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. Placethedishonahumidboxandincubatefor1hourat37^oC.
4. WashthespotbyaspiratingthePNAdsolutionandimmediatelyapply20-50mloftheTrisBuffer.Becarefulnottotouchthesurfaceorleaveitdry.
5. Aspiratethewashingbufferandcovertheareawith100-500mlof2%HSAdilutedinTrisbuffer.
6. Placetheplateintothehumidboxandincubateat37^oCfor1hourtoo.n.
7. AftergentlywashingthesurfacewithTrisbuffer,dishescanbekeptat4^oCforseveralhours.

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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