ModFit Tutorial –Cell Cycle Analysis Software · sure G2M position looks good. Let the S -phase...
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1. Start by choosing all the files to progress in batch mode. ModFit Tutorial – Cell Cycle Analysis Software NYULMC DART Cytometry and Cell Sorting ModFit Tutorial, 12/27/2016
Transcript of ModFit Tutorial –Cell Cycle Analysis Software · sure G2M position looks good. Let the S -phase...
1.Startbychoosingallthefilestoprogressinbatchmode.
ModFit Tutorial– CellCycleAnalysisSoftware
NYULMC DART Cytometry and Cell Sorting ModFit Tutorial, 12/27/2016
2.Setthebatchcontrolstonotanalyzebutwithfileprompton.Thisallowsyoutoopen filesandalterthegatesforanalysisbutwillnot trytofitthedata.This
letsyoujustlookattheunfittedhistogramsfirst.
3.Choosetheparameterforanalysis(PI-A).
4.Selectthefirsttwogatesandcreatethemwideopen (tocoverthewholeplot).Thisissoyoucanseethegeneralpositionofpopulationsandhowtheychangebetweenfiles.Wecanchangethesegateslaterif
needed.
5.WecannowsetuptheSynchWizard.
It’sgood tostartbylettingtheG0G1peakpositionandSDadjustautomatically.
G2Mposition looksgoodsononeedtochangethis.
It’salsogoodtomaintaintheS-phasedefaultoptions tostart,whicharethesimplermodels.
Underthe‘Other’tabyoucanstartwithhavingtheDebrisandAggregateschecked.
UsuallythereisnoreasontochangeModFit’s defaultssothisiswhywewanttostartwiththemfirst.
6.Thisfirstfileproducesthefollowingerror,tellingusthattheG0G1positionhasshiftedbeyondtheexpectedamountandwantsyoutotellitwhattodonext.Ifthenewvaluewereclosetotheoriginalposition(around50orso),thenyoucanletitmovetothenewposition.However,a
positionof-1doesnotmakesense,sowewillchoosetolocktheG0G1position.Oncethemodelislocked,itwillstaylockeduntilyouchangeit.
• Insomeinstancesyouwillseesubsequent filesthathaveaverydistinctG0G1peakthathasshifted totherightofleftfromthislockedpositionsoyoucanclickthemodeleditbuttontobringup theeditdialogagain.
• Ifitappearstohaveshiftedbacktotheoriginal,expectedlocationthenyoucanchoosetore-selecttheAdjustautomaticallymode.Otherwise,youcanjustdragthearrowoverthenewG0G1position.
6.IfthenormalcontrolforsynchexperimentshasawideCV,thenModFit willbeunabletogothroughtheSynchWizardproperlyandyouwillgethighRCSvaluesback.
Inthiscaseyouwillhavetoproceedmanually.
VerityalsomentionedthatiftherenoblacktrianglespresentunderneaththeG0G1andG2MpeaksthenthisisanotherindicationofCV’sthataretoohightouseSynchWizardwith.
6.YouwillhavetoexitoutofSynchWizardandclickontheMod icon.FromhereyoucantellModFit tolook forG2Masavisiblepeak.
KeepAutoaggregatesanddebrison.
RCSvaluewentfrom24.914downto1.958!
SynchWizard
1.Openallthefilesinabatch,makethegateswideopen,openSynchWizard.
2.SetthelocationforG0G1andletitadjustthepositionautomatically(thedefault).CheckandmakesureG2Mpositionlooksgood.LettheS-phaseoptionsremainthedefaultandchecktheauto-aggregateanddebrisboxes.ClickAnalyze.
3.Sometimesthesynchwizarddoesn’tdoagood jobatcalculatingaggregatesautomatically.
.
Youcanfixthisbyturning offtheauto-aggregatesandmakingyourdoublet-discriminator gatetightaround thesinglecells.
Bydoing this,itbrings theRCSvaluedownto2.601.
4.Whengoing through thesubsequent filesinyourbatchanalysisyoucaneditthebatchproperties to‘Fitwithcurrentmodel’ soyoudonothavetoclick
analyzeeverytime.YoucanalsounchecktheDatabaseandFilepromptsowhenyouswitchthrough thefilesyouarenotprompted tochoosetheparameterand
gateseverytime.
