●(Optional)PropidiumIodideSolution(#130-093-233)or7-AADforflowcytometricexclusionofdeadcells.●(Optional)DeadCellRemovalKit(#130-090-101)forthedepletionofdeadcells.●(Optional)Pre-SeparationFilters(#130-041-407)toremovecellclumps团.2.Protocol2.1SamplepreparationWhenworkingwithanticoagulatedperipheralbloodorbuffycoat,peripheralbloodmononuclearcells(PBMCs)shouldbeisolatedbydensitygradientcentrifugation,forexample,usingFicoll-Paque™.▲Note:Toremoveplateletsafterdensitygradientseparation,resuspendcellpelletinbufferandcentrifugeat200×gfor10−15minutesat20°C.Carefullyaspiratesupernatant.Repeatwashingstep.Whenworkingwithtissuesorlysedblood,prepareasingle-cellsuspensionusingstandardmethods.Fordetailsseetheprotocolssectionat/protocols.Forpreparationofcordbloodcells,bonemarrowcells,orcellsfromleukapheresismaterial,pleaserefertothesamplepreparationprotocolsat/protocols.▲Deadcellsmaybindnon-specificallytoMACSMicroBeads.Toremovedeadcells,werecommendusingdensitygradientcentrifugationortheDeadCellRemovalKit(#130-090-101).2.2Magneticlabeling▲Workfast,keepcellscold,andusepre-cooledsolutions.Thiswillpreventcappingofantibodiesonthecellsurfaceandnon-specificcelllabeling.▲Volumesformagneticlabelinggivenbelowareforupto10⁸totalcells.Whenworkingwithfewerthan10⁸cells,usethesamevolumesasindicated.Whenworkingwithhighercellnumbers,scaleupallreagentvolumesandtotalvolumesaccordingly(e.g.for2亊10⁸totalcells,usetwicethevolumeofallindicatedreagentvolumesandtotalvolumes).▲Foroptimalperformanceitisimportanttoobtainasingle‑cellsuspensionbeforemagneticlabeling.Passcellsthrough30μmnylonmesh(Pre-SeparationFilters,#130-041-407)toremovecellclumpswhichmayclogthecolumn.Moistenfilterwithbufferbeforeuse.▲Therecommendedincubationtemperatureis2–8°C.Workingonicemayrequireincreasedincubationtimes.Highertemperaturesand/orlongerincubationtimesmayleadtonon-specificcelllabeling.1.Determine确定cellnumber.2.Centrifugecellsuspension细胞悬液at300×gfor10minutes.Aspiratesupernatantcompletely.吸取上清3.Resuspendcellpelletin300μLofbufferper10⁸totalcells.4.Add100μLofFcRBlockingReagentper10⁸totalcells.5.Add100μLofCD133MicroBeadsper10⁸totalcells.6.Mixwellandincubatefor30minutesintherefrigerator(2−8°C).7.(Optional)Addstainingantibodies,e.g.,50μLofCD133/2(293C3)-PE(#130-090-853),andincubatefor5minutesinthedarkintherefrigerator(2−8°C).8.Washcellsbyadding1−2mLofbufferper10⁸cellsandcentrifugeat300×gfor10minutes.Aspiratesupernatantcompletely.9.Resuspendupto10⁸cellsin500μLofbuffer.▲Note:Forhighercellnumbers,scaleupbuffervolumeaccordingly.▲Note:FordepletionwithLDColumns,resuspendupto1.25亊10⁸cellsin500μLofbuffer.10.Proceedtomagneticseparation(2.3).2.3Magneticseparation▲ChooseanappropriateMACSColumnandMACSSeparatoraccordingtothenumberoftotalcellsandthenumberofCD133+cells.Fordetailsseetableinsection1.4.▲Alwayswaituntilthecolumnreservoirisemptybeforeproceedingtothenextstep.MagneticseparationwithMSorLSColumns▲Toachievehighestpurities,performtwoconsecutivecolumnruns.1.PlacecolumninthemagneticfieldofasuitableMACSSeparator.FordetailsseetherespectiveMACSColumndatasheet.2.Preparecolumnbyrinsingwiththeappropriateamountofbuffer:MS:500μLLS:3mL3.Applycellsuspensionontothecolumn.Collectflow-throughcontainingunlabeledcells.4.Washcolumnwiththeappropriateamountofbuffer.Collectunlabeledcellsthatpassthroughandcombinewiththeeffluentfromstep3.MS:3×500μLLS:3×3mL▲Note:Performwashingstepsbyaddingbufferaliquotsonlywhenthecolumnreservoirisempty.5.Removecolumnfromtheseparatorandplaceitonasuitablecollectiontube.▲Note:Toperformasecondcolumnrun,youmayelutethecellsdirectlyfromthefirstontothesecond,equilibratedcolumninsteadofacollectiontube.6.Pipettetheappropriateamountofbufferontothecolumn.Immediatelyflushoutthemagneticallylabeledcellsbyfirmlypushingtheplungerintothecolumn.MS:1mLLS:5mL7.ToincreasepurityofCD133+cells,enrichtheelutedfractionoverasecondMSorLSColumn.Repeatthemagneticseparationprocedureasdescribedinsteps1to6byusinganewcolumn.MagneticseparationwithXSColumnsForinstructionsonthecolumnassemblyandtheseparationrefertotheXSColumndatasheet.DepletionwithLDColumns1.PlaceLDColumninthemagneticfieldofasuitableMACSSeparator.FordetailsseeLDColumndatasheet.2.Preparecolumnbyrinsingwith2mLofbuffer.3.Applycellsuspensionontothecolumn.4.Collectunlabeledcellsthatpassthroughandwashcolumnwith2×1mLofbuffer.Collecttotaleffluent;thisistheunlabeledcellfraction.Performwashingstepsbyaddingbuffertwotimes.Onlyaddnewbufferwhenthecolumnreservoirisempty.DepletionwithCSColumns1.AssembleCSColumnandplaceitinthemagneticfieldofasuitableMACSSeparator.FordetailsseeCSColumndatasheet.2.Preparecolumnbyfillingandrinsingwith60mLofbuffer.Attacha22Gflowresistortothe3-waystopcockoftheassembledcolumn.FordetailsseeCSColumndatasheet.3.Applycellsuspensionontothecolumn.4.Collectunlabeledcellsthatpassthroughandwashcolumnwith30mLbufferfromthetop.Collecttotaleffluent;thisistheunlabeledcellfraction.DepletionwithDColumnsForinstructionsoncolumnassemblyandseparationrefertotheDColumndatasheet.MagneticseparationwiththeautoMACS®ProSeparatorortheautoMACS®Separator▲RefertotherespectiveusermanualforinstructionsonhowtousetheautoMACS_ProSeparatorortheautoMACSSeparator.▲BuffersusedforoperatingtheautoMACSProSeparatorortheautoMACSSeparatorshouldhaveatemperatureof≥10°C.▲Programchoicedependsontheisolationstrategy,thestrengthofmagneticlabeling,andthefrequencyofmagneticallylabeledcells.Fordetailsrefertothesectiondescribingthecellseparationprogramsintherespectiveusermanual.MagneticseparationwiththeautoMACS®ProSeparator1.Prepareandprimetheinstrument.2.Applytubecontainingthesampleandprovidetubesforcollectingthelabeledandunlabeledcellfractions.PlacesampletubeinrowAofthetuberackandthefractioncollectiontubesinrowsBandC.3.Forastandardseparationchooseoneofthefollowingprograms:Positiveselectionfromperipheralblood,bonemarrow,orleukapheresis:“Posseld”CollectpositivefractioninrowCofthetuberack.Positiveselectionfromcordblood:“Posseld2”CollectpositivefractioninrowCofthetuberack.Depletion:“Depletes”CollectnegativefractioninrowBofthetuberack.MagneticseparationwiththeautoMACS®Separator1.Prepareandprimetheinstrument.2.Applytubecontainingthesampleandprovidetubesforcollectingthelabeledandunlabeledcellfractions.Placesampletubeattheuptakeportandthefractioncollectiontubesatportneg1andportpos2.3.Forastandardseparationchooseoneofthefollowingprograms:Positiveselectionfromperipheralblood,bonemarrow,orleukapheresis:“Posseld”Collectpositivefractionfromoutletportpos2.Positiveselectionfromcordblood:“Posseld2”Collectpositivefractionfromoutletportpos2.Depletion:“Depletes”Collectnegativefractionfromoutletportneg1.3.ExampleofaseparationusingtheCD133MicroBeadKitCD133+hematopoieticstemandprogenitorcellswereisolatedfromnon-mobilizedhumanPBMCsusingtheCD133MicroBeadKit,MSColumns,andaMiniMACS™Separator.CellswerefluorescentlystainedwithCD34-FITC(#130-08