detroit marriott h friday september 16 sunday … 25 program 2016.pdf · 9:45 am - 10:30 am where...

DETROITMARRIOTTHOTELFRIDAY,SEPTEMBER16–SUNDAY,SEPTEMBER18,2016

Troy,MI

GLIIFCA25

TABLEOFCONTENTS

ABOUTGLIIFCA....................................................................................................................................................1

GLIIFCA2016SPONSORS..................................................................................................................................2

GLIIFCA2016CONFERENCEPROGRAM.......................................................................................................3

INDUSTRIALSCIENCESYMPOSIUMABSTRACTS.......................................................................................8

ROUNDTABLELUNCHWORKSHOPS............................................................................................................14

PRESENTATIONABSTRACTS.........................................................................................................................18

POSTERABSTRACTS.........................................................................................................................................24

INVITEDSPEAKERS...........................................................................................................................................33

GENERALINFORMATION................................................................................................................................37

DETROITMARRIOTTHOTELMAP...............................................................................................................40

NOTES....................................................................................................................................................................41

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ABOUTGLIIFCAGREATLAKESINTERNATIONALIMAGINGANDFLOWCYTOMETRYASSOCIATION,INC.

EIN#16-1545169

The Great Lakes International Imaging and Flow Cytometry Association (GLIIFCA) started in 1992 byCarletonandSigridStewartandAlexNakefffosterstheinteractionofprofessionalsfromGreatLakesregion(U.S.statesofIllinois,Indiana,Michigan,Minnesota,NewYork,Ohio,PennsylvaniaandWisconsinaswellastheCanadianprovinceofOntario)withinterestsinflowandimagecytometry.GLIIFCAorganizesaffordableannual meetings allowing the Great Lakes region cytometrists to learn about latest developments incytometryandrelatedfields,networkwithcolleagues,andsharetheexcitementabouttheirresearch.

THEHISTORYOFGLIIFCA-WRITTENBYCARLETONC.STEWART

In1992GLIIFCAbeganas... GLIFCA (theGreat Lakes International FlowCytometryAssociation - thewordImagingwasaddedin1993)andwasfirstpartofanoutreachprogramforaClinicalCancerResourceGrantthatIwaswritingatRoswellParkCancerInstituteinBuffalo,NY.Ihadbeenthinkinganddiscussingforminganorganizationthatwouldprimarilyfocusonyounginvestigatorsandtechnologistsdoingresearchincancerusing imagingand flowcytometry.Theestablished leaders ineachof these fieldswere invited to form thefirst Steering Committee. Carleton C. Stewart was elected first President, Alexander Nakeff first VicePresident, Sigrid Stewart, first Secretary/Treasurer and James Jacobberger first Educational Officer. AlanLandayandMauriceO'GormanweretobetheScientificChairsforthenextmeetingwhichwasheldOctober1-3,1993attheHotelSt.RegisinDetroit,MI.OthermembersoftheSteeringCommitteewerePaulRobinson,Waclaw Jaszcz, David Hedley, Betsy Ohlsson-Wilhelm and James Leary. This Steering Committee wasestablishedtohelpdeterminethepoliciesoftheorganization,maintainabudget,andcreatetheprogramfortheannualmeeting.Inadditiontofundingforthisorganizationbythegrant,IenvisionedparticipationbythevendorsasequalmembersofGLIIFCA.Therepsfromeachcompanywereinstrumentalinpassingthewordtotheircustomersthroughoutthecapturearea.Andyouknowtherestofthestory…

OurfirstmeetingoftheGreatLakesInternationalFlowCytometryAssociationwasheldSeptember25-27,1992attheHotelSt.Regis inDetroit,MI:Themeetingwasanoverwhelmingsuccess.Thescientificagendaincludedsessionson Immunophenotyping, tumorbiologyand instrumentation.Over130chartermembers,some of which had never attended a flowmeeting before, were able to attend because the total cost perpersonamountedtounder$150includingregistrationandaccommodations.Becauseofthecentrallocationof Detroit to the Great Lakes Regionmost people were able to carpool. Themeeting startedwith a well-attended reception on Friday evening with food and an open bar sponsored by Becton DickinsonImmunocytometry Systems. On Saturday the Flowdown, sponsored by Coulter Cytometry,was thoroughlyenjoyedbyall.Mostparticipants stayeduntil thevery end.Thegreatest single state/province representedwasOntario,Canada.USchartermemberswerefromMichigan,Indiana,Minnesota,Illinois,Ohio,NewYork,Pennsylvania,Wisconsin andNew Jersey.We even had two guests fromRussia,which trulymade this aninternationalevent.WhileourmainregionofemphasisistheGreatLakes,wewelcomeourcolleaguesfromacrossthelandtoparticipate.

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GLIIFCA2016SPONSORS

The2016GLIIFCAmeetingiskindlysupportedby:

• Abcam• ACEABiosciences,Inc.–supportfortheconferencecoffeebreaks• Affymetrix–supportfortheconferencecoffeebreaks• AgilentTechnologies• AkadeumLifeSciences–supportfortheGLIIFCATravelAwards• ApogeeFlowSystems• BangsLaboratories–supportfortheconferencecoffeebreaks• BDBioscience–supportforthePosterSessionandtheGLIIFCATravelAwards• Beckman-Coulter–supportfortheGLIIFCATravelAwardsandtheWineandCheeseHappyHour• TheBindingSite• BioLegend• Bio-Rad• Bio-Techne(R&DSystems,NovusBiologicals,Tocris)–supportforthePosterSessionandtheGLIIFCA

TravelAwards• CelseeDiagnostics• Cytognos• Cytek• EnzoLifeSciences• FlowJo,LLC• Immudex–supportfortheTranslationalandClinicalCytometrySession• Intellicyt• JacksonImmunoResearch–supportforthePosterSession• MilliporeSigma• MiltenyiBiotec–supportfortheGLIIFCATravelAwards• PropelLabs,LLC• SonyBiotechnology• Spherotech–supportfortheWineandCheeseHappyHour• Stratedigm–supportfortheconferencecoffeebreaks• Streck• Sysmex–supportfortheWineandCheeseHappyHour• ThermoFisherScientific–supportfortheGLIIFCATravelAwardsandtheWineandCheeseHappyHour• VeritySoftwareHouse

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GLIIFCA2016CONFERENCEPROGRAM

FRIDAYGLIIFCA25COREFACILITYMANAGERS'WORKSHOP

ThisworkshopoffersCoreManagersandStaff theopportunity tohear the latestonmanagementpracticesand technical topics; while meeting with colleagues who face similar challenges when running flow corefacilities.Ampleopportunitywillbeprovidedtonetworkwithattendeesdiscussing theexcitingchanges inmanagementand technologiesoccurring in flowcytometryandothercore facilities today.On the technicalside,bringyourstickiest issuesandshareyour troubleshootingandproblemsolvingstories.YourownhottopicsandquestionsarewelcomedduringtheOpenForumattheendofthishalf-dayworkshop!

12:00PM-1:00PM OpeningNetworkingLuncheon:WelcomeandIntroductions,MattCochran,MonicaDeLay,VictoriaSmith,andSallyQuataert,GLIIFCAOrganizingCommittee

1:00PM-2:00PM CRISPR,ThomasSaunders,UniversityofMichigan,AnnArbor,MI

2:00PM-2:30PM ReproducibilityinResearchDataandwhatGoodResearchPracticesMean,SallyQuataert,UniversityofRochester,Rochester,NY

2:30PM-3:00PM GRP:WhatcanCoresdoforSupportandPromotionofGRP,OpenDiscussion

3:15PM-3:45PM BreakoutSession–GroupsDiscussTopicsProposedbyAttendees

3:45PM-4:00PM PresentationofBreakoutTopics

4:00PM-4:30PM PolarisImagingFlowandGenomics,MonicaDeLay,CincinnatiChildren'sHospitalMedicalCenter,Cincinnati,OH

4:30PM-5:00PM OpenForum

OPENINGRECEPTION

Theopeningreceptionwilltakeplaceintheexhibitionroomfrom5:00PMto6:30PM.ComeandinteractwiththevendorsandfellowGLIIFCAConferenceparticipants!

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INDUSTRIALSCIENCESYMPOSIUM

SessionCo-chairs:KarenDomenico(ShrinersHospitalsforChildren,Cincinnati,OH),LouisKing(MichiganStateUniversity,EastLansing,MI)

6:20PM-6:30PM WelcomeAddress,KarenDomenico

6:30PM-6:45PM MultiplexBiomarkerProfilingusingtheFireflyTechnologyPlatform,ElnazAtabakhsh,Abcam

6:45PM-7:00PM TheCellAsicONIX2MicrofluidicSystem:PrecisionControlofyourCellCultureEnvironmentforAdvancedLiveCellImagingandMicroscopy,RobertThacker,MilliporeSigma

7:00PM-7:15PM NovelSoftwareToolsforFlowCytometryAnalysis,IreneGonzálezBarahona,Cytognos

7:15PM-7:30PM Veri-Cells™:ExceptionalControlsforSurfaceandIntracellularMarkerAnalysisbyFlowCytometry,KimCardenas,TechnicalApplicationScientist,BioLegend

7:30PM-7:45PM ParticleFlowCytometry:LightScatterMeasurementofExtracellularVesiclesand100nmSilicaBeads,AndrewCosgrove,ApogeeFlowSystemsLtd

7:45PM-8:00PM NovoCyte-SimplifyyourWorkflow,GarretGuenther,ACEABiosciences,Inc.

8:00PM-8:15PM TheSonyFX500:FluidicsExchangeableCellSorter,MichaelZordan(MarylouIngramISACScholar),SonyBiotechnology,Inc.

8:15PM-8:30PM MultiparameterCellCycleAnalysis,JamesW.Jacobberger,CaseWesternReserveUniversity,Cleveland,OH(onbehalfofThermoFisherScientific)

8:30PM-8:45PM IntroducingtheLatestAdvanceforiQueScreenerPLUS:FastisNowEvenMore“Exciting”!,DavidKennedy,IntelliCyt

8:45PM-9:00PM BDFACSMelody:TheSimpleSolutionforConsistent,QualityResults,MichaelHulsey,BDBiosciences

9:00PM-9:15PM Automated,SensitiveMicrofluidicDeviceforCirculatingTumorCellEnrichmentandAnalysis,GrantHowes,CelseeDiagnostics

9:15PM-9:30PM Microbubble-basedCellIsolation,JohnG.Younger,AkadeumLifeSciences,Inc.

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SATURDAY8:00AM-8:15AM WelcomeAddress,JosephD.Tario,Jr.

SESSION1:IMAGE-BASEDCYTOMETRY,HCSANDIMAGING

SessionChair:JosephD.Tario,Jr.

8:15AM-9:00AM DeconstructingAdipogenicNichesduringAdrenergicAdiposeTissueRemodeling,JamesG.Granneman,WayneStateUniversity,Detroit,MI

9:00AM-9:45AM DeepImagingofBoneMarrowPlasmaCellNiches,DavidM.Allman,PerelmanSchoolofMedicine,UniversityofPennsylvania,Philadelphia,PA

9:45AM-10:15AM CoffeeBreak-SponsoredbyACEABiosciences

10:15AM-11:00AM ARapid,invivoZebrafishModeltoElucidatetheMicrometastaticPotentialofHumanLungCancerCells,IndrajitSinha,AcenziaInc.andUniversityofWindsor,Windsor,ON

THE2016CARLETONANDSIGRIDSTEWARTLECTUREIntroduction:AlexNakeff

11:00AM-11:45AM Coordination-drivenSelf-assemblyofLight-emittingMetal-organicMaterials,TimothyR.Cook,StateUniversityofNewYorkatBuffalo,Buffalo,NY

LUNCHTIMEROUNDTABLEWORKSHOPS

WorkshopOrganizer:SherryL.Thornton

11:45AM-1:30PM RoundtableWorkshops

THECOFFEEBREAKSARESPONSOREDBY

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SESSION2:CYTOMETRYOFMICROVESICLESSessionChair:NancyFisher

1:30PM-2:15PM VesicleFlowCytometry:HighResolutionExtracellularVesicleAnalysis,JohnNolan,ScintillonInstitute,SanDiego,CA

2:15PM-3:00PM HepaticImmuneRegulationbyStromalCell-derivedExosomes,FrankA.Schildberg,(MarylouIngramISACScholar)HarvardUniversitySchoolofMedicine,Boston,MA

3:00PM-3:45PM InstrumentCalibrationandMeasurementStandardizationforMeasuringExtracellularVesiclesbyFlowCytometry,JoanneLannigan,UniversityofVirginia,Charlottesville,VA

3:45PM-4:15PM CoffeeBreak-SponsoredbyStratedigmandBangsLaboratories

SESSION3:CYTOMETRYRESEARCHANDINNOVATION

SessionChair:MonicaDeLay

4:15PM-5:00PM DDAOandanti-glycophorinAMarkingofMonkeyandHumanRedBloodCellstoScreenP.vivaxMalariaVaccineCandidates,BrianT.Grimberg,CaseWesternUniversity,Cleveland,OH

5:00PM-5:45PM ImmunoPETImaging:AnEmergingFrontier,NerissaViola-Villegas,WayneStateUniversity,Detroit,MI

POSTERSESSION5:45PM-7:30PM PosterSession.SponsoredbyBDBiosciencesandJacksonImmunoResearch

6:00PM-7:00PM WineandCheeseHappyHour

THEPOSTERSESSIONISSPONSOREDBY

THEWINEANDCHEESEHAPPYHOURISSPONSOREDBY

SOCIALACTIVITIES8:00PM-11:30PM GLIIFCABanquet

11:30PM-2:30AM Post-banquetScientificNetworking

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SUNDAYSTEERINGCOMMITTEEMEETING8:00AM-9:00AM GLIIFCASteeringCommitteeBreakfast

IMMUDEXTRANSLATIONALANDCLINICALCYTOMETRYSESSIONSessionChair:SusanMcQuiston

9:00AM-9:45AM TheuseofFlowCytometryfortheAssessmentofPrimaryImmunodeficiencyDisease,MauriceO'Gorman,Children'sHospitalLAandUSCKeckSchoolofMedicine,LosAngeles,CA

9:45AM-10:30AM WheredoesCytometryFitinToday’sGenomicsCrazedTwitter-feedWorld?,KeithShults,CenterforInnovation-IncellDx,MenloPark,CA

10:30AM-11:00AM CoffeeBreak-SponsoredbyAffymetrix/eBioscience

11:00AM-11:45AM SystemicMesenchymalStemCellMobilizationandMigrationFollowingAnteriorCruciateLigament(ACL)Rupture,MichaelNewton,BeaumontHealthSystem,RoyalOak,MI

11:45AM-12:30PM StudyingComplexBiologyinSolidTumors,DavidHedley,PrincessMargaretHospital,Toronto,ON

THESTUDENTTRAVELSTIPENDSWERESPONSOREDBY

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INDUSTRIALSCIENCESYMPOSIUMABSTRACTSABCAMMULTIPLEXBIOMARKERPROFILINGUSINGTHEFIREFLYTECHNOLOGYPLATFORM

ElnazAtabakhsh

ToaddresstheneedsforcirculatingmiRNAbiomarkervalidation,wedevelopedtheMultiplexedCirculatingmicroRNAassay.Thisassayenablesthedetectionofupto68microRNAtargetspersamplein96-wellformatwith readout on standard flow cytometers and analysiswith an includedbioinformatics softwarepackage.The Circulating microRNA assay combines particle-based multiplexing, using patented FireflyTM hydrogelparticles, with single step RT-PCR signal amplification using universal primers. Thus, the CirculatingmicroRNA assay leverages PCR sensitivity while eliminating the need for separate reverse transcriptionreactionsandmitigatingamplificationbiases introducedbytarget-specificqPCR.TheCirculatingmicroRNAassaycanreliablydetectasfewas1000microRNAcopiespersamplewithalineardynamicrangeof~5logs,makingtheassayideallysuitedforprofilinginserum,plasmaorurine.Furthermore,theabilitytomultiplextargets in eachwell eliminates theneed to split valuable samples intomultiple reactions.Results from theCirculatingmicroRNA assay are displayed and interpreted using our included Firefly AnalysisWorkbench,whichallowsvisualization,normalization,andexportofexperimentaldatawithonlyafewmouseclicks.Toaid discovery and validation of biomarkers, we have generated fixed panels for Oncology, Cardiology,Neurology,Immunology,andLiverToxicology.Thesecarefullycuratedpanelsincludehemolysismarkerstoassesssamplequality,aswellascriticalnormalizationfactors.Herewepresentthedatafromseveralstudiesinvestigating circulating and tumormicroRNA profiles using the Firefly CirculatingmicroRNAAssay FixedPanels. Together, this novel combination of bioinformatics tools and multiplexed, high-sensitivity assaysenablesrapiddiscoveryandvalidationofmicroRNAbiomarkersignaturesfromfluidspecimens.

Contact:[email protected]

ACEABIOSCIENCES,INC.NOVOCYTE-SIMPLIFYYOURWORKFLOW

GarretGuenther

ACEA’s NovoCyte® flow cytometer is a powerful research tool that can measure multiple parameterssimultaneously on individual cells while limiting its footprint within the laboratory. Flow cytometryrepresentsthebestmethodforstudyingfunctionalandphenotypicpropertiesofcellsub-populationsbasedon biological function and cell-surface markers. With the necessity of flow cytometry increasing,instrumentationincrementallyneedstoprovidehighqualitydatawhileenablingsimpleandeasyoperation.Novel, innovativefeatures incorporatedintotheNovoCytehelpfacilitatehighperformancedataacquisitionand analysis process, and enable researchers to do this quicker and easier. Many of the incorporated,automatic features on the NovoCyte save time and offer convenience on a day to day basis. PerformingcomplexflowcytometryusingNovoCyteissimpleandroutineforeveryone.


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AKADEUMLIFESCIENCES,INC.MICROBUBBLE-BASEDCELLISOLATION

JohnG.Younger

Bead-based and flow-cytometric cell separation methods are used daily in research, diagnostic, andtherapeutic applications. While widespread, these techniques have important limitations related tothroughput, ease of use, andmechanical damage to cells undergoing separation. AkadeumLife Sciences isworkingtoovercometheseimitationsbyusingmicrobubblesforcellisolation.

Afterbindingtocells,microbubblesenableisolationbyfloatingtargetcellstothesurfaceof liquidsamples.Thisallowsforanewseparationmethodthatis:

• Gentle–Minimalmechanicalforcesandmovementofcells• Fast–Acceleratedworkflowswithouttheneedforcolumnsorcell-by-cellsorting• Easytouse–Intuitiveproducthandlingwithlittlesetupandashortlearningcurve• Compatible–Canbeusedwithvariousdownstreamsystems,includingothercellsortingplatforms

Inthispresentation,Akadeum’sChiefTechnologyOfficerwilloutlinethestateofmicrobubble-basedmethodsandreviewmicrobubbleperformanceinanumberofcommonresearchscenarios.Additionally,Dr.Youngerwill discuss Akadeum’s Product Evaluation Program. The program allows users to assessmicrobubbles intheirworkflowwiththeassistanceofAkadeum’stechnicalteam.

Formoreinformation,pleasevisitwww.akadeum.com/technology

APOGEEFLOWSYSTEMSLTDSMALLPARTICLEFLOWCYTOMETRY:LIGHTSCATTERMEASUREMENTOFEXTRACELLULARVESICLESAND100NMSILICABEADS

AndrewCosgrove

In a typical extracellular vesicle sample themajority of vesicles are smaller than 300nm in diameter andthereforedifficulttomeasureonaconventionalflowcytometer.Flowcytometersareabletomeasurevesiclesofanysizeiflabelledwithsufficientfluorescentmolecules.Inpracticethelabelingofsuchsmallparticlesisweak and conventional flow cytometers struggle to resolve300nm silica beads fromoptical noise by lightscatter. However, recent electronic and optical developments allow the routine measurement of particleswhichscatterordersofmagnitudelessthan300nmsilicabeadsandanorderofmagnitudelessthan100nmlatexbeadswhichhaveoftenbeenusedasalowerlimitreference.Suchdevelopmentsmakelightscatteringanessentialtoolforthereliableinterpretationofextracellularvesicledatabutinfuturea100nmsilicabeadmaybeamoresuitablereferenceparticleduetoitsrefractiveindex,sizeandstability.Wepresentdatafrom100nm silica beads analysed on a commercially available flow cytometer, showing their relationship to110nm latexbeadsanddemonstrating thatparticleswhich scatter1000 times less light than300nmsilicabeads can now be measured routinely, thus permitting the study of a greater proportion of a typicalextracellularvesiclepopulationthanhaspreviouslybeenpossible.

Apogee Flow Systemsmanufacturers themost sensitive, commercially available, flow cytometer for smallparticle analysis.Our instrumentsare routinelyused for advancedextracellularvesicle analysis andas the

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only cytometer capable of measuring individual 100nm exosomes, the A60-Micro-PLUS is critical foradvancingthestudyofextracellularvesicles.


BDBIOSCIENCESBDFACSMELODY:THESIMPLESOLUTIONFORCONSISTENT,QUALITYRESULTS

MichaelHulsey


BIOLEGENDVERI-CELLS™:EXCEPTIONALCONTROLSFORSURFACEANDINTRACELLULARMARKERANALYSISBYFLOWCYTOMETRY

KimCardenas

Experimental control samples are essential in multicolor flow cytometry to assess intra and inter-experimental variation as well as to quantify changes that are attributable to a specific variable such asdiseasestateor treatment.Peripheralbloodobtained fromahealthydonor isacommoncontrol,however;selectionofadonorsample,whenavailable,canintroduceadditionalvariables,suchasdonorage,race,andgender,whichmayormaynotbe constant fromsample to sample.Furthermore, samplehandling, storageconditions and preparation technique can also vary from specimen to specimen and introduce additionalvariation. Inaneffort tominimizevariationandprovidea reliable,effectivebiological controlBioLegend®has developed Veri-Cells ™, lyophilized leukocytes for immunophenotyping. Veri-Cells™ include all humanleukocyte subsets (lymphocytes, granulocytes and monocytes) and have been validated against over 300surfacemarkers, includingmany chemokine receptors, such as CXCR5, CCR4, CCR6 and CCR7 and severalintracellularmarkers,suchasFoxP3,T-betandGranzyme-B.ThismakesthemanexcellenttoolforstainingCDmarkersutilizedtodelineatevariouscellpopulationssuchasTregs,T-helpercells,NKcellsandBcells.Inaddition,ourCustomSolutionsTeamcantailormakeVeri-Cells™tomeetyourresearchneeds;forexample,byadditionofalive/deadindicator,cellactivationcocktail,orapplicationofaselection/enrichmentprotocolpriorto lyophilization.Additionofcell linesorsortedpopulationscanalsobeaddedtomimicabnormalorrarepopulationsforstainingcontrols.Singleandmulti-testlotscanbemanufacturedandareidealformulti-site clinical trials or long-term studies that seek to investigate immunological responses and assess assayvariabilityandreagentperformance.


CELSEEDIAGNOSTICSAUTOMATED,SENSITIVEMICROFLUIDICDEVICEFORCIRCULATINGTUMORCELLENRICHMENTANDANALYSIS

GrantHowes

TheCelseeDiagnostics’ novelmicrofluidic technology enriches andanalyses circulating tumor cells (CTCs)from whole blood samples based on size and deformability. The platform design enables downstreamcharacterizationofCTCs, includingimmunochemistryforenumeration,DNAandmRNAFISH,PCRandNGS.

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Thispresentationwilloverviewthetechnology,demonstratethe85%captureefficiencyanddiscusstheuseoftheCelseePREP100toenrichandretrieveCTCsfordownstreamapplications.TheCelseePREP100deviceusesamicrofluidicchip,whichhasapproximately56,400individualcellcapturingwellswithan8µmpore.,ensuringthatsmallerbloodcells,suchasredbloodcellsandmostleukocytes,escapewhilelargerCTCsarecaptured.DownstreamanalysisofthecapturedCTCsusingimmunostaining,FISHassays,RTPCRandNGSwillbehighlighted.


CYTOGNOSNOVELSOFTWARETOOLSFORFLOWCYTOMETRYANALYSIS

IreneGonzálezBarahona

ThereisnodoubtthatFlowCytometryisanincrediblypowerfultechnologyforcharacterizationofcells.Therecentadvancesinequipmentandreagentspermitahighresolutionanalysisbymeasuringalargenumberofdifferentparametersatonce.However,theresultisacomplexdatasetwhichcanbetrickytointerpret.That’swhy we need specific software tools that help us in this task: Multiparametric Analysis to visualize ourpopulationsinamultidimensionalview;theintegrationofmultiplefilesallowingaglobalanalysis;ReferenceImagesandDatabasesstoredintheprogramforaneasycomparisonwithourcases.ThefuturewillbringtheAutomatic Gating, a tool that clusters and classifies all the events just one click away. Let’smaximize themulticolorpotentialofflow!

ContactIreneGonzá[email protected]

EMDMILLIPORETHECELLASICONIX2MICROFLUIDICSYSTEM:PRECISIONCONTROLOFYOURCELLCULTUREENVIRONMENTFORADVANCEDLIVECELLIMAGINGANDMICROSCOPY

RobertThacker

Theabilitytogrow,observeandmanipulatecomplexculturesrequiresprecisioncontroloverthecellcultureenvironment. The second generation CellASIC® ONIX2 Microfluidic System is a refined, yet powerful,automatedplatformforprecisemanipulationofmultiplekeycellcultureparameters,enablingmeasurementofcellularresponsestopre-programmedmedia,temperature,andgasenvironmentchanges.TheCellASIC®ONIX2MicrofludicSystemuseshighquality,opticallyclearmicrofluidicplatesandintuitivesoftware,whileintegratingwithabroadrangeofinvertedmicroscopestoallowcontinuous,highmagnificationobservationoflivecells—astheyreacttotheirenvironmentintime.


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INTELLICYTINTRODUCINGTHELATESTADVANCEFORIQUESCREENERPLUS:FASTISNOWEVENMORE

“EXCITING”!

DavidKennedy

Expandyourpossibilitieswhenprofilingsuspensioncellsforfunctionandphenotypewiththeadditionofaviolet-yellow-blue laser configuration for the iQue Screener PLUS. Now, application choice and assayperformance optimization are easier when using fluorescent proteins, tandem conjugates, and nextgenerationlabels.TheIntelliCytAdvantagecomesstandardwitheverymemberoftheiQueScreenerfamily–meaningyougetthepowerofaflowcytometer,andtheabilitytogowellbeyonditwith:

• Fastesttimetoinsight–process96wellsin<5minutesor384wellsin<20minutes!• Massivecontentfromminimalsample–aslittleasasinglemicroliterperwell!• ForeCytSoftware–platelevelanalytics,doseresponsecurves,standardcurves,profilemapping…it’s

allinthere!

Find out why iQue Screener PLUS is the choice of leaders in immuno-oncology, antibody discovery, andimmunetargetsscreening.


SONYBIOTECHNOLOGY,INC.THESONYFX500:FLUIDICSEXCHANGEABLECELLSORTER

MichaelZordan(MarylouIngramISACScholar)

TheFX500isamicrofluidicschipbasedcellsorterfeaturingafullyreplaceablefluidicssystem.Thisfluidicssystem includes irradiated sheath line, sample line and sorting chip and can be easily exchanged byresearchersbetweensamples.Theuniquelydesignedsheathtankcanaccommodateupto4sheathbags.

The FX500 also features comprehensive fluidics controls, advanced automation and easy to use software.PatentedCoreFinder™technologyautomatesinstrumentsetupandstreamlinesworkflow.Theopticaldesignoffersuptothreecollinearexcitationlasers-488nm,561nmand638nmandsixfluorescencedetectors.Thesixfree-formPMTsenabledetectionoffluorescencesignalsfromanylaserbasedonfiltersetting.Tosupporttrue ease of operations and save time, the software guides users through acquisition, sort control, sortmonitoringandanalysis.

Using this replaceable fluidics system, a streamlined approach can be adopted that allows sorting of cellswithoutcross-contaminatingsuccessivesamples.


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THERMOFISHERSCIENTIFICMULTIPARAMETERCELLCYCLEANALYSIS

JamesW.Jacobberger(CaseWesternReserveUniversity,Cleveland,OH)

Cytometry is well-suited for quantifying the fractions of cells in cell cycle phases. For themost part, thisamountstomeasuringDNAcontentanddeconvolvingtheresultinghistogramintoG1,S,andG2+MorG1,S,G2+early M, and late M. Considerable effort has been put to coupling DNA content with RNA, protein, orspecificepitopes–generallylimitedto1-4.Ineachcase,thenumberofcellcyclecompartmentsincreases.Inourmost recent efforts, using 6 parameters (DNA-area, DNA-peak, Light Scatter, cyclin A2, cyclin B1, andphospho-S10-histoneH3),we identifyXcompartments thataredefinedobjectivelybyarulebasedsystem.Fixingandstainingwereoptimizedmanyyearsago,andwhilewehaveexaminedmanyvariations,wehavenotdiscoveredanythingnoteworthy that improves theanalyticqualityof the resultantdata.Currently,weareworkingona“washless”stainingassay,relyingonasinglehighdilutiontominimizebackgroundstainingandacousticfocusingtorenderthesampleanalyzableoverashorttimeperiod.Theresultsarestriking.TheS/Napproachesafullywashedsample;theminimizedhandlingresultsinbettercellrecovery,andbettercellrecovery results in better definition of cell cycle compartments with low cell fractions. Additionally, the“washless” assay provides a significant labor savings.We are currently testing this approachwith clinicalsamples of limited cell numbers to detect S phase cells in peripheral blood as part of a pharmacodynamicevaluationofhypomethylatingtherapy.


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ROUNDTABLELUNCHWORKSHOPSTOPIC1:MAKINGTHETRANSITIONFROMFLUORESCENCETOMASSCYTOMETRY:UTILIZINGTHECYTOFTOPICLEADER:MATTCOCHRAN(TECHNICALDIRECTOR,URMCFLOWCYTOMETRY)DENNISONROOM1

Cytometristsareincreasinglycomfortablewithmultiparametricflowcytometry,suchthatit’snotuncommonfor researchers to be running multiple 10+ parameter panels to maximize the data they’re able toacquire.Whiletherelativesizeof traditional fluorescenceflowpanelshas increased,newtoolssuchastheCyTOFmass cytometer that alloweven further expansionof thosepanelshavebecomemoreprevalent. Inthis roundtable we'll discuss the differences researchers can expect when transitioning or considering atransition from fluorescence to mass cytometry as well as other special considerations that need to beunderstood.

TOPIC2:SIMULTANEOUSDETERMINATIONOFMRNAANDPROTEINBYFLOWCYTOMETRYTOPICLEADERS:KAHTEONGSOHANDPAULK.WALLACE(THEDEPARTMENTOFFLOWANDIMAGECYTOMETRYATROSWELLPARKCANCERINSTITUTE)DENNISONROOM1

PrimeFlowTM (Thermo Fisher Scientific) and SmartFlareTM (Merck Milipore) were recently introduced toallow for a simultaneousmeasurement ofmRNAandprotein at the single cell level usingmultiparametricflowcytometry.WithafocusprimarilyonthePrimeFlowTMassay,wewilldiscussontipsandtechniquesforasuccessful experiment, including the basics of sample preparation, quality control, analysis strategies andcertain precautions that must be taken to prevent adverse experimental outcomes. As with all otherroundtables,attendeeswithorwithoutpreviousexposuretothetechniquearewelcomeandanyquestionsregardingRNAFlowCytometrywillbeaddressed.

TOPIC3:CELLSORTING-MAXIMIZINGUSER(ANDOPERATOR)SATISFACTIONTOPICLEADER:DAGNASHEARER(MANAGER,UWCCCFLOWCYTOMETRYLABORATORY)DENNISONROOM1

We will discuss methods that the UWCCC Flow Lab has implemented to make the sorting experience asefficient and painless as possible for both users and operators. Topicswill include pre-sort consults, technotes and protocols available to users, sort request forms, communication during the sort and post-sortsurveystomeasureusersatisfaction.Examplesoftechnotes,protocols,sortrequestformsandsurveyswillbemadeavailable.

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TOPIC4:MICROPARTICLES1,MINIMALINFORMATIONFORPUBLISHINGONEXTRACELLULARVESICLESTOPICLEADER:JOANNELANNIGAN(DIRECTOR,FLOWCYTOMETRYCORE,UNIVERSITYOFVIRGINIASCHOOLOFMEDICINE)DENNISONROOM2

PublicationsonExtracellularVesicle(EV)researchareincreasingatalogarithmicrateandonecanoftenfindconflictinginformationamidstthesepublications.IfyouhavereadmultiplepublicationsonEVresearchandhavecomeawayconfusedandnotsurewhattobelieveor ifyouareplanningtopublishyourownworkinthisarea,thisworkshopisforyou.

InthisworkshopwewilldiscussthetypesofinformationthatarecriticallyimportantforaninvestigatortoprovidewhenpublishingEVresearchinorderfora)reviewerstobeabletoadequatelyassesstheaccuracyand appropriateness of themethods and conclusions and b) for the readership to be able to critique andreproducethefindings.ComplianceintheseareaswillhelptoimprovethequalityofresearchandknowledgeinthefieldofEVresearch.

TOPIC5:MICROPARTICLES2,LIGHTSCATTERVSFLUORESCENCEBASEDTRIGGERINGFORMVTOPICLEADER:JOHNNOLAN(PROFESSOR,SCINTILLONINSTITUTE)DENNISONROOM2

Cytometry-based fluorescence and light scatter measurements can provide useful information aboutindividual particles ranging from multicellular organisms to sub-cellular molecular assemblies, butextracellular vesicles (EVs) are not lymphocytes. We’ll discuss the differences between light scatter andfluorescenceforsmallandlargeparticles,andhowtoavoidrookiemistakestoensuredatafromyourlabisn’tfeaturedinthetop10listofcommonEVanalysiserrors.

TOPIC6:MICROPARTICLES3,PRE-ANALYTICALREQUIREMENTSFORCLINICALSAMPLESTOPICLEADER:FRANKSCHILDBERG(MARYLOUINGRAMISACSCHOLAR;POSTDOCTORALFELLOW,HARVARDMEDICALSCHOOL)DENNISONROOM2

We will discuss pre-analytical requirements for sample handling, storage and microvesicle isolation forclinical samples and talk about potential standard protocols. Procedures of handling and storage of bodyfluids affect numbers and composition of microvesicles; we will elaborate on how standardization is ofutmost importance to ensure reliable and comparablemeasurements ofmicrovesicles for clinical samples.This discussion will include key principles on pre-analytical issues related to sampling, sample handling,plasmagenerationandplasmafreezing/storingandoptimizationofcurrentstandardprotocols.

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TOPIC7:AMNISIMAGINGFLOWCYTOMETRY:EMERGINGAPPLICATIONSANDANALYSISTOOLSTOPICLEADER:ROBERTTHACKER(AMNISCORPORATION,MILLIPORESIGMA)DENNISONROOM3-4

Come join in on a discussion thatwill cover some of the latest applications, get your questions answeredaboutcurrentapplications,andlearnaboutthelatestfeaturesandmasksinIDEASdesignedtoenhancethedepthofyourdataanalysis.

TOPIC8:CRISPRANDTHEFLOWCYTOMETRYCORETOPICLEADER:DAVIDADAMS(MANAGER,CYTOMETRYCORE,UNIVERSITYOFMICHIGANMEDICAL

SCHOOL)DENNISONROOM3-4

CRISPR technology has brought a revolution to the science of gene editing.What does this advancementmean for the flow cytometrist and what special precautions may be required when performing protein-markedselections?

TOPIC9:MULTICOLORFLOWCYTOMETRY,BUILDINGPANELSTOPICLEADER:VICTORIASMITH(FLOWCYTOMETRYFACILITYMANAGER,UNIVERSITYOFNEBRASKAMEDICALCENTER)DENNISONROOM3-4

Fluorochromes, instrument characteristics, and knowledge of the biology being studied are vital in paneldesign.Howcanweminimizespectraloverlapanduseresolution impacttoourbenefitwhenplanningourexperiments?Whataresomeotherfactorsweneedtoconsiderwhenplanningourexperiments?

TOPIC10:CURRENTTRENDSININTEGRATINGCELLSORTINGWITHSINGLECELLTRANSCRIPTOMICSTOPICLEADER:MONICADELAY(COREMANAGER,RESEARCHFLOWCYTOMETRYCORE,CINCINNATICHILDREN’SHOSPITALRESEARCHFOUNDATION)NILESROOM1-2

This roundtable will give an overview of popular and emerging platforms for performing single celltranscriptomics(e.g.FluidigmC1,Drop-seq,etc.)thatareoftendownstreamofacellsortingworkflow.Wewill discuss strengths and limitations of each technology with additional time for attendees to shareexperiencesinworkingwiththeseorotherplatforms.

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TOPIC11:WHATISFLOWCYTOMETRYANDHOWCANIMAKEITWORKFORME?TOPICLEADER:JESSICABACK(ASSOCIATEDIRECTOR,MICROSCOPY,IMAGING,ANDCYTOMETRYRESOURCESCORE,KARMANOSCANCERINSTITUTE,WAYNESTATEUNIVERSITY)NILESROOM1-2

ThisRoundtablewillprovideanintroductiontoflowcytometry.Topicscoveredwillincludeinstrumentationbasics, data interpretation, and best practices for sample preparation. Prior understanding or use of flowcytometryisnotnecessary.

TOPIC12:MULTI-DIMENSIONALAPPROACHESFORTHESIMULTANEOUSANALYSISOFMULTIPLEPROTEINANDRNATARGETSINCELLSANDTISSUESTOPICLEADER:STEFANJELLBAUER(APPLICATIONSCIENTIST,AFFYMETRIX)NILESROOM1-2

ThisroundtablewillgiveanoverviewofAffymetrix’sportfolioofmultiplexLuminexassaystomeasureRNAandProteinwithaccuracyandprecision.LearnabouttheQuantiGenePlexassaythatallowsformeasurementof gene expression from up to 80 targets without the need for RNA purification. We will also discussimportantaspectsofmultipleximmunoassays.

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PRESENTATIONABSTRACTSDEEPIMAGINGOFBONEMARROWPLASMACELLNICHESDAVIDM.ALLMAN(PERELMANSCHOOLOFMEDICINE,UNIVERSITYOFPENNSYLVANIA,PHILADELPHIA,PA)

Long-lived plasma cells are critical for maintaining antibody-mediated immunity. Conventional wisdomsuggeststhat,tosurvive,newlyformedplasmacellsmigratetothebonemarrowwheretheymustenterandremain indefinitely in specialized size-limited regulatory microenvironments, or niches. However, little isknown about how plasma cell-supportivemicroenvironments are established, how new plasma cells gainentryintotheseniches,orhowtheseeventsultimatelypromoteplasmacellsurvivalandfunction.Toaddressthese questions we have developed methods to perform deep confocal imaging on plasma cell niches inmouse bone marrow. Our results show that mature plasma cells localize preferentially within discreetclustersadjacenttosinusoidsthroughoutthebonemarrow.Nearestneighboranalysesconfirmthatplasmacells form nonrandom associations within one another, while also demonstrating that plasma cells sharephysicalnicheswithhematopoieticstemcellsandotherrelativelysessilebonemarrowcells,segregatedfrommobile cell types including granulocytes and mature recirculating B cells. Our results suggest that bonemarrowisorganizedintofunctionallydistinctregionswhereindifferenthematopoieticcelltypesgainaccesstocell-typespecificenvironmentalcuesbyinteractingwithappropriatespecializedregion-restrictedstromalcells.

COORDINATION-DRIVENSELF-ASSEMBLYOFLIGHT-EMITTINGMETAL-ORGANICMATERIALSTIMOTHYR.COOK(DEPARTMENTOFCHEMISTRY,STATEUNIVERSITYOFNEWYORKATBUFFALO,BUFFALO,NY)

The formation of structurally complex metal-organic polygons, polyhedra, and prisms (MOPs) is greatlyfacilitated by self-assemblymethods which furnish them in single, one-pot reactions. As the chemistry ofthesematerialsmatures fromstudiesemphasizingsyntheticroutes,apriorityon functionalassemblieshasemerged.Towardsthisend,therigidorganicbackbonesthatonceservedsolelyasstructuralelementshavemorerecentlybeenexploitedfortheirabilitytoimpartinterestingphotophysicalpropertiestotheirparentMOPs.Strategiesincludetetheringpendantfluorophoresthroughcovalentcouplingchemistryandselectinginherently emissive building blocks.Molecules that exhibit aggregation-induced emissionmay similarly beincorporatedintoself-assemblydesigns,preservingtheircharacteristicallyhighquantumyieldsinthesolidstate.Suchdesignsmayremainemissiveevenindilutesolutions,owingtothestructuralrigidityofagivenMOPthatnegatesnon-radiativerelaxationpathways.Thesematerialsarebeingexploredaspotentialimagingagents,motivatedinpartbythepossibilityofagivenstructuretoservebothdiagnosticandtherapeuticroles,wherein the presence of the metal ions results in biological activity. Recent examples of emissivemetallacyclesandcageswillbediscussedwithanemphasisonpioneeringbiomedicalapplications.

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DECONSTRUCTINGADIPOGENICNICHESDURINGADRENERGICADIPOSETISSUEREMODELINGJAMESGRANNEMAN(CENTERFORINTEGRATIVEMETABOLICANDENDOCRINERESEARCH,WAYNE

STATEUNIVERSITYSCHOOLOFMEDICINE,DETROIT,MI)

Adiposetissuescontainpopulationsofcommittedprogenitorsthatrespondtodiversemetabolic,hormonal,and physical signals to differentiate into white or brown adipocytes. De novo adipogenesis involvesactivation, proliferation, and differentiation of quiescent progenitor. These events often occur within in ahighly localized region,orniche, and involve close interactionswith tissuemacrophages.Our labhasbeeninvestigatingthemechanismsbywhichadrenergicreceptoractivationtriggersdenovobrownadipogenesisin mouse perigonadal white adipose tissue. In this model treatment with a beta-3-adrenergic receptor(ADRB3) agonist triggers white adipocyte cell death, which is followed by macrophage-mediatedefferocytosis and replacement of white adipocytes with clusters of new brown adipocytes. This processoccurswithin 5 days,making it a tractablemodel for evaluatingmechanisms of progenitor recruitment invivo.RecruitmentofprogenitorsexpressingthesurfacemarkersPDGFRAandCD34requireschemotacticandmitogenic signals emanating from M2-polarized macrophages. Their close physical proximity withdifferentiating progenitors suggested that the macrophages that clear dying adipocytes might generateproadipogenic factors. Flow cytometric analysis of macrophages from mice treated with CL 316,243identifiedamacrophagesubpopulationthatcontainedelevatedlipidandexpressedCD44.LipidomicanalysisofFACS-isolatedmacrophagesdemonstratedthatCD44+macrophagescontained4-5foldhigherlevelsoftheendogenous PPAR ligands 9-HODE and 13-HODE, compared to CD44- macrophages. Gene expressionprofilingandimmunohistochemistrydemonstratedthatADRB3agonisttreatmentupregulatedexpressionofAlox15, the lipoxygenase responsible for generating9- and13-HODE.Usingan invitromodelof adipocyteefferocytosis, we found that IL-4 primed tissue macrophages accumulated lipid from dying fat cells andupregulatedexpressionofAlox15.Furthermore,treatmentofdifferentiatingPDGFRA+preadipocyteswith9-and 13-HODE potentiated brown adipogenesis. Collectively, these data indicate that macrophages recruitprogenitorstohighlylocalizedsitesoftissuerepairwheremacrophage-mediatedgenerationofPPARligandspromotesbrown/beigeadipogenesis.Currenteffortsarefocusedonselectiveisolationandcharacterizationofquiescent,transitamplifying,andactivelydifferentiatingadipocyteprogenitors.

DDAOANDANTI-GLYCOPHORINAMARKINGOFMONKEYANDHUMANREDBLOODCELLSTOSCREENP.VIVAXMALARIAVACCINECANDIDATESBRIANT.GRIMBERG(CASEWESTERNRESERVEUNIVERSITYSCHOOLOFMEDICINE,CLEVELAND,OH)

The difficulty culturing the malaria parasite Plasmodium vivax has hampered any progress towards aneffectivevaccine.Whiletherecanbesomeparasiteinvasioninvitrothesmallnumberofinvasioneventsandhighpercentageofparasitedeathmakesevaluationofinvasioninhibitionverydifficult.WehavedevelopedanovelflowtechniquetolabeltargethumanredbloodcellswithGlycophorinA(FITC)orDDAOstain,toallowfortheenumerationofnewP.vivaxinvasions.P.vivaxarrivesfrozeneitherfrompatientsamplesorfromtheATCC in the blood of Aotus monkeys which are Glycophorin A negative. Therefore, the observation of aparasitizedGlycophorinApositiveorDDAOpositiveredbloodcellcanonlybetheresultofanewinvasionevent. Coupling this event identification with high throughput rare event analysis allows for analysis ofinvasion mechanisms to be studied for the first time. Reticulocyte enriched human blood samples wereexposedtodifferentstrainsofP.vivaxwhichhaveseveralkeygeneticvariations inthe ligandtheparasitesuse tobind toredbloodcellscall theDuffyBindingProtein(DBP).New invasionsweremeasuredonaBDLSRIIusingHoechst33342to identifyDNApositiveredbloodcellswhen inculturewithandwithoutanti-

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DBPblockingantibodies.Theresultsshowedthattheabilityoftheanti-DBPantibodiestopreventinvasionofhuman redblood cellswashighlydependentupon theparasite strainsusedwhichhasbeen theorizedbutneverdefinitivelyshownuntilnow.Anti-DBPantibodiesdirectedagainsttheDBPvariantfoundintheAotusisolatedThailandstrainshoweda97.0%inhibitionofinvasion.Conversely,thesesameanti-DBPantibodiesonlyshowed1.4%inhibitionoftheP.vivaxNicaraguastrain.Theseresultsdemonstratethatpolymorphismsintheparasite’sDBPmayplayalargerroleininvasionsuccessthanpreviouslybelieved.Withthisproofofconcept in hand we are developing similar tests of P. vivax infected patient samples invading human redbloodcellsusingDDAOstainedtargethumancells.

STUDYINGCOMPLEXBIOLOGYINSOLIDTUMORSDAVIDHEDLEY(PRINCESSMARGARETHOSPITAL,TORONTO,ON)

Mostofourworkisfocusedonpancreaticcancers,whicharehighlylethal.Wemakeextensiveuseofalargeresource of patient-derivedmaterial, including primary xenografts, patient-derived primary cell lines, andsurgical samples. Despite a large sequencing effort, few patients are identified with driver mutations forwhicheffective targeted treatmentsareavailable.Thechallenge is therefore todevelopnewapproaches tounderstandingthebiologyofpancreaticcancerthatcanleadtoinnovativeapproachestotreatment.Weareapproachingthisfromtwodirections:

• Ourwholegenomesequencingdatahave foundanunusuallyhigh levelof chromosomal instability inpancreatic cancers, which likely accounts for their aggressive biology. It is hypothesized thatchromosomalinstabilityplacesgreaterdependenceonlatecyclecheckpointsthatallowrepairofbreaksduringG2,andcorrectalignmentofchromosomesonthemetaphaseplatepriortoentryintoanaphase.WearedevelopingadvancedflowcytometrytechniquesbasedonEdUpulse/chasewithcombinationsoflatecyclemarkers,inordertomeasuretheactualtransittimesthroughG2andmitosisinvivo,andtotest if drugs that block the G2 and spindle assembly checkpoints show selective toxicity tochromosomally-unstablecancers.

• We have previously shown that high levels of hypoxia occur in some pancreatic cancers, and areassociatedwith aggressive features including rapid growth and spontaneousmetastasis. To establishclinicalrelevance,wearegivingthehypoxiatracerpimonidazoletopatientspriortopancreatectomy.This allows us to establish the extent of hypoxia in the surgical specimen using antibodies topimonidazole, and to map its spatial relations to relevant markers such as signaling pathways andmetabolic regulationusinghistological sections.Wewill complete enrolmentof100patientsbymid-2016,andhavepatientoutcomedatabylate2017.Toanalyzethesamples,wearedevelopingprotocolsforveryhighcontentanalysis,usingtheinnovativetechnologyImagingMassCytometry(IMC).Similarto mass cytometry (CyTOF), the IMC uses metal tagged antibodies and ICP-MS to detect up to 40antibodies.Butinsteadoflabelingcellsinsuspension,IMCisappliedtoregulartissuesectionsthatthenundergo pulsed UV laser ablation to transfer material to the mass cytometer, and software toreconstruct imageswithresolutionsimilarto20xlightmicroscopy.TheIMCisanincrediblypowerfulplatform for studying the complex biology of hypoxia in pancreatic cancer, and aligns well with thewholegenomesequencingbeingdoneonthispatientcohort.

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INSTRUMENTCALIBRATIONANDMEASUREMENTSTANDARDIZATIONFORMEASURING

EXTRACELLULARVESICLESBYFLOWCYTOMETRYJOANNELANNIGAN(FLOWCYTOMETRYCOREATUNIVERSITYOFVIRGINIA,CHARLOTTESVILLE,VA)

Asaresultofthehighthroughputcapabilitiesandabilitytosimultaneouslymeasuremultiplemarkers,flowcytometry is currently themostcommonlyused technology formeasuringextracellularvesicles (EVs).Themajorityoftheinstrumentsusedhavebeendesignedtomeasurecellulareventswithsizeslargerthan1umandfluorescent intensitiesmuchbrighterthanthatwhichwouldbeassociatedwithEVs,whichmeasure inthe rangeof50nm-1um. Inorder to ensure thatmeasurementsofEVsby flowcytometryarepossible andaccurate,instrumentsneedtobecharacterizedintermsofsensitivityandresolution.Inaddition,inordertobe able to compare results across laboratories, it is critically important to standardize measurements bycalibratingintensitiesagainstacceptablestandardssuchasMoleculesofEquivalentFluorescenceorMESFs.Thispresentationwilldiscussusableapproachesforaccomplishingthesegoals.

SYSTEMICMESENCHYMALSTEMCELLMOBILIZATIONANDMIGRATIONFOLLOWINGANTERIORCRUCIATELIGAMENT(ACL)RUPTUREMICHAELNEWTON(DEPARTMENTOFORTHOPAEDICRESEARCH,WILLIAMBEAUMONTHOSPITAL,ROYALOAK,MI)

Asidefromincreasesinintra-articularconcentrationofcytokinesandproteases,theacutebiologicaleventsfollowinganteriorcruciateligament(ACL)ruptureremainlargelyuncharacterized.Theinvolvementofbothlocal and bone marrow-derived mesenchymal stem cells (MSCs) in the acute, intermediate, and chronicphasesafter injuryhavebecomea focus in recent literature.Whilehighly-traumaticevents suchas stroke,myocardial infarction, and longbone fractureareknown to result inmobilizationofmarrow-derived stemcells,itisunknownwhetherorthopaedicsofttissueinjuriesinduceasimilarsystemicresponse.Furthermore,no study has indicated whether mobilized, circulating MSCs migrate to the site of injury to contribute inregenerative or immunomodulatory processes. As the long-term incidence of post-traumatic osteoarthritis(PTOA)hasbeendescribedashighas80–100%followingACLrupture, themobilizationandmigrationofMSCsareattractivemechanismstocharacterizeandpotentiallyexploitinregenerativetherapies.Tothisend,the purpose of this study was to 1) characterize the endogenous mobilization of marrow-derived MSCsfollowingACL rupture; and to 2) assesswhether circulatingMSCsmigrate to the knee joint followingACLruptureinaratmodelofpost-traumaticosteoarthritis.

THEUSEOFFLOWCYTOMETRYFORTHEASSESSMENTOFPRIMARYIMMUNODEFICIENCYDISEASEMAURICER.G.O’GORMAN(CHILDREN’SHOSPITALOFLOSANGELES&KECKSCHOOLOFMEDICINE,USC,LOSANGELES,CA)

Overthepast4decades,flowcytometryhasemergedasaninvaluabletechnologyinclinicallaboratoriesandhas contributedsignificantly to thebiologicalunderstandingand thediagnosticevaluationofpatientswithsuspectedofimmunesystemabnormalities.Theunparalleledabilitytosimultaneouslyidentifyatasinglecelllevel, characteristic physical properties (light scatter), functions, and specific gene products at rates ofthousandsofcellspersecondhasresultedinthedevelopmentofalargerepertoireofdiagnostic,prognostic,andmonitoring assays. Primary immunodeficiency represents a unique opportunity for the application offlowcytometrybasedtestingwithapproximately300classifieddisordersforwhichthemajorityhaveknown

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geneticassociations.Severalofthesegeneticabnormalitiesresultintheexpressionofcellularabnormalitieswhich are readily detected by flow cytometry. Such abnormalities can be broadly grouped as (1)abnormalitiesinspecificleukocytesubset(s),e.g.absenceofBcellsinX-linkedagammaglobulinemia;(2)lossorabnormalexpressionofaspecificcell-associatedmarker(s)e.g.absenceofCD40-ligandonactivatedTcellsinX-linkedhyperIgMsyndrome,and(3)lossorabnormalexpressionofacellularfunctione.g.absenceofanoxidative burst in the granulocytes of patientswith chronic granulomatous disease. The presentationwillinvolve an overview of the primary immunodeficiency diseases in the context of the current IUISclassificationtables,areviewoftheassociatedabnormalitiesandadiscussionoftheflowcytometryassaysthathavebeendevelopedtomeasuretheseabnormalities.

HEPATICIMMUNEREGULATIONBYSTROMALCELL-DERIVEDEXOSOMESFRANKA.SCHILDBERG(MARYLOUINGRAMISACSCHOLAR;DEPARTMENTOFMICROBIOLOGYAND

IMMUNOBIOLOGY,HARVARDMEDICALSCHOOL,BOSTON,MA)

Ametabolic organ, the liver also has a central role in tolerance induction. Stromal cells lining the hepaticsinusoids, such as liver sinusoidal endothelial cells and hepatic stellate cells, are the first liver cells toencountergut-derivedandsystemicantigens, therebyshaping local andsystemic tolerance.Recent studieshave demonstrated that stromal cells can modulate immune responses by antigen-dependent andindependentmechanisms. Stromal cells impair the function of other antigen-presenting cells and interferewith T cell activation, as well as inducing suppressive regulatory T cells andmyeloid-derived suppressorcells. The immunosuppressivemicroenvironment thus created provides ameans to protect the liver fromtissuedamage;however,suchtolerizedsurroundingscanalsobeexploitedbycertainpathogens,promotingpersistent liver infections. Inrecentstudieswecouldshowthat liverstromalcellsreleaseexosomes,whichcouldbe anexciting addition to the existinghepatic immune regulatorymechanisms.Exosomesoccupyanimportantroleasmediatorsofcell-cellcommunicationwithinthemicroenvironment.Functionally,exosomeshavebeenshowntotransfermoleculesbetweencellsandmodulatethephenotypeoftherecipientcell.Usingflow cytometry and electron microscopy, we characterized the phenotype of liver stromal cell-derivedexosomes. Interestingly, we could show that these stromal cell-derived exosomes can, indeed, transportcargo.Thisresultsinphenotypicchangesinhepaticimmunecellsand,ultimately,regulatesthelocalimmunemicroenvironmentintheliver.Thesedataadvanceourunderstandingoftheimportanceofliverstromalcell-derived exosomes and their effects on hepatic immune tolerance. Our data will provide the basis fordevelopingnewimmunotherapiestargetingchronicviralinfectionandcancer.

WHEREDOESCYTOMETRYFITINTODAY’SGENOMICSCRAZEDTWITTER-FEEDWORLD?KEITHSHULTS(CENTERFORINNOVATION,INCELLDXINC.,MENLOPARK,CA)

Thecurrentmindset inbothscienceandthebusinessworldseemstorevolvearoundsilosoftechnologyinwhicheverydiseaseknowntomanmustbeforcedthruindividualsilosinhopesoffindingasolutioninthiscomplexmaze we call “cancer”. It has led to programs such as theMoon Shots Program and the currentimmuno-oncologythrustthatpromisestochangethetreatmentparadigmsinamultitudeofdiseases.Ineachcase, however, we hear the following confusion: heterogeneity! Cytometry has taught us that a prioriknowledgeisverygranularwhenintegratingmultiplecellulardescriptorsinthisworldofheterogeneity.Inthisgenomicscrazedworld,IwishtosharewithyouourexperienceusingtheQuantitativeCellularMultiplexwhichwe feel operates as an integrationpoint capable ofmany readouts fed to computermodels thatwehopecanbeappliedinthepurediagnosticsworldaswellastheCDxuniverse.Intoday’stalk,IwishtosharethegenerationofatrainingsetinbreastcancerthatwearevalidatingatMDAndersoninPA14-0887.Oncewe have established the performance of QCM, wewish to discuss its further expansion into the realm of

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immuno-oncologyandfinallydescribeourthoughtsinitsadaptationtotheworldoflungcancer(Proceedingsofthe107thAnnualMeetingoftheAmericanAssociationforCancerResearch;2016Apr16-20;NewOrleans,LA.Abstract#1372).MyanswertothequestionIpose:stepback,takeadeepbreathandlookatthecell!

ARAPID,INVIVOZEBRAFISHMODELTOELUCIDATETHEMICROMETASTATICPOTENTIALOFHUMANLUNGCANCERCELLSINDRAJITSINHA(ACENZIAINC.,TECUMSEH,ON)

The advent of effective targeted therapeutics has led to increasing emphasis on precise biomarkers foraccurate patient stratification. Here, we describe the role of ACK1, a non-receptor tyrosine kinase inabrogatingmigrationandinvasioninKRASmutantlungadenocarcinoma.Bosutinib,whichinhibitsACK1at2.7nMIC50,wasfoundtoinhibitcellmigrationandinvasionbutnotviabilityinapanelofnon-smallcelllungcancer(NSCLC)cell lines.KnockdownofACK1abrogatedbosutinib-inducedinhibitionofcellmigrationandinvasion specifically in KRAS mutant cells. This finding was further confirmed in an in vivo zebrafishmetastatic model. Tissue microarray data on 210 Singaporean lung adenocarcinomas indicate thatcytoplasmic ACK1 was significantly over-expressed relative to paired adjacent non-tumor tissue.Interestingly, ACK1 expression in “normal” tissue adjacent to tumour, but not tumour,was independentlyassociated with poor overall and relapse-free survival. In conclusion, inhibition of ACK1 with bosutinibattenuatesmigrationand invasion in thecontextofKRASmutantNSCLCandmay fulfila therapeuticnichethroughcombinatorialtreatmentapproaches.

IMMUNOPETIMAGING:ANEMERGINGFRONTIERNARISSAVIOLA-VILLEGAS(KARMANOSCANCERINSTITUTE,WAYNESTATEUNIVERSITYSCHOOLOFMEDICINE,DETROIT,MI)

The progress of targeted therapy allowed selective inhibition of oncoproteins that support growth andproliferationofmalignancies.Overthepastseveralyears,however,ithasbecomeapparentthatattenuatingone survival pathway leads to the pathological induction of another, reinforcing themalignancy. Of greatclinical benefit would be for clinicians to quantitatively assess tumor response to therapy and screen forcompensatingsignalingmechanismsinrealtimeandinanon-invasivemanner.Thisprovidesthebenefitofaltering or including a new treatment course, ultimately minimizing tumor progression and resistance.Moreover,developmentofnewdrugsunderclinical trialswillbenefit fromthesebiomarkersbystratifyingresponders from non-responders and help evaluate engagement of the drug target at the early stages oftreatment.Suchbiomarkersmayberealizedthroughpositronemissiontomography(PET).

Molecularimagingisagrowingfieldwithmajorthrustsindiseasedetection,stagingandtherapyevaluation.With 18F-FDG, the gold standard for PET imaging, limited to measuring only glucose metabolism and isindiscriminateforotheroncoproteinexpressionandamplification,ithasbecomeespeciallydifficulttodetectthese developing molecular changes. Translational research endeavors should put emphasis on thedevelopmentofbiomarkersandfunctionalimagingstrategiesthatpredictapositiveornegativeresponsetotargeteddrugs inawideselectionofmalignancies.Here,wepresentPET imagingstrategies specific to theinhibitedoncoproteintoscreenforresponsetotherapy.

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POSTERABSTRACTSPOSTER1:SHEATHPRESERVATIVE2-PHENOXYETHANOLMAYAFFECTCELLVIABILITYANDFUNCTIONALITYOFSORTEDCELLSERICVANBUREN

WayneStateUniversity,615Hudson-WebberCRC,4100JohnRSt.,Detroit,MI

Cell sorting can be particularly stressful on cells; thus, it is important to determine the ideal sortingconditions to ensure long-term post-sort viability and functionality of cells for a successful culture. Onevariable to consider is sheath fluid. Shared Resource Laboratories (SRLs) often use as sheath fluidcommerciallymanufacturedsaline solutions suchasNERLDiluent2 (D2)orpreservative-freeNERLBloodBankSaline(BBS).Whilebothoftheseoptionsmaybeinterchangeableforparticularcelltypes,ourlabhasobserveddecreasedpost-sortviabilityandfunctionalityincertaincelltypeswhenD2isusedinsteadofBBS.D2 contains ethylene glycol monophenyl ether (2-phenoxyethanol, 2-PE), a preservative which acts as abacteriostatic agent. 2-PE has also been shown to cause deleterious effects in some eukaryotes. To assessviabilityandfunctionalityofaselectionofcell typesexposedto2-PE,wehaveemployedflowcytometrytomeasure dye exclusion, cell membrane permeability, mitochondrial membrane potential, cytosolic pH,reactiveoxygenspecies,andintracellularcalciumionconcentration([Ca2+]i).


POSTER2:CHARACTERIZATIONOFAPRECLINICALMODELTOSTUDYBONEMARROW

MECHANISMSINPULMONARYHYPERTENSIONNICHOLASWANNER1,KELLYWEISS1,KIMBERLYQUISSER1,SERPILERZURUM1,2,KEWALASOSINGH1

1Departments of Pathobiology, Lerner Research Institute, 2Respiratory Institute, Cleveland Clinic,Cleveland,OH

Several studies have shown that bone marrow (BM) contributes to the development of pulmonaryhypertension(PH).OurgroupwasthefirsttoshowthatBMhematopoieticstemcells(HSC)fromPHpatientshave a skewed megakaryocyte-erythroid differentiation and were able to transfer the disease whenxenografted into NODSCID mice. Interestingly, in this cohort, patients with germline caveolin-1 (CAV-1)mutationhada severephenotype.BecauseCAV-1knockout (KO)micedevelopseverePHwhenexposed tohypoxia,weanalyzedwhetherthesemicehavesimilarBMabnormalitiesaspatients.CAV-1KOandwildtype(WT)micewere exposed tonormoxia or3weeksof hypoxia (10%oxygen) andBMcellswere isolated todelineateHSCdifferentiationusingflowcytometry.Lineageneg/lowcellsweregated,thenbasedonc-KitandSca-1expression,primitiveandmultipotentprogenitorcells(KSL)andmyeloidcommittedprogenitors(KL)were selected.Using expressionpatternsofCD135, theKSLpopulationwas further sub-grouped intoHSC,multipotent progenitors (MMP), and lymphoid-primedmultipotent progenitors (LMPP). Commonmyeloidprogenitors (CMP), granulocyte-monocyte progenitors (GMP), andmegakaryocyte-erythrocyte progenitors(MEP)weredefinedfromtheKLsubsetbasedonCD34andCD16/32expression.

KSLsubsetsintheWTandCAV-1KOweresimilarunderbothnormoxiaandhypoxia.WithintheKSLsubset,HSC was higher inCAV-1 KO mice under normoxia or hypoxia. MPP cells were lower inCAV-1KO micecompared toWTmice inbothnormoxiaandhypoxiagroups.KLsubsetswere increased inWTmicewhen

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exposed to hypoxia, but were elevated in normoxicCAV-1KO mice and remained high when exposed tohypoxia. Within the KL subset, CMP cells were lowered in WT mice when exposed to hypoxia, but werealready low in normoxicCAV-1KO mice and remained low when exposed to hypoxia. GMP cells wereincreasedunderhypoxia forWTmice,butwere low forCAV-1KOmiceundernormoxiaandhypoxia.MEPcellswere elevated inCAV-1KOmice under normoxia or hypoxia compared to a lowerMEP subset inWTmiceundernormoxiaorhypoxia.

OurdatashowthatCAV-1KOBMexhibitsskewedHSCdifferentiationsimilartopatientsmakingitasuitable,immunocompetentpreclinicalmodeltostudyBMmechanismsinPH.


POSTER3:CREATINGARESOLUTIONIMPACTMATRIX:ATOOLFORHIGHCONTENTFLOWCYTOMETRYPANELDESIGNVICTORIASMITHANDPHILIPHEXLEY

UniversityofNebraskaMedicalCenter,Omaha,NE

Inmulticolor flow cytometry, after compensation is applied, ameasurement error from fluorescence spillover becomes apparent by spreading of a negative population. This spillover spread is instrument andreagentspecificandwill impactsensitivity inagivenparameterwherethisspreadingoccurs.Therecanbedifferencesbetweenhowwellinstrumentsresolvecertainmulticolorpanels.Herewehaveusedamethodtogenerateaninstrument-specific,andfluorochromefluorochrome-specifictooltoassistwithhighcontentflowcytometrypaneldesign: theResolution ImpactMatrix.To identifyresolutionofagivenparameter,and thedegreetowhichagivenfluorochromewillimpactsensitivityinadouble-positivepopulation,wedemonstratethe generation of a resolution impact matrix for the Fortessa X50: a 5-laser, 30 parameter instrument atUniversityofNebraskaMedicalCenter.

POSTER4:DETECTIONOFNY-ESO-1MRNABYFLOWCYTOMETRYAFTERINDUCTIONWITHDEMETHYLATINGAGENTSKAHTEONGSOH1,PRAGYASRIVASTAVA2,ELIZABETHA.GRIFFITHS2,PAULK.WALLACE1

1DepartmentofFlow&ImageCytometry,and2DepartmentofMedicine,RoswellParkCancerInstitute,Buffalo,NY

Cancertestisantigens(CTA)aretumor-associatedproteinscommonlyfoundinhumanmalignanciesbutnotinnormaladulttissues,exceptforthetestis.NY-ESO-1isaCTAthatcangeneratestronginvivohumoralandcellularimmuneresponses.IthasbeenrecognizedasanattractivetargetincancervaccinetherapyandtheNCIantigenprioritizationpanelrankedNY-ESO-1inthetop10antigensforfurtherdevelopment.Duetoitslowantigenexpressionlevelinovariancancerandacutemyeloidleukemia,theefficacyofNY-ESO-1vaccinetherapy has been limited. The administration of aDNAmethyltransferase inhibitor such as decitabine hasbeen shown to upregulate themRNA andprotein expression ofNY-ESO-1, so that T-cell responses can beaugmentedduringvaccinetherapy.ThemeasurementofNY-ESO-1mRNAusing‘bulk’methodsuchasqRT-PCR may underestimate the actual expression of the gene due to sample heterogeneity, thus making itunsuitable for a discriminative analysis of the tumor environment. Branched DNA (bDNA) is a flowcytometry-basedmethodthatallows for theassessmentofmRNAusing flowcytometryonaper-cellbasis;bDNAprovidesimprovedspecificityviaauniqueprobedesignandalsogreatlyamplifiedfluorescencesignal

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intensitythroughsequentialhybridizationsteps.Usingthistechnique,wemeasuredtheinductionofNY-ESO-1mRNAafter treatmentwithdecitabine.OVCAR3 (ovarian cancer) andKG-1a (myeloblast) cell lineswereincubated for 4 days with 0.5 uM of decitabine and the expression of NY-ESO-1mRNAwas compared tocontrolsnotexposedtodecitabine.NY-ESO-1mRNAwasinducedin47±7%ofOVCAR3and36±1%ofKG1acells(n=3).AparallelstudyusingqRT-PCRconfirmedtheresultsthatNY-ESO-1mRNAwasinducedinbothcell lines. In conclusion, increased level of NY-ESO-1 mRNA in cells after treatment with decitabine wasdetectedusingthebDNAtechniqueandflowcytometry.


POSTER5:FLT-3EXPRESSIONONENDOTHELIALPROGENITORCELLSINSEVEREPREECLAMPSIAMAGGIESCHMIERER1,W.ROYOVERTON1,TATIANAARANGO1,MICHAELMCGRANE1,WADE

ROGERS1,JONNIMOORE2,TODDJOHNSON1,EMILEMOHLER2

1CytoVas,LLC,and2UniversityofPennsylvania,Philadelphia,PA

Preeclampsiaisoneoftheleadingcausesofmaternalandneonatalmorbidityandmortality.Currentmethodstopredictpreeclampsiaarenotreliableanddiagnosisisbasedonexclusion.Aneffectiveprognostictopredictpreeclampsiaanditsseveritybeforetheonsetofclinicalsymptomsisasignificantunmetneed.

Weconductedaflowcytometry-basedstudytoidentifydifferencesincellularbiomarkersrelatedtovascularhealth in pregnant women with preeclampsia (n=10) versus healthy pregnant women (n=10) and non-pregnant women (n=10). Our data demonstrates a strong correlation between severe pre-term birth inwomendiagnosedwithpreeclampsiaandexpressionofcellsurfaceproteinclassIIItyrosinekinase3(FLT3;CD135) on circulating endothelial progenitor cells (EPCs). Expression of FLT3was elevated on peripheralblood EPCs in 5 of the women diagnosed with preeclampsia. All 5 of these women progressed to severepreeclampsiaresultinginseverepretermbirths(gestationalage:24to33weeks).Onlyoneotherpregnancyresulted inaseverepretermbirth(gestationalage:33weeks).TheexpressionofFLT3onendothelialcellshasnotpreviouslybeenreportedandmayhavesignificantvalueasaprognostictest.

SourceofSupport:CytoVas,LLCandBectonDickinsonBiosciences


POSTER6:DISTINCTROLESFORMTORINGENERATINGVERSUSSUSTAININGHUMORALIMMUNITYDEREKD.JONES,BRIANT.GAUDETTE,JOELR.WILMORE,IRENECHERNOVA,ALEXANDRABORTNICK,BRENDANM.WEISS,DAVIDALLMAN

UniversityofPennsylvania,230JohnMorganBuilding,3620HamiltonWalk,Philadelphia,PA

Little is known about the role of mTOR (mammalian target of rapamycin) signaling in plasma celldifferentiationand function.Furthermore, for reasonsnotunderstood,mTOR inhibition reversesantibody-associated disease in lupus-prone (NZB x NZW)F1 mice. Here we report that induced B-lineage specificdeletion of the gene encoding Raptor, an essential signaling adaptor for the rapamycin-sensitive mTORcomplex 1 (mTORC1), or short-term rapamycin treatment abrogated the generation of antibody-secretingplasmacells.Bothstrategiesledtotheablationofnewlyformedplasmacellsinthespleenandbonemarrow

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while also obliterating pre-existing germinal centers. Surprisingly however, although perturbing mTORactivitycausedaprofoundadeclineinserumantibodiesspecificforexogenousantigenorDNA,frequenciesof long-lived bone marrow plasma cells were unaffected. Instead, mTORC1 inhibition led to decreasedexpressionof immunoglobulinbindingprotein(BiP)andother factorsneeded forrobustproteinsynthesis.Consequently blockadeof antibody synthesiswas rapidly reversed after terminating rapamycin treatment.We conclude that mTOR signaling plays critical but diverse roles in early and late phases of antibodyresponses.


POSTER7:KINETICSTUDYOFBCELL-DEPLETIONWITHANOVELMABANTI-MOUSECD20,CLONESA271G2PATRICKSEANMURPHY

BioLegendInc.,9727PacificHeightsBlvd.,SanDiego,CA

CD20 isamemberof themembrane-spanning4A family (MS4A), involvedon the transmembranecalcium-influx, activation and proliferation of B cells. CD20 is first expressed on Pre-B cells and its expression issustainedthroughoutthedifferentstagesoftheBcelldifferentiation,withtheexceptionofplasmacells.CD20is also expressed by many malignant cells from the B cell-lineage, making it an important target forimmunotherapy.Wedevelopedanovel antibodyanti-mouseCD20 (cloneSA271G2, rat IgG2b,κ),useful todepleteBcellsinvivo.Asingleintravenousdoseofantibody(0.25mg)depletedmorethan90%oftheBcellsfrom peripheral blood, spleen and lymph nodes; while in the bonemarrow, only mature B cells (CD19hiB220hi IgD+ IgMhi IL-7Ra-), but not immature B cells (CD19int B220lo IgD- IgM-/int IL-7Ra+), wereeliminated.TheBcelldepletionwassustainedforover20days;thenagradualreturnoftheBcellpopulationwasobserved,andwasfullyrecoveredaroundday40.Duringthistime,nonoticeableeffectofthisantibodyonCD20-cellswasobserved.SA271G2isanovelandusefultooltostudytheroleofBcellsoninvivomodelsofdisease,mechanismsoftheimmuneresponseandimmunotherapy;withtheadvantagesofallowingtheuseofonlywildtypemice,eliminatingtheneedofgeneticallymodifiedanimals,andofhavingafullyreversibleeffectonBcelldepletion.


POSTER8:SMALLPARTICLEFLOWCYTOMETRY:LIGHTSCATTERMEASUREMENTOF

EXTRACELLULARVESICLESAND100NMSILICABEADSANDREWCOSGROVEANDOLIVERKENYON

ApogeeFlowSystemsLtd.,7250WColfaxAve.,C103,Golden,CO

In a typical extracellular vesicle sample themajority of vesicles are smaller than 300nm in diameter andthereforedifficulttomeasureonaconventionalflowcytometer.Flowcytometersareabletomeasurevesiclesofanysizeiflabelledwithsufficientfluorescentmolecules.Inpracticethelabelingofsuchsmallparticlesisweak and conventional flow cytometers struggle to resolve300nm silica beads fromoptical noise by lightscatter. However, recent electronic and optical developments allow the routine measurement of particleswhichscatterordersofmagnitudelessthan300nmsilicabeadsandanorderofmagnitudelessthan100nmlatexbeadswhichhaveoftenbeenusedasalowerlimitreference.Suchdevelopmentsmakelightscatteringanessentialtoolforthereliableinterpretationofextracellularvesicledatabutinfuturea100nmsilicabead

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maybeamoresuitablereferenceparticleduetoitsrefractiveindex,sizeandstability.Wepresentdatafrom100nm silica beads analysed on a commercially available flow cytometer, showing their relationship to110nm latexbeadsanddemonstrating thatparticleswhich scatter1000 times less light than300nmsilicabeads can now be measured routinely, thus permitting the study of a greater proportion of a typicalextracellularvesiclepopulationthanhaspreviouslybeenpossible.


POSTER9:EXAMININGTHELINKBETWEENDIABETICRETINOPATHYANDTHESPLENICCLOCKELENIBELI1,YUANQINGYAN2,YAQIANDUAN3,TATIANASALAZAR1,JAMESDOMINGUEZ1,JUDEAL-SABAN1,SERGIOLICALZI1,REHAEMILLER1,JULIABUSIK4,MARIAB.GRANT1

1DepartmentofOphthalmology,IndianaUniversitySchoolofMedicine,Indianapolis,IN;2Departmentof Biostatistics, MD Anderson Cancer Center, Huston TX; 3Department of Physiology, IndianaUniversitySchoolofMedicine,Indianapolis,IN;4DepartmentofPhysiology,MichiganStateUniversity,EastLansing,MI

Murinemodelsofdiabeteshavebeeninstrumentalinidentifyingaroleforinflammatorycellsinprogressionof diabetic retinopathy. However, little is known about the source of the inflammatory cells in the retina.Currentthinkingisthatthesecellscomedirectlyfromthebonemarrow.Asthespleenisthelargestunitofthemononuclearphagocytesystemandinthemousecontainsalmosthalfofthebody’smonocytes,readytobedeployedwhen theyareneeded,wehypothesized that thespleen isanextramedularsourceofmyeloidcellsarrivingtotheinjuredretina.Weusedtheocularischemia/reperfusion(I/R)modeltoestablishretinalmicrovascularinjury.SplelectomizedorshammiceunderwentI/Randtheinfiltrationofmyeloidcellsintheretinawasmeasuredwithflowcytometry.Weshowthattheinfiltrationofmonocytestotheinjuredretinaofsplenectomizedmiceisreducedbyhalfcomparedtoshamcontrols,indicatingasplenicorigin.Inaddition,wedemonstrate that splenic monocytes are an early target of diabetes. We show that early in STZ-induceddiabetes (4months), disruption of diurnal rhythmsofmonocytes occurs only in the spleen andnot in thebonemarrow.We further relatecircadiandisruption in thespleen toalteredsympathetic toneandalteredexpression of Bmal-1, a key clock gene. As diabetes has been shown to affect the circadian clock, theimplications of altered splenic circadian rhythms require further investigation in the content of theprogression of diabetic retinopathy. Based on these resultswewill pursue future studies to treat diabeticcomplications,basedonalignmentof thediabeticclockeitherbyaltering light/darkcycles, chronodiets,orusingpharmacologicalagentstargetingmetabolismatspecifictimesoftheday.


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POSTER10:MULTIPLEXEDDETECTIONOFBIOLOGICALANDCHEMICALFOODCONTAMINANTSUSINGSPARK-INDUCEDBREAKDOWNSPECTROSCOPY(SIBS)CARMENGONDHALEKAR1,2,EVABIELA1,BARTEKRAJWA1,3,EUIWONBAE4VALERYPATSEKIN1,JENNIFERSTURGIS1,HUISUNGKIM4,IYLL-JOONDOH4,LARRYSTANKER5,J.PAULROBINSON1,2

1BasicMedicalSciences,CollegeofVeterinaryMedicine; 2WeldonSchoolofBiomedicalEngineering;3BindleyBioscienceCenter;4SchoolofMechanicalEngineering;PurdueUniversity;5USDA-ARS,CA

Introduction:Thesafetyoffoodresourcesisnotonlyabiosecurityissue,butalsoaconstantconcerntothegeneral population due to the adverse health effects of contaminants and the time-intensive detectionprocess.Wedevelopedasamplepreparationandanalysisplatformwhosegoalistoreducethetimeandcostofcontaminantdetectionbasedon theuseofspark-inducedbreakdownspectroscopy(SIBS).TheprinciplecomponentsoftheplatformaretheFireflysparkgeneratorandhigh-resolutionspectrometersthatcover220nmto590nmrange.Auniqueaspecttoourstudywasthereplacementoffluorochromes,typicallyusedfordetectionof contaminants,with lanthanides.Lanthanidesarenotnaturallyoccurring inbiological samples,thus reduce thepotential forbackgroundeffects.The spectral emissionproducedby lanthanides isuniquebasedonthespecies(La,Ce,Pr,Nd,Pm,Sm,Eu,Gd,Tb,Dy,Ho,Er,Tm,Yb,Lu)andinducedbyahighenergyspark pulse applied to the sample. In one sampling event, we aim to simultaneously identify differentlanthanide species in the sample, and infer thepresenceofup to four classesand twosub-classesof toxin(shiga,botulinum,ricintoxinsandaflatoxinB1andM1).MethodsandResults:ToprepareasampleforSIBS,we constructed an assay that labels organic toxins immobilized on a solid-phase surfacewith lanthanide-taggedanti-toxinantibodies.Beforeutilizing lanthanides,we tested thesystemusing fluorescentanti-toxinantibodies as this allowed us to determine the assay's efficiency using flow cytometry. We successfullyfunctionalized silicabeadswith the toxinand fluorescent antibody label,washedexcess antibody from thesample, andoptimized theprotocol topreventnon-specific bindingofmiscellaneousprotein.Theultimategoalistouselanthanide-taggedantibodiesandthelow-costSIBStechnologywearedeveloping(seeabstracton instrument) to detect toxins bound to beads. To perform SIBS on a sample, we apply 3 μl to a 10mmdiameter cutoutofWhatmanpaper.Basedonourpreliminary results, a positive correlationwasobservedbetween the concentration of beads in the sample (0.5, 0.05, 0.005, 0.0005 g/ml) and spectral emissionintensity at thewavelength characteristic to silica beads (R2: 0.98) we hypothesize a similar relationshipbetween signal intensity and amount of toxin in a sample. Conclusion: A key advantage of this rapiddetectionassayistheabilitytomultiplextheantigensofinterestandtakeadvantageoftheuniquespectralemissionofeachlanthanidetouniquelytagasmanyas15typesoftoxininasinglesample.Thistechnologyhasadvantagesoverfluorochromesbecauseofthehighresolutionoflanthanideatomicspectra.

POSTER11:STIMULATEDRAMANSCATTERINGFLOWCYTOMETRYFORLABEL-FREESINGLE-PARTICLEANALYSISKAI-CHIHHUANG1,BARTEKRAJWA2,JUNJIELI1,SHIQIYANG1,HAONANLIN1,CHIEN-SHENGLIAO1,GREGORYEAKINS1,SHIHUANKUANG1,VALERYPATSEKIN3,J.PAULROBINSON3,JI-XINCHENG1

1Department of Biomedical Engineering, College of Engineering; 2Bindley Bioscience Center;3Department of Basic Medical Sciences, College of Veterinary Medicine Purdue University, WestLafayette,IN

Flowcytometry isoneof themostwidelyusedtechnologies forhigh-throughputsingle-particleanalysis. Inmodern flow cytometry, fluorescent labeling acts as the primary approach for cellular analysis. However,

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fluorescent labeling is not suitable to all cases, especially for studying small molecules. In such cases,fluorescent labeling may significantly perturb the biological functionality. A label-free flow cytometrytechnique capable to provide chemical information of the cellswithout disrupting cellular functionwouldoffer significant value in biological and pharmaceutical research. Spontaneous Raman flow cytometryprovides the capability to noninvasively detect chemical compositions of cells, but suffers from slow dataacquisition.ThethroughputofatypicalspontaneousRamanflowcytometryis3-4ordersofmagnitudelowerthanthatofacommercialfluorescencebasedflowcytometry.Suchathroughputisfarfrombeingsufficientforpracticalhigh-speedsingle-cellanalysis.Toovercomesuchathroughputlimitation,wedevelopedthefirstmultiplex stimulated Raman scattering flow-cytometry (SRS-FC) prototype able to measure chemicalcontentsofsingleparticlesataspeedof5microsecondsperRamanspectrum.Usingmixedpolymerbeads,wedemonstratetheseparationofdifferentparticlesatathroughputashighas11,000particlespersecond–a throughput similar to that of the commercial fluorescence based flow cytometry. We also show thequantitationoflipidaccumulationin3T3-L1cellsatdifferentlipogenicstagescanbemadeusingSRS-FC.OurSRS-FC technique opens newopportunities for high-throughput and high-content chemical analysis of livecellsinalabel-freemanner.

POSTER12:INTRODUCTIONOFADVANCEDBIOTECHNOLOGYTOTHEUNDERGRADUATECURRICULUMUSINGTHEBDFACSARIAIICELLSORTERGRACEA.ALTIMUSANDDACHENGREN

DepartmentofBiomedicalandChemicalEngineering,SyracuseUniversity,309ALinkHall,Syracuse,NY

Technology is a moving target; new technology is continuously being developed, while old technology isconstantly being applied to new fields and in newways. In order to participate in our increasingly globaleconomy, engineers are expected to be familiar with the wide range of technology and instrumentationcurrently in use throughout the research and industrial fields. However, cost and availability can prohibitstudentsfromgainingexposuretothesetechnologies.

To transform engineering education,we developed new coursemodules and enhanced outreach activitiesusing a state-of-the-art core research facility. Specifically, we aimed to create synergy among teaching,research, and outreach using a core facility of flow cytometry funded through theMRI program ofNSF. Ahigh-endcellsorterandaflowcytometerwereusedtoenhanceteachingandoutreach.

POSTER13:TEMPLATE-BASEDCLASSIFICATIONFORFLOWCYTOMETRYDATA

SHUVRAKANTINATH1,ARIFULAZAD2,RYANCALVERT1,BARTEKRAJWA1,ALEXPOTHEN11PurdueUniversity,WestLafayette,IN;2LawrenceBerkeleyNationalLaboratory,Berkeley,CA

Wedescribealgorithmsfordiscoveringimmunophenotypesfromlargecollectionsofflowcytometrysamplesand using them to organize the samples into a hierarchy based on phenotypic similarity. The hierarchicalorganizationishelpfulforeffectiveandrobustcytometrydatamining,includingthecreationofcollectionsofcell populations’ characteristicofdifferent classesof samples, robust classification, andanomalydetection.We summarize a set of samples belonging to a biological class or category with a statistically derivedtemplate for the class. Whereas individual samples are represented in terms of their cell populations

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(clusters), a template consists of generic meta-populations (a group of homogeneous cell populationsobtained from the samples in a class) that describe key phenotypes shared among all those samples.Weorganize an FC data collection in a hierarchical data structure that supports the identification ofimmunophenotypes. Core algorithms used in our data analysis are available in the flowMatch package atwww.bioconductor.org.Ithasbeendownloadednearly6,000timessince2014.


POSTER14:COTHERAPEUTICIMPACTOFSTINGAGONISTSANDDNAVACCINATIONONANTI-TUMORIMMUNITY

NIKOMOSES,H.GIBSON,J.REYES,R.F.JONES,W.Z.WEI

WayneStateUniversity,110EWarrenAve.,Detroit,MI

ActivationoftheSTINGpathwayinducestheexpressionoftypeIinterferonswhichpromotethematurationof dendritic cells, professional antigen presenting cells that can primeT andB cells specific for foreign oralteredselfantigens.TheSTINGpathwayisagonizedbycytosolicDNA,adangerassociatedmolecularpattern(DAMP) thatcanbe indicativeofhostDNAdamageor intracellular infection.Thispathwaycanbe induceddirectly by treating STING-expressing cellswith cGAMP, a cyclic dinucleotide (CDN). STING activation andsubsequenttypeIinterferonexpressioninthetumormicroenvironmenthasgainedinterestasananti-tumortherapeutic. Recruitment andmaturation of DCs in the tumormicroenvironment exposes them to tumor-associated antigens (TAAs) and can promote anti-tumor immunity. We have tested the combination oflocalized STINGagonistwithDNAvaccination to create a synergistic therapeutic effect against establishedbreasttumors.OurDNAvaccineispE2TM,aplasmidencodingtheextracellularandtransmembraneregionsofHER2,whichinducescross-reactivecytotoxicTcellresponsestotheTAAratneuexpressedonthemousemammarytumorlineTUBO.IntratumoraladministrationofcGAMPaloneinTUBObearingmicehasmarginalimpactontumorgrowth.Similarly,pE2TMvaccinewith intratumoral injectionofacontrol linearanalogofcGAMPresulted inuncontrolledgrowthof establishedTUBO tumorsand limitedanti-neuT cell activation,potentially due to immune suppressive mechanisms within the tumor. The combination of local cGAMPadministration with systemic pE2TM vaccination allows anti-neu T cell induction and restricts tumorprogression.WeevaluatedthecompositionofthesetumorsandthecellspresentintheirmicroenvironmenttoelucidatethemechanismbehindthesynergyofcGAMPwithDNAvaccination.


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POSTER15:OPTIMIZINGACOMMERCIALINSTRUMENTFORTHEDETECTIONOFEXTRACELLULARVESICLES

EDWARDP.PODNIESINSKI1, JOSEPHD.TARIO, JR1,PAULK.WALLACE1,KATHARINEA.MUIRHEAD2,RICHARDB.BANKERT31RoswellParkCancerInstitute,BuffaloNY;2SciGro,Inc.,Middleton,WI;3UniversityatBuffalo,Buffalo,NY

The acquisition and characterization of extracellular vesicles (EVs) by flow cytometry has becomeincreasingly-pursuedinresearchandclinicalapplications.

Typicalmanufacture specifications for benchtop flow cytometers are currently limited to the detection ofparticles that are greater than one micron in size, such as platelets or red blood cells; which makes themeasurement of small (100 nm – 1000 nm) EVs very challenging. We identified some instrumentmodificationsthatcanbeappliedtoastandard,commercially-availableflowcytometerinordertoenhancethedetectionofEVs.

Enhancementstechniquesthatwereimplementedtomakemeasurementsbelow1000nmincludeimproveddetection efficiency (Qr) by reducing thenumberof optic elements in thedetectionpath.Also, instrumentfluidicmodificationsweremade, resulting inminimizedbackgroundnoisebyusinga small, custom-built1litersheathtankfittedwitha0.1micronsheathfilterthat injecteddirectly intothe instruments fluidvalveassembly. This modification eliminated unnecessary lengthy tubing surfaces, which serve as a potentialsourceofsmallparticulatenoise.Thechoiceofpeakheightmeasurementparameters insteadofpeakarea;andalsotheuseofa405nmexcitationlasertomeasurescatteredlightalsoimprovedthesignaldetectionofEVs.Experimentaldatawillalsobepresentedthatappliestheseenhancementstopracticalflowcytometricapplications.


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INVITEDSPEAKERSDAVIDALLMAN

DavidAllmanreceivedhisB.S.inMicrobiologyfromPennsylvaniaStateUniversityin1982.HecompletedhisPhDinImmunologyattheUniversityofPennsylvaniain1993.HeisthecurrentchairofthePh.D.programinimmunology at the Perelman School of Medicine, University of Pennsylvania and an immunologist in thedepartment of pathology & laboratory medicine. He uses an experimental evidence-based approach forteaching fundamental and clinically related immunological concepts to first-year medical students. HisresearchconcernsthemechanismsunderlyingdifferentiationwithintheBcelllineage.

TIMOTHYCOOK

TimothyR. Cookwasborn inMoretown,Vermont in1982.He studiedunder JohnP. CaradonnaatBostonUniversityandwithDanielG.NoceraatMIT(Ph.D.,2010).AftertwoyearsofpostdoctoralstudyintheStangGroup at the University of Utah, he joined the faculty as a research assistant professor investigatingcoordination-drivenself-assemblyfortheconstructionofmolecularpolygons,polyhedra,andprisms.Inthefallof2014,heestablishedagroupattheUniversityatBuffalo,SUNYasanassistantprofessorofchemistry.His lab is currently exploring inorganic self-assembly with an emphasis on designing newmolecules andmaterialsforenergyconversion,photochemistry,andhost/guestchemistry.

JAMESGRANNEMAN

James Granneman is Professor of Molecular Medicine and Genetics at Wayne State University School ofMedicine.TheGrannemanlaboratoryinvestigatesmechanismsofcellularandmetabolicplasticityinadiposetissues. Current efforts are focused on deconvolving adipogenic microenvironments in vivo, elucidatingmolecularmechanismsofadipocytelipolysis,andpreclinicalvalidationofnovelobesitytherapeutics.

DAVIDHEDLEY

David Hedley completed his higher specialist training in medical oncology, combined with a graduateprogramintumourimmunology,attheRoyalMarsdenHospital,UniversityofLondon.Hewasjuniorfacultyat the University of Sydney, Australia, 1981-89 where he was responsible for the development of flowcytometry applications to cancer biology, including the technique forDNA content analysis using paraffin-embeddedtissuethatplayedamajorroleintheearlydevelopmentofclinicalflowcytometry.Since1990hehasbeenSeniorScientist/SeniorStaffPhysicianatthePrincessMargaretHospital,andProfessorofMedicineattheUniversityofToronto,Canada,withamajorfocusonpancreaticcancer.Hislaboratorymakesextensiveuse of patient-derived xenografts that recapitulate the clinical spectrum of the disease, and develops flowcytometrytechniquestostudycomplexbiologicalprocesseslinkedtoexperimentaltreatmentdevelopment.

JOANNELANNIGAN

Joanne Lannigan,M.S. has been actively involved in Flow Cytometry for over 35 years. She served as theAssociate Technical Director of the Clinical Immunology and Flow Cytometry Laboratories at UniversityHospitalattheStateUniversityofNewYorkatStonyBrookfrom1986-1999whereshemanagedtheclinicalimmunologyand flowcytometrypatient testingservices.Since2002shehasbeena facultymember in theDepartmentofMicrobiology,ImmunologyandCancerBiologyintheSchoolofMedicineattheUniversityof

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Virginia as a Senior Research Scientist and Director of the Flow Cytometry Core Facility. Her interest inExtracellularVesiclesbegan in2011aspart of a collaborationwithDr.UtaErdbruegger, anephrologist atUVa. Together they have published multiple publications dealing with the measurement of EVs by flowcytometry, and are currently involved in the standardization efforts of the ISAC-ISEV-ISTH tri-societyconsortium.AspartofISAC’sEVInterestDevelopmentGroup,shehasorganizedmultipleworkshopsattheannualCYTOmeetings.Togetherwith JohnNolanandBobZucker shewasanAssociateeditorona recentSpecialIssueofCytometrydedicatedtothemeasurementofsubmicronparticles.

JOHNNOLAN

JohnNolanisaProfessorattheScintillonInstitute,anacademicnon-profitresearchinstitute,wherehislabdevelopsnewinstruments,reagents,andassaysforhighresolutioncellandmolecularanalysis.HereceivedB.S. degrees inBiologyandChemistry from theUniversityof Illinois, andPh.D. inBiochemistry fromPennStateUniversity.Hedidpost-doctoralworkattheNationalFlowCytometryResource(NFCR)atLosAlamosNationalLab(LANL),wherehewaspromotedtotheTechnicalStaff,becomingDirectoroftheNFCRin2000.From2004-2104,hewasonthefacultyattheLaJollaBioengineeringInstitute.HeisaFellowoftheAmericanInstitute of Medical and Biological Engineering, serves on the Editorial Boards of Cytometry and CurrentProtocolsinCytometry,andisPast-presidentofISAC.Hislab’scurrentresearcheffortsarefocusedonhighlymultiparameter cell measurements using spectral flow cytometry and the high resolution analysis ofextracellularvesicleandothernaturalandsyntheticnanoparticles.

MICHAELNEWTON

The orthopaedic research laboratory at Beaumont Hospital conducts a broad range of basic science,translational, andclinical researchspanning severaldifferent subspecialtiesoforthopaedic surgery. Iworkwithateamofotherengineers,aswellasresidents,volunteers,andsurgeonstoanswerresearchquestions.Themainfocusofourresearchisthedevelopmentandapplicationofquantitativeimagingandimageanalysistechniquestocharacterizepost-traumaticosteoarthritis,intervertebraldiscdegeneration,wearandfailureoforthopaedic implants, and other relevant issues. We utilize micro-computed tomography, MRI, andfluorescent imaging, along with novel image analysis techniques, to characterize disease progression andtreatment.Wehaverecentlydevelopedanon-invasivemodelof isolatedACLrupture inrats,whichweareutilizingtostudytheprogressionofpost-traumaticosteoarthritis.Inadditiontocharacterizationofboneandcartilagechangesviamicro-computedtomography,wehaveutilizedflowcytometryandfluorescentimagingtostudytheeffectsofACLrupture

MAURICEO’GORMAN

Dr. O’Gorman completed his Masters degree, PhD degree, and fellowship in Academic Pathology at theUniversity of British Columbia in Vancouver, Canada studying B cell differentiation and immuneabnormalities in Multiple Sclerosis. He then left Canada to pursue a career in Laboratory Medicine andcompletedafellowshipDiagnosticandClinicalLaboratoryImmunologyintheDepartmentofPathologyandLaboratory Medicine at the University of North Carolina in Chapel Hill. After completing the clinicalfellowship he was awarded a faculty position at Northwestern University as an Assistant Professor ofPediatrics and a Directorship of the Diagnostic Immunology and Flow Cytometry Laboratories at theChildren’sMemorialHospitalinChicago.Hisfocuswasonappliedresearchwherehaspublishedover85peerreviewed manuscripts, edited a text book and written over 25 book chapters. While a professor atNorthwestern University, Dr. O’Gorman completed the executive MBA program at the Kellogg School ofManagement.He leftNorthwesternUniversityafter21yearsofserviceasa fullprofessorofPathologyand

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Pediatrics and served his last 9 years as the Vice Chairman of Pathology and Laboratory Medicine. Dr.O’Gorman is currently Professor of Pediatrics and Pathology at theKeck School ofMedicine, University ofSouthernCaliforniaandservesasChiefofLaboratoryMedicineandtheDirectoroftheClinicalLaboratoriesattheChildren’sHospitalLosAngeles.Histeachingandresearchinterestscontinuetofocusaroundappliedimmunological studies inhealthanddisease.Dr.O’Gormanservesas theReviewsEditor for the JournalofImmunological Methods, has served as a section editor and author for the past four editions of the ASMManualofMolecularandClinialLaboratoryImmunologyandisontheeditorialboardofCytometry.Recentrecognitionofhisresearchandteachingincludeinvitationsaskeynotespeakeratthe14thEuroconferenceinLisbon, Spain, recipient of theWallaceH. CoulterDistinguishedLecturerAward in2014 and theAwardofMerit forcommitment toHIVEducation from in2016.Hecontinues tobeheavily involvedwithmentoringtechnologists, faculty, and studentswithin his institution. Dr. O’Gorman’s commitment to teaching and hiscontributions toclinical immunologyandclinical laboratoryscience ingeneralaredrivenbyhispassiontounderstandmechanisms of diseases in order to develop improved technologies leading to new diagnosticmodalitiesandtheoverallimprovedhealthoutcomesofchildren.

FRANKSCHILDBERG

FrankSchildbergisaresearchfellowatHarvardMedicalSchool.HeearnedhisPh.D.fromtheUniversityofBonn in Germany. His research is focused on the clinical aspects and applications of basic immunologicalresearch,specifically immunemodulationandstromalcell immunology.Frankhasaproventrackrecordinimmune regulation with over 35 peer-reviewed papers. He is investigating how stromal cells regulatedifferent immunecellpopulationsandhowthis influences the inductionandmaintenanceof toleranceandimmunity.Frankhasdevelopednewapproachestostudytheinteractionsofstromaandimmunecellsusingdifferentinvitroandinvivosystems,aswellasadvancedcytometrictools.Hisstudiesprovidethebasisforcellularandmolecularinsightsintohowourimmunesystemisregulatedandhowthisknowledgecouldbetranslated into novel therapies for cancer immunotherapy, infectious diseases, autoimmune diseases,transplantation and fibrosis. Frank is a recipient of the Klaus-Goerttler-Prize of the German Society forCytometry. He is an advisory boardmember of the German Society for Cytometry and co-organizer of itsannualconferences.FrankisanISACMarylouIngramScholaraswellasamenteeof theCytometryPartA-ISACScholarsMentorshipProgramandservesonseveralISACcommittees.

KEITHSHULTS

KeithShultshasbeenfortunatetopracticethescienceofcytometryinmanyformsoverthelast33years.Heis creditedwith co-foundingbothCytometryAssociates andEsoterixCenterof InnovationalongwithGregStelzer. Itwasduring this time that he andGregwere awarded theWallaceCoulter LifetimeAchievementAwardfortheirworkusingSSC/CD45gatingroutinesinthestudyofbonemarrow.Hecontinuedhisjourneybyfollowingthephospho-signalingpathwayastheDirectorofAdvancedCytometricApplicationsatNodalityand iscurrently theVicePresidentof Innovation for IncellDx. It ishisgoal todayandbeforeherunsoutofsheathfluidtosharehisthoughtsontheintegrativescienceofcytometryfusedwithintegrativetechniquesinthisgenomics-crazed,Twitter-feedworld.

INDRAJITSINHA

Dr.IndrajitSinhaisChiefScientificOfficeratAcenziaInc.,ahighlyscience-drivenresearchandmanufacturingcompanyinWindsor,Ontario,Canada.HehasBSc.MSc.andobtainedhisPh.D.fromUniversityofDelhi,India.HeworkedattheInstituteofMolecularandCellBiology,SingaporeaswellasWayneState/KarmanosCancerInstituteinMichiganduringhispostdoctoralresearch.Dr.Sinhahasover25yearsofexperienceinworking

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with research models mimicking human disease and drug development. He is currently focusing oninnovativeZebrafishdiseasemodelsforcancerandotherchronicinflammatoryhealthconditions.

NERISSAVIOLA-VILLEGAS

NerissaViola-VillegasisanassistantprofessorinOncologyandamemberofthemolecularimagingprogramat theKarmanosCancer Institute. She obtainedher PhD in Chemistry at SyracuseUniversity in June2009under the supervision of Prof. Robert Doyle. She was a postdoctoral scholar at Memorial Sloan KetteringCancerCenterfrom2010to2014underthementorshipofProf.JasonS.Lewiswhereshehonedherexpertiseinmolecular imaging. Dr. Viola-Villegas' research focus as an imaging scientist is to develop and validatenovel imaging tools geared to interrogate tumor biology with the long-term goal of putting these tracerstoward clinical translation. She has a broad experience in bioinorganic chemistry, preclinical imaging andradioactive and optical probe development.Herwork has resulted in the development of several positronemissiontomographyradiotracersforthedelineationandmeasurementofoncogenicbiomarkers(i.e.HER2,tumoracidosis,PSMA,CA19.9,CD3)necessaryformalignantgrowthandmetastasis.

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GENERALINFORMATIONCONFERENCEREGISTRATIONDESK• Friday,Sept.16,5:30PMto9:00PM• Saturday,Sept.17,8:00AMto7:00PM• Sunday,Sept.18,9:00AMto12:30PM

POSTERS• Friday,Sept.16,afternooninSalonABCDE• Numbersonposterscorrespondtoposterabstractnumbersintheprogram• Posterboardsize:3ftwideand4fthigh

Pleasemountoneposteroneachsideofaposterboardusingvelcroonly.

• PosterviewingfromFridayafternoontoSundayat10:30AM• Posterpresentationandjudging:Saturday5:00PMto7:30PM

EXHIBITS

ScheduledvendorswillhaveboothsintheExhibit/PosterareaatSalonABCDE.

PleaseNote:Boothset-upforSalonABCDEstartingatnoononSept.16withopeningofSalonABCDEat6:00PM.Breakdownstartingat12:30PMonSunday,Sept.18.

Alltheactivitiesotherthantheplenarysessions,roundtableluncheon,SteeringCommitteemeeting,andthebanquetwillbelocatedintheExhibit/Posterarea(SalonABCDE).

Please frequent theboothsandshowourappreciationforthegenerousfinancialsupportprovidedbythevendorswhosubstantiallyhelp"paythefreight"forthismeeting.

BREAKFASTS

FreecontinentalbreakfastprovidedforallregistrantsisprovidedintheExhibit/PosterareainSalonABCDEon:

• Saturday:7:15AMto8:15AM• Sunday:8:00AMto9:00AM

SteeringCommitteeBreakfastMeeting,Sundaymorning(8:00AMto9:00AM)inDennison1-2-3

COFFEEBREAKS• Saturday:SalonABCDE,9:45AMto10:15AMand3:45PMto4:15PM• Sunday:SalonABCDE,10:30AMto11:00AM

COREMANAGERSMEETING• Friday:Dennison1-2-3,12:00PMto5:00PM

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INDUSTRIALSCIENCESYMPOSIUM&FRIDAYRECEPTION

IndustrialScienceSymposiumpresentations:7:00PMto9:30PM,SalonFGH.

OPENINGRECEPTION• SalonABCDE,6:00PMto8:00PMand10:00PMto11:00PM• Useyourdrinkticketsforwineandbeer!

LOCATIONOFALLORALPRESENTATIONS• Saturday,SalonFGH• Sunday,SalonFGH

SATURDAYLUNCHEONROUNDTABLES• Free lunch/pop;2/3roastbeef/turkeyand1/3veggiewrap inDennison1 (Tables1-3),Dennison2

(Tables 4-6), Dennison 3-4 (Tables 7-9); Niles 1-2 (Tables 10-12); Athens (for the remainingregistrants).

• Pickupboxlunch(roastbeef,turkeyorveggiewrap)andsoftdrinkdisplayedineachroomandmovetoroundtableof10 labeledwiththetitleofdiscussiontopic -attendanceateachtabledeterminedfromsignupsheetatGLIIFCAregistrationdesk.AllotherGLIIFCAparticipantscanlunchintheAthensroom.

SATURDAYWINEANDCHEESEHAPPYHOUR• 6:30PMto7:30PMintheExhibit/PosterareaSalonABCDE• Cheeseandfreshfruittrays• Useyourremainingdrinkticketsforwineandbeer!

BANQUETTheplanningcommitteeisveryexcitedtobringafunandexcitingbanquettocelebratethe25thAnniversaryofGLIIFCA.ThebanquetwillbeablacktieaffairtoringintheSilverYearofoneofthemostcommittedandengagingregionalimaging&flowcytometrymeetingsaroundtheglobe.

The eveningwill be full of fun startingwith dinner and followed by an open bar, a DJ for dancing, dancecompetition, aphotobooth, and the2ndannual cornhole tournament.Wewillhaveprizes for thewinnersandraffleprizesthroughouttheevening.

• Freetoregistrantsandpaidguests• Commencesat8:00PMinMediterraneanRoom• Numerousfoodstations(salad,entréeanddessert)providedtominimizewaitingtime• Fullservicebaravailablefordrinks(OPENBAR!!)• DJwithdancemusicfrom9:00PMto12:00AM;requestsencouraged(getupandhavefun!)

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DRINKS• Full service bar located in the Exhibit/Poster area Salon ABCDE for Friday reception and Saturday

afternoon.Useyourdrinktickets!Softdrinksinbottles/cansarefree.• Openbarin**MediterraneanRoom**forbanquetSaturdayevening.

FACILITIES/SERVICES• MessageBoard:oneaselnexttotheGLIIFCARegistrationDesk• Xeroxcopying,faxing,etc.-askattheRegistrationDesk

CMLECREDITS• ToreceiveCMLEcredit,signapplicationformattheGLIIFCAregistrationdesk.

ADDITIONALENQUIRIES

ContactDr.AlexanderNakeff:

• email:[email protected]• cell:(313)820-6227• ...orleaveamessageattheGLIIFCAregistrationdesk

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DETROITMARRIOTTHOTELMAP

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detroit marriott h friday september 16 sunday … 25 program 2016.pdf · 9:45 am - 10:30 am where...

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