NorthTexas2017

FlowCytometryConference

December8th,2017

10amto4pm

UTSouthwesternCampus(NL12)Dallas,TX

FlowCytometryFacilitiesInfo

Institution Children's Research Institute at UT Southwestern

Baylor Research Institute UNT Health Science Center

Shared Resource Section

Moody Foundation Flow Cytometry Facility

Flow Cytometry Shared Resource Laboratory

Flow Cytometry and Laser Capture Microdissection Core Facility

Service Provided

Flow Cytometry related service: • User training • Monthly continuing education (seminars series) • Sort

services • Troubleshooting • User assisted FACS acquisition • experiment design consulting •

Panel development & validation • Data analysis • Daily and monthly

instrument maintenance • Data management • Manage

institutional license for flow cytometry data analysis software.

Other instrument service: • User training on instruments operation

and data collection • Daily and monthly instrument maintenance•

Services provided across two locations • User training •

Monthly continuing education (one-on-one & seminars) • Sort services • Troubleshooting •

User assisted FACS acquisition • Panel development & validation • Data analysis • Daily and monthly

instrument maintenance • Data management • Maintain

institutional license for flow cytometry data analysis.

Flow Cytometry related service: User training on flow cytometry analyzer acquisition and data

analysis • Sort services and user training on sorting•

Troubleshooting • experiment design consulting • Panel

development & validation • Assist on data analysis • Daily and

monthly instrument maintenance • Advanced course

(annually)

Other instrument service: User training on instruments operation and data collection • Experiment design consulting • Daily and

monthly instrument maintenance• Laser Capture Microdissection

Course (one on one basis)

Key Instruments

• 1 BD FACS Canto RUO (12-parameters/3-lasers with

FACSLoader) • 1 FACS Lyric (14 parameters/3 lasers) with universal

loader• 2 BD LSR Fortessa (18-parameters/4 lasers with HTS unit)

• 1 FACS Aria III SORP (17-parameters/5-lasers) in biosafety class II cabinet• 1 FACS Aria III

SORP (15-parameters/4-lasers) in biosafety class II cabinet • 1 BD FACS Aria Fusion SORP (20-

parameters/5 lasers) in biosafety class II cabinet •

• Leica TCS SP8 with Z-stacking

• 2 BD FACS Canto II (8-parameters/3-lasers with HTS unit)

• 1 BD FACS Canto II (8-parameters/3-lasers with loading

carousel) • 1 BD LSRII (Four-lasers/18-parameters cell

analyzers) • 1 BD LSR Fortessa (Five-lasers/18-parameters cell analyzers) • 1 FACS Aria Four-

way cell sorters (18-parameters/5-lasers) • 1 FACS Aria Four-way cell sorters (13-parameters/4-

lasers) • 1 BD Influx Six-way sorter (18-parameters / 4-lasers BSL2+

bioprotect hood) • 1 BD FACS Array (6-parameters/2-lasers) • 1

Cellometer Mini Cell Viability Counter •

• 1 BD LSRII (8-parameters/2-lasers, 488nm and 633nm) •

Beckman Coulter Cytomics FC500 flow cytometer (5-parameters/2-

lasers, 488nm, 635nm) • BD Influx Cell Sorter (7-parameters/2-lasers, 488nm and 635nm)• Sony SH800 Cell Sorter (7-parameters/3-lasers,

488nm, 562nm, 633nm) •

• Veritas Laser Capture Microdissection (molecular

devices) • IVIS Lumina XR small animal imaging (PerkinElmer) •

Contact Information

Nicolas Loof

214-648-4231

Nicolas.loof@utsouthwestern.edu

Kay Kayembe

214 820 3586

Kay.Kayembe@BSWHealth.org

Xiangle Sun

817-735-0117

Xiangle.sun@unthsc.edu

KeynoteandMainSpeakers

MassCytometryandMeasurementofGlobalImmuneCompetence

HoldenMaecker,Ph.D.,StanfordUniversity

DirectoroftheHumanImmuneMonitoringCenter

Professor(research)ofmicrobiologyandimmunology

TheInflammationStory:LeveragingFunctionalNutritiontoFacilitateMuscleRecovery

BrianMcFarlin,Ph.D.UniversityNorthTexas

AssociateProfessor,Kinesiology,HealthPromotion,andRecreation

Director,AppliedPhysiologyLaboratory

UnderstandingOxidativestressinMelanomaMetastasis

ElenaPiskounova,Ph.D.WeillCornellMedicalCollege

AssistantProfessorofCellBiologyinDermatology

DevelopmentofaModularFlowCytometryApproachtoExamineRegulatoryTCellBiology:Activation&Homing

NihanKara,Ph.D.BDBiosciences

ApplicationSupport&Development

ImmunologyApplications

Humanpluripotentstemcells:frommodelingadeglycosylationdisordertodiscoveringnovelanticancerapproaches

Yu-ChiehWang,Ph.D.UniversityNorthTexasHealthScienceCenter

AssistantProfessorofPharmaceuticalSciences

Abstracts

#1)InhibitionofNKcellcytolyticfunctionbycancerstemcellsismediatedthroughPCNA-NKp44interaction.

JosephD.Malaer1andPorunelloorA.Mathew1

Natural killer (NK) cell function is regulated through an intricate balance of signals received fromactivatingandinhibitoryreceptorsinteractingwithligandsonthesurfaceoftargetcellsallowingNKcellstoplayavitalroleintheinnateimmuneresponseagainstcancerandinfection.CancercellsmayevadeNK-mediated killing by expressing ligands for NK cell inhibitory receptors. NKp44, a member of theNatural Cytotoxicity Receptor (NCR) family, can function as an activating or inhibitory receptordepending on ligand interaction. Proliferating cell nuclear antigen (PCNA) associates with HumanLeukocyteAntigen(HLA)IandformstheinhibitoryligandforNKp44,resultingintheinhibitionofNKcellcytotoxicity.Wehave identified diffuse B cell lymphoma (DB) cells and prostate cancer cells (DU145)with cell surface PCNA have a cancer stem cell (CSC) phenotype. The CSC phenotype is described byincreased expression of CD44 and CSC transcription factors as determined by flow cytometry,fluorescence activated cell sorting, and qRT-PCR. Blocking the NKp44-PCNA interaction enhanced NKmediatedkillingofDBcells.CharacterizationofstemcelltranscriptionfactorsandcellsurfacePCNAmayprovidenovelimmunotherapeutictargetsforCSC.

#2)TheEffectofROSonNeutrophilContainmentoftheIntracellularBacteria,Listeriamonocytogenes

Busola M. Okunnu

Extracellularsuperoxidedismutase(ecSOD)iscommonlyregardedashavingaprotectivefunctionduringROS induced inflammation. However, we have shown that ecSOD activity is detrimental to the hostduringinfectionwithListeriamonocytogenes.WiththeuseofecSODcongenicmiceexpressingdifferinglevels of ecSOD activity (ecSOD HI, ecSODWT, ecSOD KO), we found that high ecSOD activity is notconducivetohostsurvivalduringListeria infection. Interestingly,althoughthe infectedecSODHImicehad a higher percentage of neutrophils present in the target organs in comparison to the ecSOD KOmice, theywere alsomore susceptible to infection. To better understand the function of neutrophilsduringinfectionwithListeria,westudiedphagosomalescapeinthecontextofvaryinglevelsofecSODactivity. We observed, in vitro, that a higher percentage of ecSOD KO neutrophils allowed forphagosomalescape.ThisalsocorrelatedwithahigherpercentageofecSODKOneutrophilsassociatingwith the bacteria in comparison to the ecSOD HI neutrophils. Furthermore, although there was nodifference in the relative amount of bacteria that escaped per neutrophil in the context of ecSODactivity, the ecSOD KO neutrophils took up, relatively,more bacteria than the ecSODHI neutrophils.Lastly, analysis ofmonocyte andmacrophage containmentofListeria showed that ecSODactivityhasminimal to no effect onmonocyte andmacrophage containment of Listeria. In conclusion, althoughecSODactivityhaslittletonoeffectonmacrophageandmonocytecontainment,lackofecSODactivityallowsforbetterphagosomalcontainmentofListeriabyneutrophils.

#3)acLDLendocytosisinfluencethedynamicsofintracellularPPAR-gammamRNAandProteinexpressioninPeripheralBloodMonocytes?

Henning,Gary,Levitt,andMcFarlin

Thepathophysiologyofatherogenesisinvolvesabroadrangeofcellularchangesthatcontributetotheonsetandprogressionofatherosclerosisandother formsofcardiovasculardisease.Suchdiseasesareassociatedwithstatesofchronic,low-levelinflammationmediatedbyleukocyteswithpro-inflammatoryphenotypes.Ourlaboratorystudiesperipheralbloodmonocytesduetotheirroleinatherogenesisandtheircontributiontochronicinflammation.PPAR-gammasignalingisanestablishedpathwayinvolvedinatherogenesis,butitspro/antiatherogeniccontributionsarelessunderstood.ThepurposeofthisstudywastoevaluatetheroleofPPAR-gammainfoamcellformationbyobservingtheeffectofmodifiedLDLendocytosis on subsequent expression of PPAR-gamma mRNA and protein expression in individualmonocytes using image-based flow cytometry. When incubated with acLDL, circulating monocytesincreasetheirproductionof intracellularPPAR-gammaprotein.Thischangeappearedtobeassociatedwith new protein production as noted by co-localization of PPAR-gammamRNA and protein. To ourknowledgethisisthefirststudytoreportthatmodifiedLDLcantransientaltertheexpressionofPPAR-gammaatthesinglecelllevel,whichhasimplicationofatherogenesis.

#4)Determiningcellularheterogeneityinhumanprostatewithflowcytometryandsingle-cellRNA-sequencing

HenryGH,MalewsakaA,RoehrbornCG,StrandDW

Theprostateisacomplexheterogeneoustissue,broadlycomposedofstromaandepithelia.Historically,epitheliaweredividedintoneuroendocrine,basal,luminal,andtransientintermediatelayers,basedonpoorly defined immunohistochemical markers. Stromal populations were simply characterized as amixtureofsmoothmuscleandfibroblasts.Thecellularcompositionoftheprostatechangesdramaticallyin diseases such as benign prostatic hyperplasia and cancer, but the identity of these cells has beendifficulttodescribewithtraditionalapproaches.WeareusingsinglecellRNA-sequencingtopredictcelltypes/stateswithnovelmarkersforfutureconfirmationinsitu.

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Agenda

StartTime EndTime Duration Speaker Institution

Breakfast Startingat9:30am

Welcome 9:50am 10:00am 10Min NicolasLoof CRIatUTSW

MainSpeaker#1 10:00am 10:30am 30min ElenaPiskounovaWeillCornellUniversity

Abstract#1 10:30pm 10:50pm 20min JosephD.Malaer UNTHSC

Technicaltalk 10:50am 11:05am 15min BioLegend BioLegend

Coffeebreak 15min

MainSpeaker#2 11:20am 11:50am 30min Yu-chiehWang UNTHSC

Abstract#2 11:50am 12:10pm 20min BusolaM UNTHSC

Abstract#3 12:10pm 12:30am 20min Henning UNT

Food&Drinks/VendorShow

30min

MainSpeaker#3 1:00pm 1:30pm 30min NihanKara BD

Abstract#4 1:35pm 1:55pm 20min GervaiseHenry UTSW

KeynoteSpeaker 2:00pm 2:45pm 45min HoldenMacker Stanford

CoffeeBreak 15min

MainSpeaker#4 3:00pm 3:30pm 30min BrianMcFarlin UNT

Technicaltallk#2

3:30pm 3:45pm 15Min BioRad BioRad

ClosingandAwards

10min KayKayembe BSWRI