Purpose

Thepurposeofachemotaxisassayistodeterminewhetheryourproteinorsmallmoleculeofinteresthaschemotacticactivityonaspecificcelltype.ChemotaxisistheABIlityofaproteintodirectthemigrationofaspecificcell.ThisassayisbasedonthepremiseofcreatingagrADIentofthechemotacticagentandallowingcellstomigratethroughamembranetowardsthechemotacticagent.Iftheagentisnotchemotacticforthecell,thenthemajorityofthecellswillremainonthemembrane.Iftheagentischemotactic,thenthecellswillmigratethroughthemembraneandsettleonthebottomofthewellofthechemotaxisplate.

Mediaandcultureconditions:RPMI164010%FBS(notheat-inactivated)5mg/mlgentamycin(1:100ofthelabstock)

Materials

• Neuroprobe96-wellchemotaxisplate5mmporesizeCAT#ChemoTx#101-5
• Favoritechemokineofchoice(100mg/ml)
• Fixationmedia:2.5%FBS,3%formaldehyde,inL15media
• SmallglassFACStubes
• 96-wellroundbottomplate
• 12wellor8wellmulti-channelPipette
• Multi-channelpipettedishtoholdthefixationmedia

Procedure

1. Countcells.Add10mlofcellsto90mloftrypanblueinonewellofa96-wellplate.Take10mlandloadontoahemacytometer.Counttheouter4squaresandthemiddlesquare.Dividethisnumberby5togetthenumberofcellspersquare.Onesquareisequalto1x10⁴cells/mlofa1:10dilution.Therefore,multiplythenumberby10andthisgivesyouthenumberofcells/mlofculture.

   Example:thereare25cellsin5squares=5cells/square.Thisistheequivalentof5x10⁴cells/mlofa1:10dilution.Multiply5x10⁴time10=5x10⁵cells/mloftheoriginalculture.

2. Spinthecellsdownat1400RPM,4minutes.
3. RemovethesupernatantandresUSPendthecellto1x10⁷cells/mlinfreshculturemedia.Leavethecellsoniceuntilyouarereadytousethem.
4. Make1:10serialdilutionsofchemokineforthebottomchamberofthechemotaxisplate.Youwillneed30mlperwell.Igenerallyusetheplateincolumnsratherthanrows.Therefore,Iusuallymake7serialdilutions(10mg/mldownto0.01ng/ml)andusethelastwellforabuffercontrol.
5. Loadthechemotaxisplate.Thechemotaxisplateisa96-wellplatefromNeuroprobeconsistingofabottomplate,amembrane,andacover.Youneedtoaddenoughchemokinetothebottomwellsothatitlooksasifitisalmostoverthetopofthewell.Whenyouplacethemembraneontopofthewells,thisslightexcessofmediawillformasealbetweenthemembraneandplate.Dependingonyourspecificpipette,thisvolumeamountsto28-30ml.Makesureyouaddthemediasuchthattherearenosmallbubblesformed.Bubbleswillinhibitmigrationofcellsacrossthemembrane.
6. Inaddition,toonecolumnoftheplateyouwillneedtomakeapercentinputstandardcurve.Thisisforlateranalysistodeterminethepercentofchemotaxisforyoursamples.Add25mlofculturemediatoeachwellofonecolumn.Take25mlofthecellsuspensionandpipetupanddowninthefirstwellofthecolumn.Thiswellisnowtheequivalentof50%ofthecellinput.Continuetodilute1:1allthewaydownthecolumnuntilyoureachthelastwell.These1:1dilutionsresultinastandardcurvewiththefollowingpercentinputvalues:50,25,12.5,6.25,3.13,1.56,0.78,and0.39.Anexampleofatypicalchemotaxisassayisattachedtothisprotocol.
7. Placethemembraneoverthebottomplate.Thereare4pegs(oneoneachcorner)thatthemembraneslidesonto.Makesurethemembraneisfirmlyseatedonthepegs.Youcantellbecauseallofthewellswithchemokineshouldformasealwiththemembrane(thewellwilllookdarkerwhensealedproperly).
8. Add25mlofthecellsuspensiontothetopofeachwell.Thereisahydrophobiccoatingsurroundingthemembraneforeachwellwhichallowsthecelldroplettoformaroundsphere.Besurethatyoudon"ttouchthemembranewiththetipofyourpipette.
9. Putthecoveroverthecelldropletsandcarefullyplaceina37^oCtissuecultureincubatorfor2hoursand15minutes.
10. Afterchemotaxis,gentlyremovetheplatefromtheincubator.Pryoffthemembraneusingthethinendofametalspatula.Itisimportanttoremovethemembraneinonefluidmotion.Don"tmovethemembraneupanddownduringtheprocessofremovalbecausethiscausesasmallamountofsuctionresultingindrawingsomecellsthroughthemembrane.Youcantellifthishashappenedbylookingatthewellsafterremovalofthemembrane.Iftherearecellsfloatingratherthanlyingflatonthebottom,thenyouhaveaccidentallydrawnsomecellsthroughthemembrane.
11. Usingamulti-channelpipette,add50mloffixationmediatoeachwellandpipetupanddown.Transfertheentirevolume(~80ml)toaglassFACStubewhichhasbeenplacedina96-wellroundbottomplate.
12. Countsamplesfor15secondseachontheFACScan.Usethepercentinputwellsasastandardcurvetodeterminethepercentchemotaxisforeachsample.

ExpectedResults

Dependingonthechemokine,youshouldexpectabellshapedcurvewiththemaximalchemotaxisfrom1-10ng/mlanddecreasingchemotaxisathighconcentrations(1-10mg/ml).

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