InvitroexpansionofhumanPanTcells -1-

IntroductionTlymphocytes(Tcells)playacentralroleintheadaptive immune system by controlling avarietyofcellularresponsesdefendingthehostagainst pathogens and tumor development.For example, cytokine secretion by Thelpercells suppresses or stimulates immuneresponsesandleadstoantibodyproductionbyB cells, isotype switching, and macrophageactivation. Cytotoxic T cells however directlykillcancercells, virus-infected cells, orotherwisedamagedcells.Theircrucial impacton immuneresponsesanddistinct role in theprotectionagainstdiseasemakeTcellsafocusof many researchers studying immuneregulation.Itisallthemoreimportanttoprovideareliableworkflow for the isolation and cultivation ofhuman Pan T cells directly from peripheralbloodmononuclear cells (PBMCs) that is fullycompatiblewithyourdownstreamapplicationofchoice.Pan T cells are isolated directly from humanPBMCsbymagneticenrichmentusingthePanT Cell Isolation Kit, human and subsequentlyactivated and expanded with the T cellActivation and Expansion Kit, human. T cellpurity, proliferation, and expression ofactivation markers is assessed by flowcytometryatdifferenttimepoints.

Workflow

PBMCisolationIsolationofhumanPanTcellsAnalysisofpurityaftercellseparationCFSElabelingforanalysisofcellproliferationActivationandexpansionofhumanPanTcellsFlowcytometryanalysisofactivationmarkers(e.g.day2)Analysisofcellproliferationandexpansionrates(e.g.days3,6,8,10,13,and14)RemovalofMACSiBeadParticles(optional)

18min

45min

Isolation,cultivation,andexpansionofPanTcellsfromhumanPBMCsInvitroexpansionofhumanPanTcells

14d

90min

8min

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Materials

IsolationofPBMCsusingFicoll-Paque™• PBS-EDTA:Phosphate-bufferedsaline

(PBS),pH7.2,with2mMEDTA.▲Note:EDTAcanbereplacedbyothersupplementssuchasanticoagulantcitratedextroseformula-A(ACD-A)orcitratephosphatedextrose(CPD).

• Ficoll-Paque™(ρ=1.077g/mL)• 50mLconicalcentrifugetubes

Tcellcultivation

• TexMACS™Medium(#130-097-196)• HumanIL-2IS,premiumgrade

(#130-097-744)• 24-wellplate(e.g.Gas-permeable

CulturePlate#150-000-362)▲Note:WiththeGas-permeableCulturePlate,upto2.5´107cells/well/mLcanbestimulatedasopposedto1´107PBMCs/well/mLinstandard24-wellplates.

Optional:• RPMI1640medium• 100´L-Glutaminestocksolution

(200mM)• 100´penicillin/streptomycinstock

solution• 2-Mercaptoethanol• Fetalbovineserum(FBS)

Buffer(standardwashanddilutionbuffer)

• autoMACS®RinsingSolution(#130-091-222)

• Bovineserumalbumin(BSAStockSolution;#130-091-376)

Magneticcellseparation

• PanTCellIsolationKit,human(#130-096-535)

• LSColumns(#130-042-401)• MACS®SeparatorforLSColumns(e.g.

MidiMACS™Separator;#130-042-302)

• MACSMultiStand(#130-042-303)

CFSElabelingforanalysisofcellproliferation• 5(6)-CarboxyfluoresceindiacetateN-

succinimidylester(CFSE;MW557.46,e.g.fromSigma-Aldrich;#21888-25MG-F)

• Dimethylsulfoxide(DMSO;e.g.CryoMACS®DMSO10;#170-076-303)

• Phosphate-bufferedsaline(PBS),pH7.2

• HumanABserumTcellactivationandexpansion

• TCellActivation/ExpansionKit,human(#130-091-441)

Analysis of phenotypic and activation T cellmarkers

• Antibodies,human:· CD2-PE(#130-091-115)· CD3-FITC(#130-080-401)· CD69-APC(#130-092-159)· CD25-PE(#130-091-024)· CD3-APC-Vio®770(#130-096-610)

• MACSQuant® Analyzer, MACSQuantAnalyzer10(#130-096-343),orotherflow cytometers equippedwith violet(405nm),blue(488nm)andred(635nm)lasersabletodiscriminateFITC,PE,andAPCfluorescence▲Note:TheMACSQuantVYBcannotbeused.

• MACS Chill 96 Rack (# 130-094-459),when using MACSQuant Analyzer orMACSQuantAnalyzer10

• MACSQuantCalibrationBeads(#130-093-607),whenusingtheMACSQuantAnalyzerorMACSQuantAnalyzer10

RemovalofMACSiBead™Particles

• MACSiMAG™Separator(#130-092-168)

InvitroexpansionofhumanPanTcells -3-

MaterialpreparationTcellmedium▲MakesuretoaddIL-2freshlytotheTcellmediumforcellexpansion.▲InsteadofTexMACS™Medium,RPMIsupplementedwithFBS(10%finalconcentration),100´L-Glutaminestocksolution(1%finalconcentration),2-mercaptoethanol(0.01mMfinalconcentration)andhumanIL-2(50IU/mL)canbeusedforTcellcultivationandexpansion.▲Additionofpenicillin/streptomycintotheTcellmediumisoptional(e.g.100×penicillin/streptomycinstocksolutiontoafinalconcentrationof1%).PrepareTexMACSMediumsupplementedwithhumanIL-2(50IU/mL).CFSEstocksolutionforcelllabeling

• Preparethe10mMCFSEstocksolutionby dissolving, e.g., 5.57 mg of CFSE(MW557.46)in1mLofDMSO.

• UseCFSEatafinalconcentrationof1µM by diluting the stock solution1:10,000(e.g.1µLCFSEstocksolutionper10mLcellsuspensioninPBS).

• Aliquotsofthestocksolutionshouldbestored at –20 °C or below. Avoidrepeatedfreeze-thawcycles.

Buffer(standardwashanddilutionbuffer)PrepareasolutionofPBS,pH7.2,2mMEDTAand 0.5% BSA by diluting MACS® BSA StockSolution1:20withautoMACS®RinsingSolution.Reconstitution of Human IL-2 IS, premiumgradeIt is recommended to reconstitute lyophilizedHumanIL-2IS,premiumgradewithdeionizedsterile-filteredwatertoafinalconcentrationof0.1–1.0mg/mLinavolumeofatleast100µL.▲Note:Furtherdilutionsshouldbepreparedwith0.1%BSAorhumanserumalbumin(HSA)inPBS.

• TheED₅₀is≤0.2ng/mLcorrespondingtoa

specific activity of ≥5.0×106 IU/mg(calibratedwithNIBSC86/504)or≥1×10⁷IU/mg(calibratedwithProleukin®).

• Recommended stock concentration: 0.1mg/mL by reconstituting a 10 µg vial ofHumanIL-2IS,premiumgradewith100µLdeionizedsterile-filteredwater.

• Thisresultsinafinalactivityof500IU/µL.• Upon reconstitution aliquots should be

storedat–20°Corbelow.Avoidrepeatedfreeze-thawcycles.

• To obtain a cell culture mediumsupplementedwith50 IU/mL,add1.0µLreconstituted Human IL-2 IS, premiumgrade freshly to 10 mL cell culturemedium.

LoadingofAnti-BiotinMACSiBead™Particles▲ Resuspend Anti-Biotin MACSiBead™Particles (contained in the T Cell Activation/ExpansionKit)thoroughlybyvortexingbeforeuse,toobtainahomogenoussuspension.▲ Anti-Biotin MACSiBead Particles aresupplied without preservative. Removealiquotsunderasepticconditions.▲ It is recommended to load Anti-BiotinMACSiBead Particles in batches of 1×108particles. Loaded AntiBiotin MACSiBeadParticles are stable for up to 4monthswhenstoredat2–8°C.1. Pipette 100 µL of CD2-Biotin, 100 µL CD3-Biotinand100µLCD28-Biotin intosealable2mLtubeandmixwell.▲Note:Thisantibodycombination,withafinalantibodyconcentrationof10µgperantibodyper1mLof loadedAnti-Biotin MACSiBead Particles, is optimized forachievingmaximalTcellactivation.2.ResuspendAnti-BiotinMACSiBeadParticlesthoroughlybyvortexing.3. Remove 500 µL Anti-Biotin MACSiBeadParticles(1×108AntiBiotinMACSiBeadParticles)andaddtoantibodymix.4.Add200µLbuffertoadjusttoatotalvolumeof1mL.▲Note:Anti-BiotinMACSiBeadParticlescanbeloadedin a flexible manner with biotinylated antibodies orligands other than those supplied in the T CellActivation/Expansion Kit, human. If desired, add otherbiotinylated antibodies or ligands at appropriateconcentrationsandadjustwithbuffertoatotalvolumeof1mL,accordingly.

InvitroexpansionofhumanPanTcells -4-

5. Incubate for 2 hours at 2–8 °C underconstant, gentle rotation by using theMACSmix™ Tube Rotator (# 130-090-753) atapproximately4 rpm (slowestpermanent runprogram).6. The loaded Anti-Biotin MACSiBead™Particles(1×108particles/mL)arenowreadytouse. Do not remove the loaded Anti-BiotinMACSiBead Particles from the antibody mix.Storeat2–8°Cforupto4months.AntibodypanelforphenotypingFreshly prepare the followingmastermix foreachsample:

• 80µLbuffer• 10µLofeachantibody:

· CD3-FITC· CD2-PE

• Store master mix in the dark in therefrigerator (2−8 °C)untiluse.Donotstoreforextendedperiods.

AntibodypanelforactivationmarkersFreshly prepare the followingmastermix foreachsample:

• 70µLbuffer• 10µLofeachantibody:

· CD69-APC· CD3-APC-Vio®770· CD25-PE

• Store master mix in the dark in therefrigerator (2−8 °C)untiluse.Donotstoreforextendedperiods.

▲Note:HighlyactivatedTcellsmightdown-regulateCD3tosomeextent.Alternatively,CD4andCD8stainings(e.g.withCD4-VioBlue®;#130-097-333andCD8-VioGreen™;#130-096-902) can be used to properly gate on T cellsubpopulations.Methods1. Isolation of human PBMCs using Ficoll-Paque™▲ The peripheral blood or buffy coat shouldnotbeolder than8hoursand supplementedwithanticoagulants(e.g.heparin,EDTA,citrate,ACD-A,orcitratephosphatedextrose(CPD)).1. Dilute cells with 2–4× the volume of PBS-EDTA.▲Note:Themoredilutedthebloodsample,thebetterthepurityofthemononuclearcells.

2. Carefully layer 35 mL of diluted cellsuspension over 15 mL of Ficoll-Paque in a50mLconicaltube.3. Centrifuge at 400×g for 30–40 minutes at20°Cinaswinging-bucketrotorwithoutbrake.4. Aspirate the upper layer leaving themononuclear cell layer (lymphocytes,monocytes,andthrombocytes)undisturbedattheinterphase.5.Carefullytransferthemononuclearcelllayertoanew50mLconicaltube.6.FilltheconicaltubewithPBS-EDTA,mix,andcentrifuge at 300×g for 10minutes at 20 °C.Carefullyremovesupernatantcompletely.7.Forremovalofplatelets,resuspendthecellpelletin50mLofPBS-EDTAandcentrifugeat200×g for 10–15 minutes at 20 °C. Carefullyremovethesupernatantcompletely.▲Note:This stepwill increase thepurityof the targetcellsinthesubsequentMACS®CellSeparation.8. Repeat step 7. Most of the platelets willremaininthesupernatantuponcentrifugationat200×g.9. Resuspend cell pellet in an appropriateamountofPBS-EDTAandproceedtomagneticlabeling.▲ Note: PBMCs may be stored in the refrigeratorovernight in PBS containing 0.5% BSA or autologousserum. Do not store cells longer than one day in therefrigerator. Wash at least once before proceeding tomagnetic labelingandresuspendcells inanappropriatebuffer. For details see MACS Cell Separation Reagentsdatasheets.2.Magneticlabeling▲Workfast,keepcellscold,andusepre-cooledsolutions(2–8°C).▲Volumesformagneticlabelinggivenbelowareforupto10⁷totalcells.Whenworkingwithfewercells,usethesamevolumesasindicated.Whenworkingwithhighercellnumbers,scaleupallreagentvolumesandtotalvolumesaccordingly.▲Foroptimalperformanceitisimportanttoobtainasingle-cellsuspensionbeforemagneticlabeling.

InvitroexpansionofhumanPanTcells -5-

1.DeterminecellnumberofPBMCs.2.Resuspendcellpelletin40µLofbufferper107totalcells.3.Add10µLofPanTCellBiotin-AntibodyCocktail(containedinthePanTCellIsolationKit,human)per107totalcells.4.Mixwellandincubatefor5minutesintherefrigerator(2−8°C).5.Add30µLofbufferper107totalcells.6.Add20µLofPanTCellMicroBeadCocktailper107totalcells.7.Mixwellandincubatefor10minutesintherefrigerator(2−8°C).8.Proceedtosubsequentmagneticcellseparation.▲Note:Aminimumof500µLisrequiredformagneticseparation.Ifnecessary,addbuffertothecellsuspension.3.Magneticseparation▲ Always wait until the column reservoir isemptybeforeproceedingtothenextstep.▲ChooseanLSColumnandasuitableMACS®Separator.1.PlaceLSColumninthemagneticfieldofasuitableMACSSeparator.FordetailsrefertotherespectiveMACSColumndatasheet.2.Preparecolumnbyrinsingwith3mLofbuffer.3.Applycellsuspensionontothecolumn.Collectflow-throughcontainingunlabeledcells,representingtheenrichedTcells.4.Washcolumnwith3mLofbuffer.Collectunlabeledcellsthatpassthrough,representingtheenrichedTcells,andcombinewiththeeffluentfromstep3.5.(Optional)Removecolumnfromtheseparatorandplaceitonasuitablecollectiontube.Pipette5mLofbufferontothecolumn.Immediatelyflushoutthemagnetically

labelednon-Tcellsbyfirmlypushingtheplungerintothecolumn.4.Analysisofpurityaftercellseparation▲Thisstepisoptional.Additionalantibodiescanbeincludedintheanalysisaccordingtotherespective needs. For additional antibodiesvisitwww.miltenyibiotec.com.1. Remove a small aliquot from the fractionrepresentingtheenrichedTcells(e.g.50µL).2.Determinecellnumber.3. Centrifuge cell suspension at 300×g for 10minutes.Aspiratesupernatantcompletely.4. For each sample resuspend up to 106nucleatedcells in100µLphenotypingmastermix (see Material preparation, sectionAntibodypanelforphenotyping).5.Mixwellandincubatefor10minutesinthedarkintherefrigerator(2−8°C).▲Note:Highertemperaturesand/or longer incubationtimesmayleadtonon-specificcell labeling.Workingonicerequiresincreasedincubationtimes.6.Washcellsbyadding1−2mLofbufferandcentrifuge at 300×g for 10 minutes. Aspiratesupernatantcompletely.7.Resuspendcellpelletinasuitableamountofbuffer (e.g. 500 µL) for analysis by flowcytometry.▲ Note: Add propidium iodide according to themanufacturer’sinstructionsbeforeflowanalysis.5. CFSE labeling for analysis of cellproliferation▲CFSEisaddedtothefreshlyisolatedPanTcell suspension to assess cell proliferation atany given time point and for every conditiontested.Therefore,pleasedonotuseantibodiesforflowanalysisconjugatedtofluorochromesthat exhibit the same spectrum as CFSE(λex492nm; λem517nm). Alternatively, aseparate well in the cell culture dish can beused for CFSE labeling. Please make sure toprepare at least one additional well per

InvitroexpansionofhumanPanTcells -6-

condition (e.g.stimulatedandnon-stimulatedcontrol).▲Cellproliferationisusuallyanalyzedondays6, 8, and 10. However, please feel free tochoose time points that are appropriate foryour experimental needs (see section 8.Analysis of cell proliferation and expansionrates).▲Toassessexpansionrates,pleasealsodeterminecellnumbers,e.g.,ondays3,6,8,10,13,and14using,e.g.,aNeubauerChamberorthecountingfunctionoftheMACSQuant®Analyzer10(seesection8.Analysisofcellproliferationandexpansionrates).1.Determinecellnumber.2.Centrifugecellsuspensionat300×gfor10minutes.Aspiratesupernatantcompletely.3. Resuspend cells to a final concentrationof2×107cells/mLinPBS.4. Add the CFSE stock solution to a finalconcentrationof 1µM to the cell suspension(e.g.add1µL10mMCFSEstocksolutionto10mLcellsuspension).5.Mixwellandincubatefor10minutesat37°C.6. Add one volume of human AB serum,mixwell and incubate for 5 minutes at roomtemperature(e.g.add10mLhumanABserumto10mLcellsuspension).7.Centrifugecellsuspensionat300×gfor10minutes.Aspiratesupernatantcompletely.8. Resuspend cells to a final concentrationof2×107cells/mLinTexMACS™Mediumwithoutsupplements.9.Repeatsteps7and8towashcellstwice.▲ Note: After the last wash resuspend cells in freshTexMACSMediumsupplementedwithIL-2.10. Cells are now ready for activation andexpansion.Proceedtosubsequentstep.

6.Tcellcultivation,activation,andexpansionwithMACSiBead™Particles1.Resuspend loadedAnti-BiotinMACSiBead™Particlesthoroughlyandtransfer10μL(1×10⁶loaded Anti-Biotin MACSiBead Particles) per2×10⁶cellsintoasuitabletube.▲Note:IfunloadedMACSiBeadParticleswillbeusedfornegative control experiments, replace the loaded Anti-BiotinMACSiBeadParticleswith10μL (1×10⁶beads)ofunloadedAnti-BiotinMACSiBeadParticlesper2×10⁶cells.2. Add 100–200 µL of TexMACS MediumwithoutsupplementstotheloadedAnti-BiotinMACSiBead Particles and centrifuge at 300×gfor5minutes.3.AspiratesupernatantandresuspendloadedAnti-Biotin MACSiBead Particles in 100 µL offresh TexMACS Medium supplemented withIL-2.4. Resuspend cells at a density of 2×10⁶ cellsper900µLofTexMACSMediumsupplementedwithIL-2.5. Add the prepared Anti-Biotin MACSiBeadParticles from step 3 to the 900 µL of cellsuspensionandmixwell.6. Dilute cells with TexMACS Mediumsupplemented with IL-2 to a final density of1×106cellspermLpercm2andaddthemixturetoasuitablecellculturevessel(e.g.2×10⁶cellsin2mLperwellofa24-wellplate).7. Incubateat37°Cand5–10%CO₂forupto3days.▲Note: Inspectculturesdaily,andaddfreshTexMACSMediumsupplementedwithIL-2ifrequired.8. At day 3 gently pipette cell suspension upanddowntobreakupclumps.9.SplitcellsuspensionintotwoequalpartsandaddTexMACSMediumsupplementedwithIL-2.Incubateat37°Cand5–10%CO2.10. Split cell suspension again whenevernecessary (e.g. every 2–3 days or when cellsreach80%confluency)intotwoequalpartsandaddTexMACSMediumsupplementedwithIL-2.Incubateat37°C,5–10%CO2.

InvitroexpansionofhumanPanTcells -7-

▲Note:TheidealstartingcelldensityforTcellexpansionis1–2×106TcellspermL.Inspectthecellculturedaily.Dependingontheexpansionrate,itmightbenecessarytosplittheculturemorefrequentlythanevery2–3days.IfIL-2interfereswithdownstreamexperiments, it canbeomitted.However, omissionwilllowerthecellviability.11.Atday14,resuspendTcellsat2.5×106cellsper mL in fresh TexMACS™ MediumsupplementedwithIL-2.12. For longerexpansionperiods, restimulatethe cells by adding one loaded Anti-BiotinMACSiBead™ParticlepertwocellsTothisend,count the cells and add 12.5 μL loaded Anti-BiotinMACSiBeadParticlesper2.5×106Tcells. 13. Further cultivate cells and repeat steps 8and9every2–3days.▲Note: Inspect the cultures daily. Depending on the

expansionrate,itmightbenecessarytosplitculturesmorefrequentlythanevery2–3days.

14. Proceed to downstream application, e.g.analysisofcells.▲Note: Removal ofAnti-BiotinMACSiBeadParticles isnotrequiredforimmunofluorescentstaining.ForassayswhereTcellsarerequiredtoreturntoafullyrestingstateprior to further stimulation, Anti-Biotin MACSiBeadParticles should be removed at least 24 hours beforerestimulation (see section 9. Removal of MACSiBeadParticles).7.Flowcytometryanalysisofactivationmarkers▲ForanalysisofTcellactivation,samplesaretaken 48 hours after stimulation and stainedforearlyactivationmarkersCD25andCD69todetermine the proportion of activated cellsamongviableCD3+Tcells.▲HighlyactivatedTcellsmightdown-regulateCD3 to some extent. Alternatively, CD4 andCD8 stainings (e.g.withCD4-VioBlue®;#130-097-333 andCD8-VioGreen™; # 130-096-902)can be used to properly gate on T cellsubpopulations.▲Additionalantibodiescanbeincludedintheanalysisaccordingtotherespectiveneeds.ThisspecificpaneliscompatiblewithCFSElabeling.Whenmodifyingtheantibodypanel,makesureto use fluorochrome-conjugated antibodiesthat do not interfere with CFSE analysis (seesection 5. CFSE labeling for analysis of cell

proliferation) or use separate wells for CFSElabeling. For additional antibodies visitwww.miltenyibiotec.com.1. Removea small aliquot from samples (e.g.stimulatedsampleandunstimulatedcontrol).2.Determinecellnumber.3. Centrifuge cell suspension at 300×g for 10minutes.Aspiratesupernatantcompletely.4. For each sample resuspend up to 106nucleated in 100 µL phenotypingmastermix(see Material preparation, section Antibodypanelforphenotyping).5.Mixwellandincubatefor10minutesinthedarkintherefrigerator(2−8°C).▲Note:Highertemperaturesand/or longer incubationtimesmayleadtonon-specificcell labeling.Workingonicerequiresincreasedincubationtimes.6.Washcellsbyadding1−2mLofbufferandcentrifuge at 300×g for 10 minutes. Aspiratesupernatantcompletely.7. Resuspend cells in a suitable amount ofbuffer (e.g. 500 µL) for analysis by flowcytometry.▲ Note: Add propidium iodide according to themanufacturer’sinstructionsbeforeflowanalysis.8.Analysisofcellproliferationandexpansionrates1.Formonitoringcellproliferationsamplesof25–50µLaretakenatappropriatetimepoints(e.g.days6,8,and10),diluted1:2withbuffer,and CFSE dilution is measured on theMACSQuant® Analyzer 10. At least 20,000eventsarerecorded.▲ Note: Time points can be modified to meetexperimentalneeds.2.Fordeterminationofthecellexpansionratesamples are takenat appropriate timepoints(e.g.days3,6,8,10,13,14)andthecountofviable cells is measured on the MACSQuantAnalyzer10.FordeathcellexclusionPIisaddedpriortoflowcytometricacquisition.Expansionrateiscalculatedafterwards.

InvitroexpansionofhumanPanTcells -8-

9.RemovalofMACSiBead™Particles(optional)▲Forsomeexperiments(e.g.restimulationofT cells) it is recommended to remove theMACSiBead™Particlesfromthecellsuspension.1. Harvest cells and pool cells fromwells thatweretreatedunderthesameconditions.Washempty wells with cold buffer to rinse out theremainingcellsontheplate.2.Determinecellnumber.3.Washcellswithcoldbuffer.4.Resuspendcellsinbufferatadensityofupto2×107/mLandvortexthoroughly.5. Place the tube in themagnetic field of theMACSiMAG™Separator.6.AllowtheMACSiBeadParticlestoadheretothewallofthetubefor4minutes.7.WiththetubestillplacedintheMACSiMAGSeparator, carefully remove the supernatantcontaining the cells depleted of MACSiBeadParticles.Transfercellstoanewtube.8.Removethetubefromtheseparatorandaddthesamevolumeofbufferasbefore.9.Vortexsample,placetubeintheMACSiMAGSeparatorandrepeatsteps5–7.10.Collectedcellscannowbefurtherprocessedasrequired.

Figure1:FlowcytometryanalysisofT cellpurityaftermagnetic separation. Separation of untouched T cellsfromhumanPBMCsbydepletionofnon-TcellsusingthePan T Cell Isolation Kit (human), an LS Column, and aMidiMACS™ Separator. Cells were labeled with CD2-PEandCD3-FITCandanalyzedbyflowcytometryusingtheMACSQuant®Analyzer.

Figure2:FlowcytometryanalysisofactivationmarkersofhumanPanTcells.(A)PanTcellswereisolatedfromhumanPBMCsusingthePanTCellIsolationKit,human.Cells were fluorescently stained with CD3, CD25, andCD69antibodies48hoursafteractivationusingtheTCellActivation/ExpansionKit,human(TCAE)andTexMACS™Medium. Non-stimulated cells served as a control. (A)FlowcytometryanalysisofCD25andCD69performedontheMACSQuant Analyzer 10. (B) Frequencies of CD69+andCD25+CD69+PanTcellswithandwithoutstimulationusingtheTCellActivation/ExpansionKit,human(n=9).

InvitroexpansionofhumanPanTcells -9-

Figure 3: Analysis of cell proliferation. (A) CellproliferationofhumanPanTcellswasanalyzedbyCFSElabelingatdays6,8,and10afteractivationwiththeTCellActivation/Expansion Kit, human (TCAE). CFSE dilutionwas determined using the MACSQuant® Analyzer 10.Gray: cells stimulatedwith TCAE; black: non-stimulatedcontrol.(B)Proliferationrate6daysafteractivationwiththeTCellActivation/ExpansionKit,human(n=9).

Figure 4: Analysis of cell expansion. For thedeterminationoftheexpansionrate,samplesweretakenatthetime-pointsindicatedandthecountofviablecellswas measured with the MACSQuant Analyzer 10. Fordeathcellexclusion,PIwasaddedpriortoflowcytometricacquisition.Expansionratewascalculatedafterwards.

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