ThisprocedureallowsdetectionofspecificcelladhesiontoglycolipidsresolvedonTLCplates.Thestepsinclude:(1)HPTLCofglycoconjugates,(2)coatingthechromatogramwithpolymer,(3)preblockingthechromatogramtoreducenon-specificcellbinding,(4)mountingthechromatogramintheacrylicchamber,(5)addinglabeledcells,(6)incubating,(7)removalofnon-adherentcellsbycentrifugation,and(8)fixationofadherentcellsfordetection.Adherentcellsneverpassthroughanair/liquidinterfacepriortofixation,allowingdetectionofspecificcelladhesionevenifadhesivestrengthislow.

Materials:

Protocol:

1.Prescored10x10cmHPTLCplatesarebrokenalongonescoretogeneratea5cmx10cmplate.GlycolipidsareappliedandTLC"sdevelopedusingstandardprocedures.Chromatogramsarethendriedthoroughly(50°C,1hr.)andallowedtocoolpriortopolymercoating.

2.Polyisobutylmethacrylate(seepg.99)ispreparedasa10%(w/v)solutioninchloroformwhichisdiluted1/100intorapidlystirringhexanetogeneratea1mg/mlstockwhichcanbestoredatroomtemperature.Thesolutionisfurtherdilutedimmediatelybeforeusewithadditionalhexanetoafinalconcentrationof2-200µg/mldependingonthecelltype.DevelopedanddriedTLCplatesaredippedsequentiallyfor30sec.eachinhexane,theninPIBMsolution,thenallowedtoairdry.

3.ThePIBM-coatedplateisimmersedinmediumuntilwet,thentransferredintomediumcontainingblockingagent0.5mg/mlBSA)for30min.,thenintomediumwithoutblocker.

4.Thepreblockedplateisplacedsorbentsidedownonthespacersoftheadhesionchamber.MediumisPipettedontothebackoftheTLCplateandtherubbergasketappliedsuchthatairbubblesareexcludedfromtheplate-gasketcontactarea.Thegasketandacryliccoverarefixedinplacewiththescrewssupplied.

5.AnymediumtrappedinthechamberisdecantedthroughtheluerlockopeningsattheendoftheChamber.Cellsuspension(10⁵-10⁶cells/ml)inmediumcontaining1-5mg/mlBSAispipettedintooneoftheopeningsuntilthechamberisfullandfreeofairbubbles(chambercapacity15ml).Theendopeningsaresealedwithluerlocks,andthesealedboxisplacedwithTLCsorbentsideupsuchthatthecellssettleontotheplatesurface.Theboxisplacedonamicroplatecentrifugecarrierandcentrifugedat30xg,for3min.tobringcellsintocontactwiththeTLCplatesurface.

6.Thechamberisimmersed(sorbentsideup)inawaterbathtypicallyat37°Cfor30min.toallowadhesiontooccur.

7.Thechamberisremovedfromthewaterbath,inverted,placed(sorbentsidedown)inacentrifugecarrier,andcentrifuged(250-500xg,10min.,4°Ctoremovenon-adherentcellsfromtheTLCplatesurface.

8.Whilestillinverted,thechamberisfullyimmersedinanice-coldtubofPBS,thecoverandgasketremoved,andtheTLCplateisgentlyremoved,righted(whileimmersed)andplacedinthetransferdish.Thefluid-filleddishisthenusedtotransfertheTLCplatethroughtwo150mmPetridishesfilledwithPBStowash.

9.Stillinthetransferdish,theplateisplacedinanempty150mmPetridishandgentlyoverlaidwith100-150mlof2%glutaraldehydeinPBSatambienttemperaturefor30min.

10.TheTLCplateisremovedfromthetransferdish,washedbyimmersioninPBSandplacedverticallytodry.

11.ThethoroughlydriedplateisplacedonX-rayfilmandexposed.Afterexposurefor1-24hr.thefilmisprocessedtodetectthepositionofadherentcells.

Note:Ifglycolipidsarepresentatsufficientconcentration(>500pmol/lane)theymaybedetectedontheTLCplatesafterautoradiographybyincubatingtheplateinCoomassieBlue(0.3mg/ml)inmethanol:water(1:5)for30min.followedbydestaininginthesamesolventmixturefor5min.CoomassieBluetreatmentalsostainsadherentcellswhichcanbevisualizedbylight-fieldmicroscopyatthesitesofautoradiographicbandstoconfirmthattheradiolabeldetectedcorrespondstointactcelladhesion.

References:

(1)Swank-Hill,P.,Needham,L.K.,andSchnaar,R.L.(1987)Carbohydrate-specificcelladhesiondirectlytoglycosphingolipidsseparatedonthin-layerchromatographyplates.AnalyticalBiochemistry,163:27-35

(2)Stroud,M.R.,Handa,K.,Ito,K.,Saylan,M.E.K.,Fang,H.,Levery,S.B.,Hakomori,S.,Reinhold,B.B.,andV.N.Reinhold(1995)Myeloglycan,aseriesofE-selectin-bindingpolylactosaminolipidsfoundinnormalhumanleukocytesandmyelocyticleukeminHL-60cells.BiochemicalandBiophysicalResearchCommunications,209:777-787.