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LselectinPNAd Interactions under Flow Conditions.
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Purpose

Themainpurposeoftheflowchamberassayistovisualizeandmeasureinteractionsbetweenflowingcellsexpressingagivenadhesionmoleculeontheirsurface,andtheirreceptor,eitherdirectlycoatedontheflowchamberlowerwallorexpressedonacellmonolayer.

ThepresentprotocolwillfocusontheinteractionsbetweenL-selectinexpressedonneutrophilsandPNAdcoatedontotheplasticsurface.

ForneutrophilpurificationseePurificationofhumanmononuclearcellsandneutrophils

Materials

  • PurifiedhumanPNAd(fromtonsillysates).
  • 50mMTrispH9.5,0.1%n-octyl-b-D-glucopyranosil,0.02%sodiumazide.
  • 2%Humanserumalbumin(HSA).
  • Polysterenedishes.
  • 9.5gHanks’balancedsaltsdilutedin1L20mMHepessolution.AdjustpHto7.4.

Procedure

CoatingwithPNAd

  1. DissolvepurifiedPNAdin50mMTrispH9.5,0.1%n-octyl-b-D-glucopyranosil,0.02%sodiumazidebuffer,totherequiredconcentration.
  2. Place10-30mlofPNAddilutiononthecenterofapolysterenedish.WithapermanentMarkermakeaspotontheouterfaceofthedishtolocatethecoatingareathroughouttheexperiment.
  3. Placethedishonahumidboxandincubatefor1hourat37oC.
  4. WashthespotbyaspiratingthePNAdsolutionandimmediatelyapply20-50mloftheTrisBuffer.Becarefulnottotouchthesurfaceorleaveitdry.
  5. Aspiratethewashingbufferandcovertheareawith100-500mlof2%HSAdilutedinTrisbuffer.
  6. Placetheplateintothehumidboxandincubateat37oCfor1hourtoo.n.
  7. AftergentlywashingthesurfacewithTrisbuffer,dishescanbekeptat4oCforseveralhours.

Flowchamber

  1. Assembletheflowchambertothepolysterenedish,placingthecoatedareaonthecenterofthechamber.
  2. Setthepumpconnectedtotheoutlettubeofthechamberatarefillingmodeof2ml/min.Fillthechamberwiththerunningbuffer,Hank’sbuffer,makingsurethatnoairbubblesareremainigintothechamber.Toavoidbubblegrowthinthechamber,makesurethebufferisatroomtemperatureandthatthevacuumefficientlyshieldsthechambertothedish.
  3. Startinfusionofpurifiedneutrophilsintothechamberathighrate.Oncetheneutrophilsareinthemicroscopicfield,reducetheshearto0.4dyn/cm2,andprogressivelyincreaseitdependingonthepurposeoftheexperiment.
  4. Imagescanberecordedontoavideotape,andanalyzedoff-line.

A10Xobjectiveisrecommendedforvisulizationofneutrophils.

Expectedresults

DependingonPNAdcoatingsitedensitystableorrollingortransienttethers(atlowerdensities)shouldbedetectedatshearstressesrangingbetween0.8to1.5dyn/cm2.

Nofirmadhesionshouldbeobserved.Adherentcellscouldbeduetounspecificinteractionsofneutrophilswiththesurface(strongerblockingconditionsshouldbetested)ortocellactivation(performtheexperimentimmediatelyafterneutrophilisolationandkeepneutrophilsonice).

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