Immunophenotyping (CD marker labeling) PBMC’s with Orflo’s … GO-PBMC... · Orflo Application...

O r f l o A p p l i c a t i o n N o t e 1 1 / 2 0 1 6

Immunophenotyping(CDmarkerlabeling)PBMC’swithOrflo’sMoxiGO–NextGenerationFlowCytometer

Introduction/BackgroundPeripheralBloodMononuclearCell(PBMC)purifications are a critically important cellpreparation in a broad range of researchandclinicalstudiesincludingsuchprofoundapplications as HIV research,1 cancerimmunotherapy,2 cord blood banking,3regenerative medicine,4 and fundamentalstudies of cytokine-based immuneresponses5.PBMCs are commonly extracted fromperipheral blood, bone marrow andumbilical cord samples through densitygradient centrifugation or magnetic beadseparations. The approaches are designedtopurifytheleukocytes(PBMCs)ofinterestfrom other cellular components,particularly red blood cells (RBCs).Characterizing the viability and basiccompositionofthefinalcellisolatesiscriticalduetothevariablesassociatedwith:

• Theefficiencyofthesampleextraction,handling,andpurificationprocess.• Thediversityofthesourcesamples(i.e.peripheralbloodvs.cordbloodvs.bonemarrow).• Potentialcelltrauma/necrosisduetothecommonneedtocryopreserveor“biobank”samples.

Beyondtheinitialhealthofthepurifiedsamples,researcherssubsequentlyfocusonimmunophenotypingof the resulting samples to both identify sub-populations of PBMCs for quantification as well as formonitoringthevaryingexpressionlevelsofthecharacteristic“clusterofdifferentiation”(CD)markersthatuniquelycharacterizethesesub-populations. Specifically, fluorophore-conjugatedantibodiesareusedtotarget thevariousCDreceptor/markersandflowcytometry iscommonlyappliedtothequantificationofthemarkerexpressionlevelsandtothedifferentiationofsub-populations.BeyondCDmarkerexpression,precisemonitoringofcountsandcellsizescanbeessentialtotheanalysis.Onesuchexampleisthecriticalimportanceof cell size to treatment efficacy in theextraordinarilypromising researcharenaof chimericantigen receptor T (CAR-T) cell cancer immunotherapy.6 Sizing, in particular, is an area in whichconventional flowcytometrysystemshavetraditionalstruggled,relyingonlight-scatteringasopposedtomoreprecisesizingtechniques,suchasCoulterPrinciplesizing.Inthisapplicationnote,wedemonstratehowOrflo’snewest,“NextGenerationFlowCytometer”,theMoxiGO (Figure 1), can be applied towards the characterization of PBMC preparations. The Orflo Moxi GOsystem is a simple, rapid, and effective flow cytometric platform that can be applied to awide range ofcellular analysis, including PMBC immunophenotyping, counts, purity checks, viability, and sizing. ThesystemcombinestheCoulterPrinciple,therecognizedgoldstandardforprecisecellsizingandcounts,withsimultaneous fluorescent measurements using a green (532nm) laser and 561nm/LP-filtered PMTdetection. The fluorescence configuration is ideal formanyof themost common fluorophores includingphycoerythrin(PE,forimmunolabeling),prodiumiodide(PI,forviability),andRFP(tdTomato/dsRed,,fortransfectionefficiencymeasurement). TheMoxiGOutilizes adisposable flow-cell architecture,doesnotrequirewarming-up, runs test in under 10 seconds, and does not require cleaning/shutdownprotocols.Theresultisanaffordableflowcytometerthatdelivers“AssaysonDemand,”includingforPBMCanalysis.

Figure1–Orflo’sMoxiGO–NextGenerationFlowCytometer. Imageshowsuserloadingasampleintothetwo-testdisposableflowcell.

O r f l o A p p l i c a t i o n N o t e 1 1 / 2 0 1 6

Immunophenotyping(CDmarkerlabeling)PBMC’swithOrflo’sMoxiGO–NextGenerationFlowCytometer

Hereweprovideexampledataandprotocol(s)fortheapplicationoftheMoxiGOtothecharacterizationofPBMCsamples.AuniquefeatureofMoxiGOisthatthesystemallowsPBMCanalysis,tobothflowexpertsandnovices,rightintheirculturehoodorattheirlabbenchtop.Inthisregard,theease-of-useandreadyavailabilityofthesystemwillgreatlyenableresearchers’abilitytorapidlycharacterizeandstudyPBMCs.This, inaddition to theaffordabilityof the system,willundoubtedlyestablish theMoxiGOasa requiredtoolforanylabthatisinvolvedinstudiesthatutilizePBMCs.ExampleData–ResultsandDiscussion In this application note,we appliedOrflo’sMoxi GO to the study of Ficoll-PaqueTM purified PBMCs fromhumanperipheralblood. Thesystemwasapplied togeneratingexamplecharacterizationsof thePBMCsamplebydemonstratingsystemcountsandsizing,PBMCpurityanalysis,andbasicphenotypingbyusingphycoerythrin(PE)-conjugatedantibodytargetstotargetanarrayofCDmarkers.Figure2showsatypicalPMBCtestoutputontheMoxiGO.Afterabrief<10secondtest,aninitialsystemoutput of a scatter plot (Figure 2a) of the 561nm/LP fluorescence vs. Coulter-Based sizing is displayed.Fromthis screen, theusercanquicklysizegate (bluemarker/dots,Figure2a)onsub-populations. Theexampleshows the (predominantly) lymphocytepopulation in thesizegatedregion (Figure2a,betweenbluemarkerlines)andafluorescence(Figure2a,red)gatepositionedtodistinguishtheCD4+(“upper”)vs.CD4- (“lower”) lymphocytes. The black boxes underneath the scatter plot provide the precise, Coulter-Principle-based, concentrations and sizes of the lymphocyte sub-populations as well as the associatedmedian fluorescence intensity (MFI) of each population. To the right of the size-gated region, the(predominantly)monocytes population can be uniquely size-identified from the lymphocyte population.As these are not included in the size-gated region, themonocyte statistics are not included in the blackboxes below (Figure 2a and Figure 2b only). Figure 2b shows the fluorescence (PE) histogram for thelymphocyte-gatedregion,clearlyshowingthefluorescenceseparationintheCD4+vs.CD4-populationsaswell as the relevant populations statistics. Using the touch-screen, the analyzed regions can be quicklyadjustedbytouchinganddraggingthegatemarkers(similartoasmartphoneinterface).InFigure2c,therightsizemarkerwasmovedtothefarrightofthesizerangeandthedisplaywaschangedtoshowthesize

Figure2–User-generatedscreenshots (on-unit) showing thestandardMoxiGOoutput forpurifiedPBMCs. a.) Scatterplotof fluorescence(example shows PE-CD4) vs. size. User can gate by size (bluemarkers) to define region for statistical analysis (concentration, size,MFI),fluorescence (red line)for fluorescence threshold,andnoise (not shown) to reducefile sizesb.)FluorescencehistogramviewshowsthePEfluorescenceforeasyvisualizationof+vs.–populations(PE-CD4exampleshown,gatedonlymphocytes).c.)SizehistogramviewhighlightsthepreciseCoulterPrinciplesizingoflymphocytes(leftpeak)andmonocytes(rightpeak).

O r f l o A p p l i c a t i o n N o t e 1 1 / 2 0 1 6

Immunophenotyping(CDmarkerlabeling)PBMC’swithOrflo’sMoxiGO–NextGenerationFlowCytometer

histogramfor thatregion. With thatview, the finesize-discriminationof the impedance-baseddetectionapproachisclearlyhighlightedintheresolutionofthe(labeled)lymphocytevs.monocytepopulations.Beyond concentration and cell sizing, Figure3 shows someof the keyparameters that theMoxiGO canmeasureinordertodeterminetheoverallpurityandcharacteristicsofthePMBCisolation.First,thecellswerestainedwithpropidiumiodide(PI,firstimage)toassessthecellviabilityfollowingtheprocedure.Asacellimpermeantdyethatincreasesfluorescenceupto50xuponbindingtoDNA,PIiswidelyusedinflowcytometrytodeterminecellviability.Inthiscase,the97%viabilityisvalidationthatthecellstoleratedtheFicollseparationprocedurewell.Additionally,asindicatedbytheyellowarrow,(Figure3,firstimage),thedegreeofRBCcontaminationcanbequalitativelyevaluated. ThePE-CD45label(Figure3,secondimage)providesawaytolabelallleukocytesinthesampletoverifythatthedistinctcellpopulationsare,infact,leukocytesasintended.Inthethirdimage(Figure3),agranulocyte-specificlabel(PE-CD66)wasappliedto verify that the level of granulocyte contamination is lowaswouldexpected for aproperlyperformedisolation. Finally, a CD41/61 label (Figure3, fourth image)was applied to identify the level of plateletcontamination (yellow rectangle) as well as the activated platelet binding to the monocyte population(yellowcircle). UsingstainssuchasthoseshowninFigure2and3, thequalityofPBMCisolationcanbeeasilyandquicklyevaluated.Resultscanthenbespecificallyappliedinfeedbacktoaspectsoftheisolationprotocoltooptimizetheoverallpurificationprotocol/process.Figure 4 shows several example results for the application of theMoxi GO to phenotyping of the PBMCsamplethroughPEconjugatedantibodyCD-markerstaining.ForeachCDmarkerlabel,theMoxiGOscatterplotoutput ispresentedasthetopimage. AfluorescencehistogramoverlayoftheCD-markeroutputvs.theoutputoftheassociatedisotypecontrolsampleisshownbelowthescatterplotimage.Alltheimageswereeasilygeneratedonunitbytheuser. Theoverlayhistogram, inparticular,highlightstheMoxiGO’sabilitytoeasilyandpowerfullyprovidedirectdatacomparisonson-unit. TheexamplesinFigure4showidentificationofthefollowingsub-populations:AllLymphocytes(CD2), TLymphocytes(CD3),CD4+TCells(CD4,sizegated),CD8+T-Cells(CD8,size-gated),monocytes(CD11b),andB-Cells(CD19).SummaryInthisapplicationnote,weprovideaprotocolanddatashowinghowtheMoxiGOcaneasilybeappliedtothecharacterizationofPBMCsamples.TheMoxiGO(Figure1)isaflowcytometerwitha532nm(green)

Figure 3 – Basic PBMC purity and characteristic checks. (PI) Viability stain for PBMC health shows cells tolerated blood draw and Ficollprocedurewell(>97%viable).YellowarrowpointstoresidualRBCpopulation.(CD45)Leukocyte(WBC)genericlabel–confirmsthegatingofpredominantly leukocytes (>90%). Note: Lower left cluster “tail”would be residual RBCs, plateletmultiplets, and other debris. (CD66b)Granulocytelabelshowsverylittle(~2%)granulocytecontamination.(CD41/61)Platelet-specificlabel.Increasedscatterabovenoisecluster(yellowbox)reflectssmallvolumeplateletlabeling.Fluorescentleukocytes(>6µm,yellowcircle)reflectbindingofplateletstothosecells,withaparticularaffinitytomonocytes.

O r f l o A p p l i c a t i o n N o t e 1 1 / 2 0 1 6

Immunophenotyping(CDmarkerlabeling)PBMC’swithOrflo’sMoxiGO–NextGenerationFlowCytometer

laser

Figure4–ExamplesofCDmarkerlabelingofPBMC’s. LabeledtoidentifyCDReceptor(clone). Foreachmarker,ascatterplotofthefluorescenceoutputvs.Coulter-basedsizingisshown(top)andanon-unithistogramoverlayofthemarkeroutput(red)vs.theisotypecontroloutput(gray)isshown(below).

O r f l o A p p l i c a t i o n N o t e 1 1 / 2 0 1 6

Immunophenotyping(CDmarkerlabeling)PBMC’swithOrflo’sMoxiGO–NextGenerationFlowCytometer

and561nm/LPPMTcollectionfilter,ideallysuitedforcapturingemissionspectrafromPhycoerythrin(PE),Propidium Iodide (PI) and RFP (dsRed or tdTomato) cellular labels/stains. One of the most powerfulfeaturesoftheMoxiGOinstrumentistheease-of-useandversatilityincollectionofdata.Withanabilitytorun tests without the need for system warm-up, maintenance, or shutdown procedures, it is uniquelysuitedforquickimmunophenotypingofsamplesincludingdesignofexperiment(DOE)andkinetic-analysisstudies.Inaddition,it’ssmallfootprintandaffordablepriceenableresearcherstoplacetheMoxiGOintheculture hood or lab benchtop, allowing formore immediate and frequent flow analysis of their systemsover (potentially) longperiods of time. ForPBMCanalysis, this easy access allows for great power andflexibilityinperformingpuritychecksandCDmarkerphenotyping.FinallytheMoxiGOtouchscreenGUIisdesignedtomakeevencomplexflowanalysisaccessibletoresearchers,regardlessoftheirflowexpertise.ThesefeaturesshouldestablishtheMoxiGOasastapleinanylabperformingPMBCstudiesorothercell-basedflowcytometrytechniques.References

1. Furtado, M. R. et al. Persistence of HIV-1 Transcription in Peripheral-Blood Mononuclear Cells in Patients Receiving Potent Antiretroviral Therapy. N. Engl. J. Med. 340, 1614–1622 (1999).

2. Kochenderfer, J. N. et al. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19. Blood 116, 4099 LP-4102 (2010).

3. Haase, A. et al. Generation of Induced Pluripotent Stem Cells from Human Cord Blood. Cell Stem Cell 5, 434–441 (2009).

4. Bocelli-Tyndall, C. et al. Bone marrow mesenchymal stromal cells (BM-MSCs) from healthy donors and auto-immune disease patients reduce the proliferation of autologous- and allogeneic-stimulated lymphocytes in vitro. Rheumatol. 46, 403–408 (2007).

5. Duchmann, R. et al. Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease (IBD). Clin. Exp. Immunol. 102, 448–455 (1995).

6. O’Sullivan, D. & Pearce, E. L. Targeting T cell metabolism for therapy. Trends Immunol. 36, 71–80 (2015).

MethodsPBMCisolationfromPeripheralBloodHumanperipheralbloodsampleswerecollectedintosodiumcitrateorsodiumheparincoatedvacutainers.The blood was then processed with Ficoll-Paque Premium (GE Healthcare, 17-5442-02) following themanufacturer’s protocol. Briefly, 2ml blood was diluted 1:1 with HBSS (Sigma, H6648) and carefullylayeredover3mlofficollmediumin15mlcentrifugetubes.Multipletubeswerepreparedandcentrifugedat400xg(40min,18°C).ThePBMClayerwasextractedwithapasteurpipetteandwashed(100g,10min18°C)twicebeforefinalre-suspensioninstainingbuffer(BioLegend#420201).PBMCViabilityStainPBMC’s were stained with Orflo’s Moxi Cyte Viability Reagent (Orflo #MXA055) by adding 15µL of thePBMC final suspension to 135µL of viability reagent and incubating the mixture (5min/roomtemperature/dark). Following incubation, 75µl of the cellswas run on theMoxiGOusing the “ViabilityAssay.”CDmarkerLabelingProtocolAllcellswerelabeledfollowingthe“MoxiGO–CellSurfaceImmunolabelingProtocol”detailedbelow.PE-conjugatedantibodiesusedinthisapplicationnoteweresourcedasfollows:

O r f l o A p p l i c a t i o n N o t e 1 1 / 2 0 1 6

Immunophenotyping(CDmarkerlabeling)PBMC’swithOrflo’sMoxiGO–NextGenerationFlowCytometer

Target(Clone) Manufacturer Cat.#CD2(RPA-T2.10)

TonboBioSciences 50-0029-T025CD3(OKT3) BioLegend 317308

CD4(OKT4) BioLegend 317409

CD8(Hit8A) BioLegend 300907

CD11b(ICRF44) TonboBioSciences 50-0118-ST05CD19(HIB19) TonboBioSciences 50-0199-ST05

CD41/CD61(A2A9/6) BioLegend 359805CD66b(G10F5) BioLegend 305105

PE-conjugatedisotypecontrolsweresourcedasfollows:

Isotype(Clone) Manufacturer Cat.#IgG1,κ(MOPC-21)

Biolegend 400111IgG2a,κ(MOPC-173) BioLegend 400213

IgG2b,κ(MPC-11) BioLegend 400311

DataAnalysisandOutputAllimagesfortheapplicationnoteweregenerateddirectlyfromtheMoxiGOunitusingthe“File|Print|ExportScreenshot”functionalityavailableforeachtest.HistogramOverlays(Figure4)weregeneratedusingtheMoxiGo’son-unit“TestCompare/Overlay”functionality.Off-unitanalysis(notuseddirectlyintheapplicationnote)wasperformedbyexportingtheMoxiGOFCS3.1testfilesintoFlowJoX(MacOSX).

O r f l o A p p l i c a t i o n N o t e 1 1 / 2 0 1 6

Immunophenotyping(CDmarkerlabeling)PBMC’swithOrflo’sMoxiGO–NextGenerationFlowCytometer

MoxiGO–CellSurfaceImmunolabelingProtocol

PrimaryAntibodyStainingProtocol

1. Suspendcellsatdensityof2-5x106cells/mlincellstaining.(Note:ForPBMCSamples,thiswouldbefollowingthelastwashofthePBMC’s,re-suspendthepelletdirectlyintothestainingbuffer).Combinepelletsifnecessary.VerifycountsusingtheMoxiGOinstrument.

2. Aliquot100μlofcells(2-5x105totalcells)per1.5mlmicrocentrifugetubeforeachsampletype.

(Optional)BlockFc-Receptors(recommendedforreducingnonspecificstainingofantibodies)

i. Add1testvolume(typically5μl)ofFcReceptorBlockingSolution(BioLegendcat#422301)toeach100μlvialofcells.

ii. Incubatefor10minutesatroomtemperature.3. Quickspin(e.g.500xg,5seconds)antibodyvialsformaximumvolume.4. Addappropriateantibodytoeachmicrocentrifugetube.5. Vortexeachtubegently.6. Incubatefor15-20minat4°C,protectfromlight.7. Wash2Xwith2mlofCellStainingBufferbycentrifugationat350xgfor

5minutesat≤18°C.8. FORDIRECTLYCONJUGATEDANTIBODIES:

Re-suspendpelletin1mlofcellstainingbuffer(ideallytarget2e5–5e5cells/ml)andadd20μlofMoxiCyteFlowReagent.Invert10xtomix.AnalyzewithMoxiGOusingthe“OpenFlowCytometry”app.FORPURIFIEDANTIBODIES:Re-suspendpelletinresidualbuffer(typically40-50µl)andaddCellStainingBufferto100μltotalvolume.ProceedtoSecondaryAntibodyStaining.

SecondaryAntibodyStaining

9. Add1testvolume(typically2-5μl)of2°Ab(conc.0.2μg/ml)to100μlcells(≤0.5μgpermillioncellsin100μl).

10. Incubatefor15-20minutesat4°C,protectfromlight.11. Wash2xwith2mlofCellStainingBuffer.12. Re-suspendpelletin1mlofCellStainingBuffer(ideallytarget2e5–5e5cells/ml).13. Add20μlofMoxiCyteFlowReagent.Invert10xtomix.14. AnalyzewithMoxiGOusingthe“OpenFlowCytometry”app.

O r f l o A p p l i c a t i o n N o t e 1 1 / 2 0 1 6

Immunophenotyping(CDmarkerlabeling)PBMC’swithOrflo’sMoxiGO–NextGenerationFlowCytometer

MoxiGOCellSurfaceImmunolabelingProtocol-CoreProduct/ReagentList

• Cat#MXG001–OrfloMoxiGONextGenerationFlowCytometer• Cat#MXC030–MF-S+Cassettes• Cat#MXA080–OrfloFlowReagent• CellStainingBuffer(E.g.BioLegendcat#420201orPBSwith0.5%BSA,0.1%Azide)• PBS(anyformulation,e.g.Gibco,Cat#10010023)• Phycoerythrin(PE)labeledantibody(e.g.PEanti-humanCD4-BioLegendcat#300507)–Note:

SubstituteAntibodiesthatarecompatiblewith532nmexcitationand561nm/LPemissioncanalsobeusedinsteadofPE.

• (Optional)HumanTruStainFcX-FcReceptorBlockingSolution(BioLegendcat#422301)

http://www.orflo.com