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Antibodies-Online/IngoFlowEx® Kit/ABIN3072914/100 tests

Application
FlowCytometry(FACS)
Options
Bulkdiscount
Supplier
SupplierProductNo.
PurposeIngoFlowEx®KitisdesignedforquantificationofphagocyticactivityofhumangranulocytesandmonocytesbymeasuringtheingestionoffluorescentlylabeledE.colibacteriainhumanheparinizedwholebloodusingflowcytometry.
BrandIngoFlowEx®
SampleTypeBloodCells
Components
  • E.coli-FITC(Ready-to-Use)
    Onevialcontaining1mloffluorescein-labeledhumanplasmaopsonizedE.colistrainK-12insUSPension,thevialisintendedfor100reactions.
  • QuenchingSolution(Ready-to-Use)
    Onevialcontaining20mlofbufferedsolutionoftrypanblue,darkbluesolution,thevialisintendedfor200reactions.
  • 10xLysingSolution(10xconcentratedsolution)
    Onevialcontaining60mlof10xconcentratedtransparentsolutionoffixation-lysingreagent.Eachvialisintendedtoprepare600mlof1xLysingSolution,i.e.200reactions.
  • 25xWashBuffer(25xconcentratedsolution)
    Onevialcontaining80mlofconcentratedwashbuffer.Eachvialisintendedtoprepare2litersof1xWashbuffer,i.e.222reactions.
  • DNAStainingSolution(Ready-to-Use)
    Oneambervialcontaining60mlofbufferedpropidiumiodidesolution.Eachvialisintendedfor200reactions.
Materialnotincluded
  • Deionizedwater(dH2O),approximately0.6literforonekit
  • Phosphatebufferedsaline(PBS),approximately2liters
  • Suitable5mLtesttubesforbloodstaining(e.g.12x75mm)
  • Racksforthetesttubes
  • Centrifugewithsuitablerotorfor5mLtubes
  • AutomaticPipetteswithdisposabletips
  • Vortex
  • Thermalincubatororwaterbath(37°C)
  • Containerwithice(icecubesorcrushedice)suitableforplacingarackwithtubes
  • Flowcytometer-bluelaserexcitation488nm,lightemissionat525nm(FITCchannel)and617nm(PEchannel).
  • Cylindersandglassbottlesforthedilutionof25xWashBufferandof10xLysingSolution
BackgroundPhagocytosisisapartoftheinnatedefensemechanismsinwhichphagocytesingestextracellularparticulatematerial.TheABIlityofphagocytosisisacharacteristicfeatureoftheso-calledprofessionalphagocyticcells(neutrophilandeosinophilgranulocytes,monocytesandmacrophages).Theprocessincludesparticlebinding,itsingestionandsubsequentdegradation.Plasmaproteins(C3bandantibodies)assistthephagocytesinparticlerecognitionandingestionbycoveringthesurfaceofforeignparticleswithreADIlyrecognizableligands.

ThetestisbasedonmeasuringthefluorescenceofcellsthatingestedFITC-labeledE.coli.AsampleofheparinizedbloodismixedwithfluorescentE.coliandincubatedat37 °C.AcontrolreactionwithoutE.coliisperformedinparallelwitheachreactionwithE.coli.Thisnegativecontrolisusedtosetthediscriminationbounderybetweenthephagocytingandthenonphagocytingcells.Thenon-ingestedbacteriaareseparatedbyrepeatedwashes.Thefluorescenceofremainingextracellularandsurfaceboundbacteriaisquenchedwithtrypanblue,avitaldyedoesnotcrossthecellularmembrane.Samplesarethensubjectedtoerythrocytelysisandfixed.FinallythecellularDNAisstainedwithpropidiumiodide.DNAstaininghelpstodefinenucleatedcellsfromdebrisandclumpsofbacteria.BacteriausedinthetestwereopsonisedwithhumanABplasma,theresultsarenotprimarilydependentonopsonizingactivityofthetestedbloodsample.
ApplicationNotes
  • Bloodmustbecollectedintoatubecontainingheparin.AnticoagulantsEDTAandcitratenegativelyaffectsresultsoftheanalysis.
  • Bloodsamplesshouldbemeasuredwithin8hoursaftercollection.
  • Theflowcytometershouldbecalibratedonaroutinebasisusingfluorescentmicrobeadstoensurethestablesensitivityofdetectors.
  • Donotusereagentsaftertheexpirationdatestatedonviallabels.
  • Avoidcontaminationofreagents.
  • Useprotectiveglovesandfollowproceduresforhandlingpotentiallyinfectiousmaterials.
  • Avoidcontactofsampleswithskin,eyesandmucousmembranes.
SampleVolume50μL
ReagentPreparation

Preparationofreagentsbeforemeasurement

  • WashBuffer
    Dilute25xWashBufferwithPBSbeforeuse.Allowthebuffertocoolto2-8°C.Assignwiththedateofpreparationandstoreat2-8°Cforupto4weeks.
  • LysingSolution
    Dilute10xLysingSolutionwithdeionizedwater.Assignwiththedateofpreparationandstoreat2-8°Coratroomtemperatureforupto4weeks.
  • E.coli-FITC
    Beforeusethoroughlymixthecontentofthevialusingvortexmixer.

SamplePreparation

Pipettingprotocol

  • Allow1xWashBuffertocoolto2-8°C.
  • Allow1xLysingSolutiontoreachroomtemperature.
  • PlaceE.coli-FITC,QuenchingSolutionandDNAStainingSolutiononice.
  • Placearackfortesttubesonice.

Foreachbloodsamplepreparetwotubes:
  • thereactionwithE.coli
  • thecontrolreaction(withoutE.coli)
  1. Pipette50μLofbloodintothetesttubes.
  2. Placethetubesonicefor10minutes.
  3. Add10μLofE.coli-FITCtothetubeintendedtoperfomreactionwithEcoliandmixusingavortexmixer.Returnthetubetoice.
  4. Donotaddanythingintothecontroltubes.
  5. Transferthetubes(includingthecontroltubes)intoathermalincubatorsetto37°C(orawaterbathsetto37°C).
  6. Incubatefor30minutes(10minutesifusingthewaterbath).
  7. Returnthetubestoiceandincubatefor5minutes.
  8. Add100μLofQuenchingSolution(2-8°C)andmixwell.Keepthetubesonice.
  9. Add3mLof1xWashBuffer(2-8°C)andcentrifugethetubesat200gfor5min.
  10. Removethesupernatantbyaspirationintoacontainerwithanappropriatedisinfectant.Donotdisturbthepellet.
  11. Repeatwash(steps9and10).
  12. Resuspendthepelletintheresidualvolumeof1xWashbuffer.
  13. Add2mLof1xLysingSolution(roomtemperature),mixandincubatefor20minutesatroomtemperatureinthedark.
  14. Centrifugethecellsat200gfor5min.
  15. Removethesupernatantbutdonotdisturbthepellet.
  16. Add3mLprecooled1xWashbufferandcentrifugeat200gfor5min.
  17. Removesupernatantandresuspendthepelletin300μLofDNAStainingSolution(2-8°C).
  18. Incubatefor10minutesoniceinthedark.
  19. Measurewithflowcytometerwithin2hoursafteraddingtheDNAStainingSolution.

AssayProcedure

Settingupthecytometerbeforethefirstrun:(Optional)

Thecolorcompensationforspectraloverlapmaybeassessedwithperformingasetofsinglereagentstainedbloodsamples.Toperformsinglestainedreactionsfollowtheassayproceduredescribedinsection8.Useonlyonekitcomponentatatime.

  1. bloodandEcoli-FITC(omitQuenchingSolutionandDNAStainingSolution)
  2. bloodandQuenchingSolution(omitE.coli-FITCandDNAStainingSolution)
  3. bloodandDNAStainingSolution(omitE.coli-FITCandQuenchingSolution).
Usethesemeasurementstoadjustthecytometersettings.

Analysisofsamples
SetagateinPEchannelandacquireasufficientnumberofcellularPE-brightevents(atleast10,000events).Thisgateexcludesdebrisandclumpsofbacteriafromfurtheranalysis.VisualizethePEchannelgatedeventsintheside-scatter(SSC)versusforward-scatter(FSC)dot-plotandsetgatesaroundthegranulocytesandmonocytes.ThenbringthegatedsubpopulationstohistogramsinFITCchannel.Usethecontrolreactionhistogramtoplacethediscriminationline.Asinglediscriminationlinesettingisusuallyapplicableforalltestedsampleswithintherun.Calculatethepercentageofpositivecells(thisparameteriselswerecalledphagocyticactivity).

RestrictionsForResearchUseonly
Storage4°C
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