Thewoundhealingassayallowstheresearchertostudycellmigrationandcellinteractions.Insomecasesalsosinglecellmigrationcanbeanalyzed.ThisassaycanbeimagedusingNikonmicroscope3ortheOlympusCell^R/Scan^Rsystem.NormallyNikonmicroscope3isusedbutifthereisdemandwecanalsowriteaprotocolfortheOlympussystem.AnautomationisavailableforNikonmicroscope3toimagemultiplepositionsinamultiwellplate.Atthemomentwecanimage60positions.Normallyweusea10xPhasecontrastobjective,butrecentlywepurchaseda4xobjectivethatalsocanbeused.CO2isavailableoryoucanaddsomeHEPEStoyourmedium.Don"tforgettopre-heatthemicroscopesystemforseveralhoursorovernightbeforeyoustarttheexperiment.

WoundimagesafterT=0h,T=12handT=23h

Protocol

1.Seedyourcellsinmulti-wellplatesandcultureuntilconfluent.Wenormallyuse6wellplates,butaplatewithalargernumberofwellsispossIBLe.Ifyouusedifferenttreatmentsordifferentmutantsyoumighthavetoadapttheseedingdensity.Itisimportantthatalltheculturesareconfluentatthestartoftheexperiment.2.Usinga(yellow)Pipettetipmakeastraightscratch,simulatingawound.Oftenwemakeascratchkeepingthepipettetipunderanangleofaround30degreestokeepthescratchwidthlimited.Thisallowsimagingofbothwoundedgesusingthe10xobjective.3.CheckthereisenoughmemoryavailableontheHD;selectMaCintoshHD,goto"File"--->"Getinfo".Theamountyouneeddependsonthenumberofpositionsyouimageandthenumberoftimepoints.For18positionsand144timepoints7.2GBisenough.4.Starttheautomation"BF-timeseries-focusdrive".5.Doubleclick"Saveimagedocument"(nexttoyellowbox)andselectthesuBDirectoryyouwanttosaveyourfilesin.6.Click"run"tostartrunningtheautomation.Youwillbeaskedafewquestionsaboutthenumberoffilesetc.Thenumberoftimepointsyoucansaveinonegodependsonthetimeintervalsbetweentimepointsandthetotalnumberoftimepoints!MaximalnumberofpositionsonNikon3is60.7.Selectthepositionsyouwanttoimage.Weoftentake3imagesperwell.Thefirstimageyoucanfocususingthefocusknobonthemicroscope.AfterthatUSEONLYTHEFOCUSKNOBONTHEJOYSTICKUNITtofocusyourimage!!8.Whenallpositionsaresetthesystemwillstartimaging.

Analyzeyourdata

Separatethedifferentpositionsstoredinsinglefilesinonefileperpositionusingtheautomation"separate-timeseries"andsaveas*.tiffiles.Don"tforgettoselectthedirectoryyourfilesarestoredandtoselectadirectorytosavethenewfilesaswellastochangethefileextensionofthenewfilesfrom*.lifto*.tif.Opena*.tiffileinImageJFollowthenextsteps:-Process---->FindEdges-Process---->Sharpen

-Image---->Adjust---->Threshold;SelectBlack/White.Uppersliderto0(left).Setlowersliderinsuchawaythatitisclearwherethecellsare.Youmightwanttocheckatimepointwherethewoundispartiallyclosedtoseeifthedifferencebetweencellsandwoundarestillvisible.

-Process---->FindEdges-Image---->LookupTables---->InvertLUT

-AnalyzeParticles.Size:selectanumberthatmakesenseifyoulookatatimepointwherethewoundispartiallyclosed(oftenavaluebetween100000and50000000;dependentonimagesize).Circularity:0.00-1.00.Show:Outlines.Flag:Summarize.-SaveboththeSummarizeddatafileandthefilewithoutlines.Thedatafilewillshowthepercentageofwoundareainyourimageoneverytimepoint.

Outlineofthewound

-ThedatafilecanbefurtheranalyzedusingExcel.-Theoutlinefilecanbeusedtoanalyzeifthewoundareafoundcorrespondswiththerealwoundbymergingthisfilewiththeoriginal*.tiffileusingImage---->Color---->RGBMerge(orusingthePlugin"RGBGrayMerge").Ifnecessaryrepeattheanalysiswithadifferentsettingatthe"Threshold"or"AnalyzeParticles".

Mergedimagetocheckaccuracyoftheanalysis.