LselectinPNAd Interactions under Flow Conditions._实验方法...
来自 : 蚂蚁淘
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
- DissolvepurifiedPNAdin50mMTrispH9.5,0.1%n-octyl-b-D-glucopyranosil,0.02%sodiumazidebuffer,totherequiredconcentration.
- Place10-30mlofPNAddilutiononthecenterofapolysterenedish.WithapermanentMarkermakeaspotontheouterfaceofthedishtolocatethecoatingareathroughouttheexperiment.
- Placethedishonahumidboxandincubatefor1hourat37oC.
- WashthespotbyaspiratingthePNAdsolutionandimmediatelyapply20-50mloftheTrisBuffer.Becarefulnottotouchthesurfaceorleaveitdry.
- Aspiratethewashingbufferandcovertheareawith100-500mlof2%HSAdilutedinTrisbuffer.
- Placetheplateintothehumidboxandincubateat37oCfor1hourtoo.n.
- AftergentlywashingthesurfacewithTrisbuffer,dishescanbekeptat4oCforseveralhours.
Flowchamber
- Assembletheflowchambertothepolysterenedish,placingthecoatedareaonthecenterofthechamber.
- Setthepumpconnectedtotheoutlettubeofthechamberatarefillingmodeof2ml/min.Fillthechamberwiththerunningbuffer,Hank’sbuffer,makingsurethatnoairbubblesareremainigintothechamber.Toavoidbubblegrowthinthechamber,makesurethebufferisatroomtemperatureandthatthevacuumefficientlyshieldsthechambertothedish.
- Startinfusionofpurifiedneutrophilsintothechamberathighrate.Oncetheneutrophilsareinthemicroscopicfield,reducetheshearto0.4dyn/cm2,andprogressivelyincreaseitdependingonthepurposeoftheexperiment.
- Imagescanberecordedontoavideotape,andanalyzedoff-line.
A10Xobjectiveisrecommendedforvisulizationofneutrophils.
Expectedresults
DependingonPNAdcoatingsitedensitystableorrollingortransienttethers(atlowerdensities)shouldbedetectedatshearstressesrangingbetween0.8to1.5dyn/cm2.
Nofirmadhesionshouldbeobserved.Adherentcellscouldbeduetounspecificinteractionsofneutrophilswiththesurface(strongerblockingconditionsshouldbetested)ortocellactivation(performtheexperimentimmediatelyafterneutrophilisolationandkeepneutrophilsonice).
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