Donkey AntiGoat IgG/FITC抗体 FITC标记的驴抗羊IgG_价格厂家供应...
- Harvestcellsfromtissue,preparingasinglecellsUSPension.RedbloodcellsmayberemovedbylysisordensitygrADIent:RedbloodcellsfrommurineperipheralbloodoraspleencellsuspensioncanbelysedusingBDBiosciencesPharmingen"sPharMLyse™(Cat.No.35221E)solution.Add2.0mlof1XLysingSolutiontothespleencellsuspensionorper200µlofmurineperipheralblood.Gentlyvorteximmediatelyafteraddingthelysingsolution.Incubateatroomtemperature,protectedfromlight,for15min.Centrifuge200xgfor5min.Carefullyaspirateanddisposeofsupernatant,withoutdisturbingthepellet.Resuspendpelletin1Xcoldwashbuffer(PBS/0.1%NaN3/1.0%fetalbovineserum).Centrifugeat350xgfor5min.Finally,resuspendcellpellettoaconcentrationof2x107cells/ml(i.e.,106cellsper50µl).
- DiluteprimarymAbs(e.g.,unconjugated,biotinylated,orFluorochrome-conjugatedmAbs)topredeterminedoptimalconcentrations(seeStainingTips)inwashbufferanddelivertothewellsofaU-bottommicrotiterplateinavolumeof50µl.
- Deliver106cellsin50µltoeachwellalreadycontaining50µlofmAb(or50µlwashbufferfornegativecontrols).Mixbygentlyvortexingortapping.
- Incubateat4°Cfor20-40mininthedark.
- Wash2Xwith200µlwashbuffer(or3Xifabiotin-conjugatedprimaryantibodyisused).Aftereachcentrifugation,350xgfor5min,aspiratewellsorflickplatetoremovesupernatant.Vortexgentlyortapplatetoloosenpelletpriortoaddingnextwashordilutedsecondaryreagent.
- Ifasecond-stepreagentisneeded,resuspendcellpelletin100µlofappropriatelydilutedsecondaryreagent(e.g.,fluorochrome-conjugatedavidin,streptavidin,anti-Igallotype,anti-Igisotype,polyclonalanti-Ig).Forexample,diluteantibodyto~1µgper100µlinwashbufferandaddthistoeachwellcontainingtheloosenedcellpellet.
- Incubateat4°Cfor20-40mininthedark.
- Wash2Xwith200µlwashbuffer,asinStep5.Use100µlwashbuffertotransfercellpelletsto0.4mlaliquotsofwashbuffer(finalconcentration~106cellsin0.5ml)intubesappropriateforflowcytometer.AcquiresampledataonflowcytometerassoonaspossIBLeafterstaining.(PleaseseeStainingTip5.)
- Determineoptimalconcentrations(brighteststaining/lowestbackground)ofeachprimaryandsecondaryantibodybytitrating,inapreliminaryexperiment,between1.0µgand0.1µgantibodyper100µlwashbufferfor106cells.
- Whenperformingmulti-colorlabeling,directly-conjugatedmAbscanbeaddedsimultaneously,ratherthansequentially.Forinstrumentset-up,pleaseseedescriptionin"ProcedureforSettingCompensationforMulti-ColorFlowCytometricAnalysis".
- ForreducingFcgII/IIIR-mediatedantibodybinding(orbindingofSAv-PEorSAv-Cy-Chrome)whichcouldcontributetobackground,theuseofanti-mouseCD32/CD16(MouseBDFcBlock™;CatNo.553141/553142)oranti-ratCD32(RatBDFcBlock™;Cat.No.550270/550271)isrecommended.BDFcBlock™canbeaddedtocells(~0.25µgpermillioncells,3-5min,4°C)andneednotbewashedoutpriortoadditionofprimarymAb.ItisimportanttoverifythatnosecondaryreagentwillbindtheBDFcBlock™.Pleaseseedescriptionin"TheUsesofBDFcBlock™inImmunophenotypingofMouseandRatLeukocytes".
- Forverylow-densitycellsurfaceMarkers(e.g.,cytokinereceptors),athree-stepprotocolmayamplifythestaining:usepurifiedprimaryantibody(steps2-4ofaboveprocedure),biotinylatedanti-Igforthe2ndstep(steps6-7,above),andfluorochrome-conjugatedavidinorstreptavidinasthe3rdstep(repeatsteps6-7).WefindthatSAv-PEandSAv-BDCy-Chrome™are"brighter"thanFITCconjugatesandmayprovideevenbetterdiscriminationoflow-densityantigens,especiallyinthepresenceofBDFcBlock™,formousecells.(PleaseseeStainingTip3.)
- Wehavefoundthatfreshly-isolatedleukocytesandcelllinesmaywaitforanalysisinwashbufferat4°C,withoutfixation,forupto18hrpost-staining,withoutlossofviABIlity.Activatedlymphocytesmayloseviabilityrapidly,anddatashouldbecollectedwithin5hrpost-staining.Topreservecellintegritybeyondthesetimelimits,paraformaldehydefixationmaybenecessary;however,itispossiblethatthequalityofstainingmaybediminishedbysuchfixation.Wedonotrecommendfixationofstainedcells,exceptwhenthepossibilityofexposuretobiohazardousmaterialexists.
- Everyexperimentmustincludecontrols.Negativecontrolsaresamples`ofthesamecellpopulationtreatedexactlyasthetestsample,butwiththeomissionormodificationofoneofthestainingsteps.Examplesofnegativecontrolsareunstainedcells,cellsexposedtothe2ndstepreagentalone,orcellsexposedtoisotypecontrolswhicharethesameisotypeandformat(e.g.,purified,biotinorfluorochrome)astheprimaryantibodyandtitratedinparallel.Formulti-colorstaining,single-colorstainedcontrolsshouldbeincluded.Toidentifymarkersonanunknownornovelcelltype,positivecontrols(i.e.,cellswhichareknowntoexpresstheantigenofinterest)shouldbeincludedineachexperimentandshouldbehandledexactlyasthetestsamples.
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