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OverviewThisassayusesasimpleapparatustostudychemotaxisofleukocytesorothermigratorycells.Theapparatusconsistsoftwomulti-wellchambersseparatedbyafiltercontainingporesofuniformsize.AsolutioncontainingachemokineorchemotacticfactorisplacedinthebottonchamberandacellsUSPensionisplacedintheupperchamber.Cellscanmigratethroughthepores,acrossthethicknessofthefilter,andtowardthesourceofchemoattractant(thelowerchamber).Cellsthatmigratedacrossthefilterandattachedtotheundersidearecounted.DataisoftenexpressedintermsofMigrationIndex:thenumberofcellsthatmigratedinresponsetoagoNISTrelativetothenumberofcellsthatmigratedrandomly,thatis,tobufferonly. Procedure Title|Overview|Procedure|Solutions|BioChemicals|Hints|PrintableVersionA.FilterPreparation(seeHint#2)1.Thisprocedureuses25x80mmpolycarbonatefilters,polyvinylpropylene(PVP)free,poresize3,5,8or10μm(PoreticsorNeuroProbes)(seeHint#3).Ifusingleukocytes,skiptoSectionB.2.Prepare3to5mlofRatTailCollagenSolution.3.PourtheRatTailCollagenSolutionintoashallowvesselandsubmergethefiltersinthissolutionwithforceps(seeHint#4andHint#5).4.Incubatefor2hrat37°C.5.RemovethefilterfromthesolutionwithforcepsandallowtodryonaKimwipe.B.CellPreparation1.Growadherentcellsto80%confluence.Forcellsgrowninsuspension,skiptostep6.2.Washcellsoncewith2to3mlper10cmdishofTrypsin/EDTASolution.Aspiratesolutionimmediately.3.Add2mlofTrypsin/EDTASolutionper10cmdish.Incubatefor2minutesatroomtemperature.4.Add1mlofCellDissociationBuffer.Incubatefor2minutesatroomtemperature.5.Add6mlofSerumContainingMedium.6.Trituratethecellsvigorouslyuntilasingle-cellsuspensionisachieved.7.Countthecells(seeProtocolID#1934).8.Pelletthecellsbycentrifugationat1000rpminatable-topcentrifuge(800-1,000Xg)for5minutes.Aspiratethemediumandresuspendthecellpelletin20mlofChemotaxisBuffer.9.Pelletthecellsasinstep8.AspiratethemediumandresuspendthecellsinChemotaxisBufferat0.5to1X106cells/ml.Maintaincellsat37°Cuntilthechemotaxischambersarereadytouse.C.PreparationofAgonistSolution(s)1.Dilutetheagonists(chemokinesorotherchemoattractants)tothedesiredconcentrationsinatleast200μlChemotaxisBuffer(seeHint#6).Remembertoincludeanegativecontrol-ChemotaxisBufferwithnochemoattractant.2.Degasthesolutionundervacuumfor10minutestominimizetheformationofairbubblesunderneaththefilterduringtheexperiment.3.Equilibratetheagonistdilutionsto37°C.D.AssaySetUp1.Theassayisperformedwitha12or48multi-wellmicRochemotaxischamberfromNeuroProbe(cat.no.AP48).Inadditiontothestepsdescribedbelow,adetailedmanualisprovidedwiththechamber.2.Washthechambersextensivelywithwateranddryundercompressedair.3.Thebottomportionofthechambercontainswells.Filleachwellwith28μlofachemoattractantdilution.Ensurethatbubblesarenotpresentandthatthemeniscusisabovethewell.4.Lowerthefilter(shinysidefacingup)ontothewells.Todothis,holdthetwoendsofthefilterwithforcepsandcarefullylowerituntilthemiddleportionofthefiltertouchestheliquidinthewells,thendroptheends.Onceyouhavedroppedthefilter,donotmoveitaround.5.Carefullyplacethegasketontopofthefilterandthenthetopportionofthechamberapparatus.6.Securetheapparatuswiththescrewsprovided.Donottightenexcessively.7.Add50μlofthecellssuspendedinChemotaxisBuffertoeachwellontheupperchamber.Toavoidcreatingbubbles,touchthePipettetiptothesideofthechamber(don"ttouchthefilter)andexpelthecellsallatonce.8.Placethechamberonalevelsurfaceina37°C,5%CO2incubator.Incubatefor30minutesto3hours(seeHint#7).E.Fixing,StainingandCountingofMigratedCells1.Holdtheassemblytogetherwithyourhandsandremovethescrews.Turntheentireassemblyupsidedown,andpullthecellsourcechamber(nowonthebottom)andgasketawayfromtherestoftheassembly.Thefiltershouldbestucktothegasket.Thesidenowfacingupcontainsthemigratedcells.2.Grabtheedgesofthefilterwithforcepsandliftitoffofthegasket.Washtheupperside(nowfacingdown)ofthefiltertoremoveexcesscellsasdescribedinthemanualfromNeuroProbe.3.AllsolutionsforfixingandstainingthecellsareprovidedintheDiff-QuickStainSet(FisherScientific).Useforcepstotransferthefilterbetweensolutions(seeHint#4).4.SubmergetheentirefilterinFixSolutionfor4minutes.5.TransferthefiltertoSolution#1andincubatefor5minutes.6.TransferthefiltertoSolution#2andincubatefor5minutes.7.TransferthefiltertoddH2Otoremoveexcessstain.8.Transferthefilterontoaglassslidewiththemigratedcellsfacingup;allowittodry.9.Useamicroscopesetat200xmagnificationtocountthenumberofstainedcellsin3to5fields.Foreachagonistcondition,amigrationindexiscalculatedbydividingthenumberofcellsthatmigratedinresponsetotheagonistbythenumberofcellsthatmigratedinresponsetoChemotaxisBufferalone(seeHint#8). Solutions Title|Overview|Procedure|Solutions|BioChemicals|Hints|PrintableVersionRPMI1640medium(Gibco) 
Diff-QuickStainSet(FisherScientific) ChemotaxisBuffer 25mMHepes,pH7.41%(v/w)Endotoxin-freeBovineSerumAlbuminprepareinRPMI1640medium RatTailCollagenSolution(seeHint#1) 50μg/mlrattailcollagen,Type1(seeProtocolID#1960)0.2MAceticAcidprepareinRPMI1640medium SerumContainingMedium 10%(v/v)serum(suchasfetalbovineserum)inRPMI1640medium CellDissociationBuffer(GIBCO) Mg2+andCa2+free Trypsin/EDTASolution(GIBCO) 0.5g/literTrypsin0.2g/literEDTA BioReagentsandChemicals Title|Overview|Procedure|Solutions|BioChemicals|Hints|PrintableVersionCellDissociationBufferTrypsinEndotoxin-freeBovineSerumAlbuminRPMI1640RatTailCollagenEDTAAceticAcidFetalBovineSerumHEPES ProtocolHints Title|Overview|Procedure|Solutions|BioChemicals|Hints|PrintableVersion1.Collagenshouldbestoredunderacidicconditions;itisinsolubleatneutralpH.2.Whenusingimmunecellssuchasneutrophils,donotcoatthefilter.Thisprotocolcanbeusedtostudythemigrationofadherentcells,suchasHEK-293cells.CoatingthefilterswithRatTailCollagenwasfoundtobeimportantforthiscellline.Forothercelllines,determineempiricallywhethercoatingisnecessary.3.Theporesizeofthefilterdependsonthetypeofcellbeingused.ForHumanneutrophils,usefilterswith5μmpores;forMouseneutrophils,usefilterswith8μmpores.HEK-293cellsusefilterswith10μmpores.4.Nevertouchthefilterswithbarehands.5.Thefiltershaveatendencytofloatonthesolutionanddryoutduringthecoatingprocedure.Toavoidthis,rockthesolutionevery10-15minutes.6.Chemokinesaregenerallyusedinaconcentrationrangefrom0.01nMto100nM.However,theoptimalconcentrationrangeforaspecificchemokineorchemotacticfactorvaries.Theliteratureshouldbeconsultedfortheappropriateconcentrationrangeforafactorofinterest.7.Theincubationtimedependsonthetypeofcellbeingused.Immunecellssuchasneutrophilsrequireshorterincubationtimes(30minutes),whileadherentculturedcellssuchasHEK-293cellsrequirelongerincubationtime(3hours).8.Achecker-boardanalysisdistinguisheschemotaxis(thedirectedmigrationofcellsinresponsetoagrADIentofchemotacticfactor)fromchemokinesis(increaseinthespeedorfrequencyofmovementinducedbyafactor).Thiscanbeachievedwiththesameapparatusandprocedureoutlinedhereexceptthatmigrationismeasuredundernon-gradientconditions,(i.e.,withanequalconcentrationofchemoattractantintheupperandlowerchambers).ThisassayvariationwasoriginallydescribedbyZigmondetal.(seeCitation#1). 1.Zigmond,S.H.,andHirsch,J.G.Leukocytelocomotionandchemotaxis.Newmethodsforevaluation,anddemonstrationofacell-derivedchemotacticfactorJ.Exp.Med1973;137:387-410.