Download - Orflo Application Note 8/2017 · Orflo’s newest, “Next Generation Flow Cytometer”, the Moxi GO II (Figure 1), is ideally and uniquely suited to enabling yeast monitoring in

O r f l o A p p l i c a t i o n N o t e 8 / 2 0 1 7

MonitoringYeastCounts,Viability,andMetabolicActivityinBrewingwithOrflo’sMoxiGOII–NextGenerationFlowCytometer

Introduction/BackgroundHistoryThe documented history of brewing datesback over 5,000 years and is generallythought to have transpired for severalthousand more years before that.1,2Regardless of the exact inception time,brewing has grown into a global industrywithastaggeringestimatedmarketvalueof$530billion(USD,2016).3 Despiteits longhistory,theprocessofmakingbeerremainsahighlynuancedoneinitsgoalofachievinga consistent result from a deceptivelycomplex biological system. The techniquerequires the management of numerousvariables related to environmentalconditions, ingredients, and generalrecipes/protocols. Not surprisingly, giventhesizeofthemarketandcomplexityoftheprocess, over 440,0004 scholarly articleshavebeendedicatedtobrewing inordertobetter understand the contributing factors with the overall goal of improving both quality andconsistencyoftheendresult.ImportanceofYeastCounts/ViabilityAtthecoreofthebrewingprocessistheconversionofsugarintoalchoholbyyeast. Beyondtheinitialselectionoftheyeaststrain,theunderstandingoftheshiftingcharacteristicsoftheyeastinthe wort, relative to the constantly-changing environmental conditions, is critical. At a bareminimum, brewers need to maintain proper concentrations of yeast throughout the process byadding,or“pitching”,yeastatvarioustimepoints.Ensuringthecorrectpitchingofviableyeastcellsinto thewortcanhavepronouncedeffects,withunder-pitchingresulting in longer fermentations(andcostsassociatedwiththelossintimeandthroughput)andover-pitchingresultinyeastdeath,filtration issues, and taste issues.5 The simplest approach to pitching ignores yeast viability bysimplyaddingafixedamount(weightorvolume)ofyeastslurryperliterofbrew.However,asidefromthesmallestcraft/homebrewers,mostbrewershavecometorealizetheimportanceofexactyeastcellcountsandviabilitymeasurementsintheachievementofbatch-to-batchrepeatiblity.YeastCellCountingandViabilityTechnologyOnecommonlyusedapproachtocountingyeastcellsinvolvestheuseofamicroscopewithahemocytometer,aglasschamberwithapreciselycalibratedfluidicvolumetricregions.Thehemocytometerallowsfortheconversionofmanual/visualcountsintocellconcentrationsandthedeterminationofcellviabilitythroughstaining,typicallyusingmethyleneblue.Hemocytometersinbrewinggainedtractionduetotherelativelylowinitialcost.But,theapproachisnotoriouslyinaccurateandhighlyvariableinitsoutputwithissuessuchas:variableuserinterpretationandcountconsistencydiscrepancies,chamberloadingbiases,focusing-inducedvariability,andstatisticallysmallsamplesizes.Recently,attemptshavebeenmadetoautomatethatprocessthroughsoftwareintheformof“imagingsystem”counters.However,thosesystemsarefundamentallyflawedinthattheyinheritedtheweaknessesoftheunderlyinghemocytometerapproachandhaveevenexascerbatedthemastheyattempttorigidlyapplyautomatedrulesto

Figure 1 – Orflo’sMoxi GO II –Next Generation FlowCytometer. Image showsuserloadingasampleintothetwo-testdisposableflowcell. TheMoxiGOIIisaconfigurable flow cytometer with a 488nm laser and up to two fluorescencerecording channels (2 PMT’s with emission filters at 525/45nm filter and561nm/LP,respectively).



analyzingcomplex2Dimagesofcells.Specifically,thesmallsizeoftheyeastchallengestheresolutionoftheimagingsystemsoftwareas“debris”particlesinthewortareinvariablyrecognizedascells,introducingerrorandvariabilitytothemeasurements.BrewerswithlargerbudgetsoftenresorttousingCoulterCounters(e.g.CoulterZ2)topreciselymeasuretheiryeastcounts.Thosesystemshavelongbeenestablishedasthe“goldstandard”inparticlecountsandsizing,providinghighly-accurateandrepeatiblecountsthroughtheelectricalmeasurementoftheexactsize(andconcentrations)oftensofthousandsofcellsinamatterofseconds.Theaccuracyofthesizinginformation,inparticular,allowsfortrackingoftheyeastsizesthroughoutthebrewinglifecycle,afactorwithdemonstratedimportanceinthebrewingprocess.6However,themainchallengewithCoulterCountershadbeentheabsenceofviabilityinformationonthesampleand,untiltherelativelyrecentintroductionoftheOrfloMoxiZsystem,thehighcost.Finally,atthehighestendofthedataqualityspectrum,flowcytometersarebeginningtoemergeasanewgoldstandardinyeastcountingandviabilityforcontrolofbrewingprocesses.5However,historically,theextraordinarilyhighcostsandcomplexityofthesesystemshavemadethematoolforonlytheglobalbrewersoracademics.

EsteraseActivityasaMeasureofYeastHealth/StressTheviabilitymethodsdescribedaboveareallbasedonthesimpleprincipleofcellmembraneexclusion.Briefly,ascellsdie,ruptures/holesforminthemembranethatallowfortheentryoflargermoleculesthatordinarilywouldbeblocked.Inthismanner,viabilitydyessuchasmethyleneblueandpropidiumiodide(PI)areexcludedfromtheinteriorof“live”cellsandallowedtoenter“dead”cells.Theresultisthatthedeadcellsaccumulatethedyesandareeithershadedwiththedye’scolor(methyleneblue)orbrightlyfluoresce(PI).Thisistheapproachtraditionalusedbybrewerstoassessthe“viability”stateofyeast.However,theexclusionofthedyedoesnotreflectthemetabolic(activity)stateofthecellwhichiscrucialtounderstandingwhetherthecellisactivelyconvertingsugartoalchoholorifitisstressed/dormant.And,thecell“viability”(asdefinedbypermeability)isanend-stagemarker,notprovidingbrewerstheopportunitytointerveneinadjustingtheenvironmenttoprevent(oftenwidescale)cellloss.Asasupplementtomembraneintegrity,onecommonmeasurementofcellmetabolism(oftenreferredtoas“vitality”)istousemolecules(e.g.5-carboxyfluoresceindiacetate,acetoxymethylesterorCFDA-AM)thatloadintoallcellsandareconvertedintofluorescentdyesbytheactionofesteraseenzymes,anenzymethathasbeenfoundinalmostallcells.Inthisway,theactivityofacellcanbequantitativelymonitoredwithhighercellularfluorescencereflectinghighercellmetabolisms.Adropinesteraseactivitycanserveasapotentialsignaltobrewerstoadjustoperatingconditions.And,beyondthat,esteraseactivityalsoholdsparticularsignifcanceinbrewingbeyondjustmetabolicactivity.Specifically,esterasehasbeenshowntobeimportantinhelpingtobreakdowntheesters,thatcannegativelyimpacttaste,inhighgravitybrewing.7,8Whilemicroscopicapproachescanbeappliedtoesterasevisualization,forproperquantitificationofthefluorecenceandstatisticalanalysis,flowcytometryistherequiredtechniqueforthistypeofanalysis.

Orflo’sMoxiGOII-CombinedFlowCytometerandCoulter-PrincipleCounterOrflo’snewest,“NextGenerationFlowCytometer”,theMoxiGOII(Figure1),isideallyanduniquelysuitedtoenablingyeastmonitoring inbrewing. TheMoxiGOII is theonlysystemthatcombinestheprecisionofCoulterPrinciplebasedcellsizingandcountswiththeflexibilityofflowcytometryforviabilityandmoreadvancedbrewinganalysis.ThesystemcombinestheCoulterPrinciple,therecognized gold standard for precise cell sizing and counts, with simultaneous fluorescentmeasurements using a 488nm laser, coupled with two PMT detection channels, one filtered todetectat525/45nmandtheotherat561nm/LP.Thisfluorescenceconfigurationisidealformanyofthemostcommonfluorophoresincludingpropidiumiodide(PIforviability),CFDA-AM(esteraseactivity), and tetramethylrhodamine (TMRE,mitochondrial potential). TheMoxi GO II utilizes adisposable flow-cell architecture, does not requirewarm-up, runs test in under 10 seconds, and



doesnotrequirecleaning/shutdownprocedures.Itslowcostandverysmallfootprintmakeiteasyforbrewers toacquireone for theirbreweryandplace it convenientlynear the tanks for instantfeedback. In this way, the system can allow brewers to very easily track count, viability, andadvancedhealthdataovertime.Furthermore,thetouchscreeninterfaceandOSweredesignedtobe highly intuitive for users with no required flow cytometry experience. In this app note, wepresentdatafortheMoxiGOIIsystemasitwasappliedtomeasuringtheviability,esteraseactivity,andmitochondrialpotentialforanactivebrewatalocalSanDiegocraftbrewery.ExampleData–ResultsandDiscussionThegeneralcountandsizingperformanceofthetechnologyunderlyingtheMoxiGOII(comparedto higher endCoulter Counters9,10aswell as viability performance versus an imaging systemandmore expensive flow cytometer11) had been established in prior publications. In the data thatfollows,wefocusonthespecificapplicationoftheMoxiGOIItoanalyzingyeastsamplesfromanactual brewing environment. Figure 2 shows example counts and viability (PI-basedmembranepermeability) output for San Diego Super Yeast (WLP090,White Labs) pulled from a local craftbrewerybrighttank.Figure2a.showstherepresentativeMoxiGOIIviabilityoutputwithascatterplotdisplayofPI fluorescenceversusCoulterPrinciplecellsizing. In thecourseofa~10secondtest, thesystemclearlydisplaysthesampleviability(Figure2a,yellowbox,94.0%)aswellasthecounts and precise sizing information (black boxes below the scatter plot). To highlight thecapabilityofthesystemtodiscriminateliveversusdeadcells,halfofthesamplewasintentionallyheat-killed and processed by the system. While the live and heat-killed samples were runseparately, Figure 2b and 2c highlight the capability of the user to use the system to easilyoverlay/compare twosavedtestresults. Specifically,Figure2ashowstheuser-generatedsystemscreenshotsoftheoverlayofthetraditionalscatterplotoutput(PIfluorescencevs.CoulterSizing)forthetwosamples:Healthy(graydots)andheat-killed(coloreddots)yeast.Thisoverlayformatclearlyshows thedramatic increase in fluorescenceof theheat-killed(dead,coloredscatterplot)yeastsampleasthePIentersthecell,bindstheDNA,andincreasesfluorescence(~40xincrease)–clearlydifferentiatinglivefromdeadcells.ThatincreaseinfluorescenceisalsoreadilyvisualizedinFigure2bthatprovidesthetestcompare/overlayforthePIfluorescencehistograms(graycurve=

Figure2–a.)RepresentativeMoxiGOIIviability(PI)scatterplot(PIfluorescencevs.Coultersizing)outputforalive/healthybrighttank yeast sample. The 94.0% (yellow box) would be a typical, high, viability for such a sample. b.) The system is capable ofgeneratingcomparison/overlaysofMoxiGOIIViability(PI)datafortwotests.Hereahealthybrighttankyeast(grayscatterplot)sampleisoverlaidwithaheat-killed(coloredscatterplot)sample,showingtheclearshiftinPIfluorescence.c.)ThisdatacanalsobedisplayedasPIfluorescencehistogramswithlive/healthyyeast(graycurve)andheat-killedyeast(redcurve).



healthyyeast,redcurve=heat-killedyeast). In thisway,notonlydoesthesystemallowfor thequick (10 second measurement) of sample viability information, it also lets the brewer rapidlycompare that data to earlier time points to provide clear visualization of the sample viabilityprogression.The same yeast samples were incubated with CFDA-AM to measure the esterase activities(metabolicactivity/cellhealth)oftheyeastsamples.CFDA-AMisacell-permeantmolecule(passesreadilyinsidethecell)thatisnon-fluorescentinitsintactstate.However,thenaturalactivityoftheesteraseenzyme that ispresent invirtuallyallhealthy/activecells,will cleave theacetoxymethylester (AM)moiety from themolecule allowing the CFDAmolecule to fluorescewhen stimulatedwiththe488nmlight(MoxiGOIIlaser).AsCFDAisalsocharged,itisretainedwithinthehealthycells(intactcellmembranes)causingwholecell fluorescencethat iseasilyquantifiedontheMoxiGO. Figure 3a shows an example of the CFDA fluorescence vs. Coulter Sizing scatter plot for ahealthy yeast bright tank sample. Aswith the viability assay, the highpercentage of fluorescentcells (96.6%, Figure 3a, yellow box) would be expected for a healthy yeast samples. However,unlikethePI-basedviabilityapproach,themedianfluorescenceintensity(MFI,Figure3a,blackboxtolowerrightofthescatterplot)oftheCFDAwouldbeexpectedtoshiftinamannerproportionaltothehealthofthecell.Inthismanner,therelativevalueoftheCFDAfluorescenceversusknown-healthy and/or known-dead samples can provide valuable, relative, information on the potentialdeclineofayeastsample,likelybeforethecellsstained“dead”withPI.Anexampleoverlayfortheheat-killed (colored scatterplot) vs. healthy yeast (gray scatterplots) is shown in Figure3b andFigure3cprovides theoverlaidCFDA fluorescencehistograms for the twosamples(graycurve=healthy yeast, red curve = heat-killed yeast). In Figure 2c, note the CFDAMFI decreases from1010mVto just23.3mV,a changeof43x. In this sample, thosevalueswoulddefine thedynamicrangeofpossibleactivitystatesbetweenaclearly-killedyeastsampleanda100%healthysample,providingapowerfultooltoquantifytheintermediatesintheactivityofyeastcellsinthetank.Inadditiontothesecoreassaycapabilities,oneofthepowerfulfeaturesoftheMoxiGOIIisthatitis

Figure 3 -a.)RepresentativeMoxi GO II (CFDA-AM)scatterplot (CFDA fluorescencevs.Coulter sizing)output for a live/healthybright tankyeast sample. CFDA-AMprovidesameasureof cellmetabolicactivitybyreflecting theactivityof theconstitutivecellenzyme,esterase. So, the96.6%(yellowbox) level is typical forahealthy tanksample. b.)The system is capableofgeneratingcomparison/overlaysofMoxiGOIIViability(CFDA)datafortwotests.Hereahealthybrighttankyeast(grayscatterplot)sampleisoverlaidwithaheat-killed(coloredscatterplot)sample,showingthecleardecreaseinCFDAfluorescence(esteraseactivity)fortheheat-killedsamplec.)ThisdatacanalsobedisplayedasCFDAfluorescencehistogramswithlive/healthyyeast(graycurve)andheat-killedyeast(redcurve).



a true, open-platform flow cytometer. As such, brewers can use it to explore other fluorescentapproaches to categorizing their yeast state. As new research is being continuously releasedregarding the state of yeast in brewing samples, it is likely that new flow cytometry analysisapproaches,withthepotentialtorevolutionizebrewing,willalsobeidentified.Oneexampleofanarea that researchers are investigating is the relative contribution of yeast mitochondria to thebrewing process and outcome. While it has long been assumed that mitochondrial activity isnegligible in theanaerobicbrewingenvironment,evidencesuggests theymaystill serveacriticalroleinbrewing.12Asthemitochondrialpotentialhasbeenlinkedtothelevelofflavor-contributingfatty acids in sake fermentation13, we applied the Moxi GO II to the measurement of themitochondrialpotentialintheyeastsamplesinthisstudyasademonstrationoftheflexibilityofthesystem. Specifically, tomeasuremitochondrialpotentialweappliedadye, tetramethylrhodamine(TMRE)thatisknowntopermeatecellsandaccumulateinmitochondriainproportionthelevelofcharge (transmembranepotential) across themitochondrialmembrane. In Figure 4,wepresentthe results of this analysis. Figure 4a shows the TMRE fluorescence (Mitochondria potential)versusCoulterSizingscatterplotoverlays for thehealthy(grayscatterplot)yeastsampleversustheheat-killed(coloredscatterplot).Figure4brepresentstheTMREfluorescence-onlyhistogramsfor the two samples (gray curve = healthy yeast, red curve = heat-killed yeast). In this case, anexpected decrease is observed for the dead cells. While themitochondrial potential for yeast inbrewing would not likely provide value in a live/dead assay or its traditional use towards themeasure of aerobic respiration, it might eventually serve as a metric in the isolation of certainundesirableflavorsinthefinaloutput.DatasuchasthissimplyhighlightsthepotentialversatilityandimpactoftheMoxiGOIIinenablingmoreadvancedbrewinganalysisinthefuture,beyondthecoreMoxiGOIIassaysofcounts,viability,andmetabolicactivity.SummaryInthisapplicationnote,wedescribetheapplicationoftheMoxiGOIItowardsmonitoringofyeast

Figure4–Comparison/OverlaysofMoxiGO IIMitochondrialpotential (TMRE)data forahealthybrighttankyeast(grayscatterplot,grayhistogram)samplevs.aheat-killed(coloredscatterplot,redhistogram)yeastsampledisplayedasa.)Scatter/dotplotofTMREfluorescencevs.Coulter-BasedCellSizeandb.)TMREFluorescencehistogram. TMREprovidesameasureofmitochondrialpotentialasituptakenintomitochondriainproportiontothemitochondriacharge.



in brewing. Data is presented tohighlight the capabilities of the instrument to support the corerequirementofhighlyaccurateyeastcountsandviabilityaswellastheversatilityoftheinstrumentinmeasuringotherparameters suchasesteraseactivityandmitochondrialpotential. Oneof themostpowerfulfeaturesoftheMoxiGOIIinstrumentistheease-of-useandversatilityincollectionof data. With an ability to run tests without the need for system warm-up, maintenance, orshutdownprocedures,theMoxiGOIIisideallysuitedformonitoringthetimecourseofyeasthealththroughoutthebrewingprocess.ThisisfurtherfacilitatedbytheMoxiGOII’ssmallfootprintwhichenablesbrewersplacetheMoxiGOIIrightbythebrewingtanksformoreimmediateandfrequentflow analysis of their systems over (potentially) long periods of time. Finally the Moxi GO IItouchscreenGUIisdesignedtomakeevencomplexflowanalysisaccessibletoallusers,regardlessoftheirflowexpertise.Thesefeatures,combinedwiththesystem’slowpricepoint,shouldestablishtheMoxiGOIIasastapleinbrewerythatisseriousaboutbetterunderstandingandcontrollingthebrewingprocess.References

1. Wang, J. et al. Revealing a 5,000-y-old beer recipe in China. Proc. Natl. Acad. Sci. 113, 6444–6448 (2016). 2. Hornsey, I. S. A history of beer and brewing. 34, (Royal Society of Chemistry, 2003). 3. Zion Market Research, https://globenewswire.com/news-release/2017/01/26/911240/0/en/Global-Beer-

Market-will-reach-USD-750-00-Billion-in-2022-Zion-Market-Research.html 4. https://scholar.google.com/scholar?q=brewing&btnG=&hl=en&as_sdt=0%2C5 5. Boyd, A. R. et al. A flow-cytometric method for determination of yeast viability and cell number in a

brewery. FEMS Yeast Res. 3, 11–16 (2003). 6. Shimizu, C., Araki, S., Kuroda, H., Takashio, M. & Shinotsuka, K. Yeast cellular size and metabolism in

relation to the flavor and flavor stability of beer. J. Am. Soc. Brew. Chem. 59, 122–129 (2001). 7. Peddie, H. A. B. Ester formation in brewery fermentations. J. Inst. Brew. 96, 327–331 (1990). 8. Grondin, E. et al. Physiological and biochemical characteristics of the ethyl tiglate production pathway in

the yeast Saprochaete suaveolens. Yeast 32, 57–66 (2015). 9. Orflo Technical Note: Comparison of Moxi Z Cell Counter with an Imaging Cell Counter and a High-End

Coulter System - http://www.orflo.com/v/vspfiles/documents/AppNotes_Compare12-1.pdf 10. Orflo Technical Note: Moxi Z Performance – Precision and Accuracy that match the “Gold Standard” of

Counting Technologies - http://www.orflo.com/v/vspfiles/documents/AppNotes_Performance12-1.pdf 11. Orflo Technical Note: Monitoring Cell Viability with Orflo Technologies Moxi Flow System -

http://www.orflo.com/v/vspfiles/documents/Moxi%20Flow%20-%20Viability%20App%20Note%20V4_06212013.pdf

12. O’Connor‐Cox, E. S. C., Lodolo, E. J. & Axcell, B. C. Mitochondrial relevance to yeast fermentative performance: a review. J. Inst. Brew. 102, 19–25 (1996).

13. Sawada, K. & Kitagaki, H. Residual mitochondrial transmembrane potential decreases unsaturated fatty acid level in sake yeast during alcoholic fermentation. PeerJ 4, e1552 (2016).

MethodsInitialCellConcentrationAdjustmentsCellsweresampleddirectlyfromabrighttanksamplecontainingandunspecifiedbatchofbeerusingSanDiegoSuperYeast(WhiteLabs,#WLP090).TheSamplewasdiluted1000xbyserialdilution.First,100µLoftanksamplewasaddedinto~10mLofOrfloDiluent(OrfloTechnologies,Cat#MXA006)fora100xdilution.The1000xdilutionwasthenrealizedbymakinganadditional10xdilutionbytaking1mlofthe100xsampleanddilutingitinto9mlofOrfloDiluent.DilutedconcentrationswereverifiedontheMoxiGOIIsystem.



HeatKilledYeastCellControlsToachieveadead-cellcontrolforthevariousassays,aheat-killedyeastsamplewasgenerated.Waterwasbroughttoaboilinanelectrickettleandtransferredtoabeakerandallowedtocoolfor1min.A5mlsampleofthedilutedyeastwasaliquotedtoa15mlcentrifugetubeandplacedinthehotwaterbeakerfor10minutes.CellAssaysAllyeastcellassaysfollowedthecorrespondingOrfloProtocols(includedbelow).ThePIviabilityassayusedthe“OrfloMoxiGOII/MoxiFlowViability(PI)StainingProtocol”usingtheOrfloFlowReagentoptionalstep.Theesterase-activitywaspreparedusingthe“MoxiGOII-CellHealth/Vitality/EsteraseActivitywithCFDA-AM”protocol,followingthestepsforCFDA-AMinclusion.ThefinalCFDA-AMincubationconcentrationwas5µM,theincubationtimewas30minutes,andtheoptionalOrfloFlowReagentwasadded.Themitochondrialpotentialassaywasimplementedaccordingtothe“MoxiGOII–MitochondrialMembranePotential–TMREStaining”protocol.ThefinalTMREconcentrationwas300nM,theincubationtimewas20minutes,andtheoptionalOrfloFlowreagentwasadded.MoxiGOTestsandDataOutputAlltestswereperformedonaMoxiGO488nm(OrfloCat#MXG002)system.Theviabilityassays,andinitialcellconcentrationdetermination,wererunusingthe“ViabilityAssay”functionalitybuiltintothefirmwareusingthe561nm/LPfilter(OrfloCat#MOG002).TheCFDA-AMassaywasrunusingthe“OpenFlowCytometry”functionality,“Medium”fluorescentgain,andthe525/45nmfilter(OrfloCat#MXG001).Note:TheMoxiGOusedinthisstudyissimplythe1PMT/channelversionoftheMoxiGOIIsystem.OtherthanthePMTconfiguration,thesystemhasthesamearchitectureastheMoxiGOIIsoperformancewouldbeequivalent.TheTMREassaywasrunusingthe“OpenFlowCytometry”functionality,“Medium”fluorescentgain,andthe561nm/LPfilter(OrfloCat#MXG002).Datacomparisons/overlayswereallperformedon-unitusingthebuilt-insystemfunctionalityforcomparingsavedtests.Screenshotswereallgenerateddirectlyfromtheunitusingthebuilt-in“Print”functionalitytoexportdatascreenshotsinbitmap(BMP)format.FinalimagecroppingandarrangementwasperformedusingPhotoshop(Adobe)andIllustrator(Adobe).

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MoxiGOII/MoxiFlowViability(PI)StainingProtocol

MoxiGOII/MoxiFlow–Viability(PI)StainingProtocol

Overview:Thisviabilityprotocolisbasedonthesimpleprincipleofcellmembraneexclusion.Briefly,ascellsdie,ruptures/holesforminthemembranethatallowfortheentryoflargermoleculesthatordinarilywouldbeblocked.Inthismanner,viabilitydyessuchsuchaspropidiumiodide(PI)areexcludedfromtheinteriorof“live”cellsandallowedtoenter“dead”cells.Theresultisthatthedeadcellsaccumulatethedyesandbrightlyfluoresce(PI).PIisparticularlyattactiveasitexhibitsa~40xincreaseinfluorescenceuponbindingwithDNA(afteritentersthecell)resultingalargesignalincrease,relativetotheshadinglevelchangeassociatedwithTrypanBlue.

Reagentsrequired:

§ DilutionBuffer(e.g.PBS)§ PropidiumIodide(PI)stocksolution-1mg/ml(e.g.,ThermoFisherCat#P3566)§ Optional/Recommended:OrfloFlowReagent(OrfloCat#MXA080)

Viability(PI)Staining

1. Dilutecellsto1-5x105cells/ml(recommendedrange)usingDilutionBufferNote:Asaruleofthumb,forsampleswithunknownconcentrations,startwitha10xdilution.

2. Add2μLof(1mg/ml)PIstocksolutionforevery1mLofcellsolution(finalPIconcentrationof2μg/mL).

3. Incubatefor5minutesinthedarkatroomtemperature(25°C).4. Optional/Recommended:Add20µLofOrfloFlowReagentpermlofcellsample.5. AnalyzewiththeMoxiFloworMoxiGOIIusingthe“ViabilityCount(PI)”appwithin10

minutes.


MoxiGOII-CellHealth/Vitality/EsteraseActivitywithCFDA-AM

Overview:Traditionally,cell“viability”measurementsrelyonthesimpleprincipleofcellmembraneexclusion(i.e.largermolecules,suchasdyes,areexcludedfromtheinteriorofcells).However,thesimpleexclusionofadyedoesnotnecessarilyreflectthemetabolicstateofthecellwhichmanyresearchersregardsasamorecriticalmetricofcellviabilityor“vitality.”Towardsthisend,severaldyes(e.g.5-CFDA)areavailablethataredesignedtomeasuretheesteraseenzymeactivity,afamilyofenzymesbelievedtobecommontoallcells.Tomeasurethisactivity,thedyesareconjugatedtoacetoxymethylestermoieties(e.g.5-CFDA-AM)thatservetosimultaneouslyassistinthemembranepermeabilityofthedyesaswellasquenchthedyesfluorescence.Onceloaded,theconstitutiveactivityoftheesteraseenzymecleavestheAMfunctionalityofthemoleculecausingittofluoresceandslowingtherateofeffusionfromthecell.Theresultisameasureablecellularfluorescencethatcanbequantifiedandcorrelatedtothemetabolicstateofcells.Thisprotocoldescribestheimplementationofthis“CellHealth”or“Vitality”assayontheOrfloMoxiGOIIsystem.

Instrument/Cassettes:

§ OrfloMoxiGOIINextGenerationFlowCytometer(OrfloCat#MXG102)§ CompatibleOrfloCassette(anyofthefollowingwouldwork):

o TypeCST(OrfloCat#MXC040)o MF-S(OrfloCat#MXC020)o MF-S+(MXC030)

Reagents:• 5-CFDA,AM(e.g.ThermoFisher,Cat#C1354)• PhosphateBufferedSaline(PBS,e.g.Thermo10010023)• DMSO(anybrand,e.g.Sigma#D8418)• PropidiumIodide(PI)stainingsolution(e.g.ThermoP3566(1mg/mlPI))• DMSO(anybrand,e.g.Sigma#D8418)• Optional/Recommended:OrfloFlowReagent(OrfloCat#MXA080)

CFDA-AMDyeStockSolutionPreparation

§ Createa1mMstocksolutionoftheAMdye:o CFDA-AM

§ Add9.39mLDMSOto5mgCFDA-AMstock§ Mixtoensureitfullydissolvesandaliquot(e.g.200µLvials)tominimizethe

potentialforhydrolysiswithrepeatedopening/closingandfreezethawcycles.

Protocol:1. DilutecellswithPBStogetsampleintotheoptimalconcentrationrangefortheMoxiFlow.

Finaltargetconcentration:~1e5–5e5cells/mL.2. Foreachsampletobestained,add500µLofdilutedcellsintoamicrocentrifugetube.Note:Itis

usefultoprepareanegativecontrolbyheat-killingasample(60-70°C,10min).Forcompensation,preparethreetubes:

a. PIonlysamplewiththeheatkilledcellsb. CFDA-AMonlysamplewiththeHealthy/Live(control)cellsc. CFDA-AMandPISamplefortargetcells


MoxiGOII-CellHealth/Vitality/EsteraseActivitywithCFDA-AM

3. Add2.5µLof1mMCFDA-AMstockforafinalconcentrationof5µMCFDA-AM.Pleasenotethat,fordifferentyeastcelllines,itmaybenecessarytoperformatitrationoftheCFDA-AMconcentrationtodeterminetheoptimalstainingconcentrationtopreventrailingofthesystem(toobright)ortoachievesufficientsignal(todim).

4. Add1µLof1mg/mlPItotheappropriatetubes(finalconcentration2µg/ml,3µM)5. Vortexgently,Incubatesamplesat37°Cfor15mininthedark.Longerincubationtimescanbe

appliedforincreasedCFDA-AMsignals.Note:Iflongertimesareused,wewouldrecommendaddingthePI5-10minutespriortotheendoftheCFDA-AMincubation.

6. Optional/Recommend:Add10µLOrfloFlowReagenttoeachsampleandinversionmix.7. RunonMoxiGOIIusingthe“CellHealth(Calcein-AM&PI)”appimmediately:

a. Adjustsizegatestodefinethecellpopulation.b. Touch“X/Y”toselectaPMTvsPMTdisplayoftheCFDA-AM(PMT1)vs.PI(PMT2)

fluorescence.c. Adjustforspilloverasappropriateusingthe“Comp”buttonfunctionality.


MoxiGOII-MitochondrialMembranePotentialTMREStaining


Overview:Tetramethylrhodamine,ethylester(TMRE)isacell-permeantfluorescentdyethat,duetoit’scationicnature,isreadilysequesteredbyactivemitochondria.TMREhasapeakexcitationof549nmbutcanbeexcitedbyeitherthe488nmor532nmlaser.Thepeakemissionis574nm,requiringtheuseofthe561nm/LPchannelontheMoxiGOIIinstrument.

Instrument/Cassettes:§ OrfloMoxiGOIINextGenerationFlowCytometer(OrfloCat#MXG102)§ CompatibleOrfloCassette(anyofthefollowingwouldwork):

o TypeCST(OrfloCat#MXC040)o MF-S(OrfloCat#MXC020)o MF-S+(MXC030)o

Reagents/Components:

§ Tetramethylrhodamineethylesterperchlorate(e.g.SigmaCat#87917)§ CellStainingBuffer(BioLegendcat#420201orPBSwith0.5%BSA,0.1%Azide).Note:

StockPBS(anyformulation,e.g.Gibco,Cat#10010023)canbeusedbutmighthaveslightlylowersignal-to-noiseratios(higherbackground)fortheassay.

§ DMSO(anybrand,e.g.Sigma#D8418)§ Optional/Recommended:OrfloFlowReagent(OrfloCat#MXA080)

Mitochondria-TMRELabelingProtocol

1. MakeinitialstocksolutionsofTMREbysequentialdilutionofTMRE(SigmaCat#87917)asfollows:

a. 10mMstockTMRE:25mgdissolvedTMREin4.85mlDMSOb. 10µMstockTMRE:10µLof10mMTMREstockin990µLDSMO

2. Dilutecellstoaconcentrationof~2e5-3e5cells/mlwithCellStainingBuffer.Notes:a. Cellscanbelabeleddirectlyininculturemediaiftheyarealreadyatthecorrect

concentration(orareunder-concentration).b. Cellsneedtobeinasinglecellsuspensionfortesting.Detachmentwith

Accutase/Accumaxisrecommendedwithpipettetriturationtobreakapartclusters

3. Aliquot500µLofcellsuspensions(itisusefultogenerateanFCCP-treated(i.e.100µM),SigmaCat#C2920,sampleasanegativecontrol)intoseparatepolypropylenemicro-centrifugetubes(SantaCruz,Cat#sc-200271)–Note:donotusepolystyrene(PS)asTMREcanbindsignificantlytoPS.

4. Add2.5µLof10µMTMREstocksolutiontoeachvialtoachievea50nMfinalTMREconcentrationandgentlyvortextodisperse.NOTE:Athighoverlyhighconcentrations,TMREhasaquenchingeffect.Consequently,optimalTMREconcentrationisdependentoncelltypeandsampleprepandcanvaryfrom20nM–200nM.Initialtitrationtestsshouldbeperformedforoptimalconcentrations.Asaninitialguess,startwith50nMTMRE.

5. IncubateTMRE/Cellmediaat37°Cfor15-30minutesinthedark.6. Post-incubation,incubatecellsforanadditional15min(RT/Dark).7. Optional/Recommended:Add20µLOrfloFlowReagentpermlofcellsandinversionmix

sample.



8. Followingdilution,immediatelyrunthesamplesontheMoxiGOIIsystemusingthe“OpenFlowCytometry”assaywithPMT2(561nm/LP)selectedandthe“Default”gainsetting.

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