LEGENDplex™...NP Th Cytokine Mi and Match Subpanel 6 Storage Information Recommended storage for all original kit components is between 2 C and 8 C. DO NOT FREEZE Beads, Detection

LEGENDplex™Mul�-Analyte Flow Assay Kit

Enabling Legendary Discovery™

For Accurate Quantification of Multiple Human Th(T helper Cell) Cytokines from Cell Culture Supernatant,

Serum, Plasma and Other Biological Samples

Please read the entire manual before running the assay

BioLegend.com

LEGENDplex™Mul�-Analyte Flow Assay Kit


Non-Human Primate (NHP) Th Cytokine Panel Mix and Match Subpanel

Please read the entire manual before running the assay.

BioLegend.com

For Research Purposes Only. Not for use in diagnostic or therapeutic procedures. Purchase does not include or carry the right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of BioLegend is strictly prohibited.

It is highly recommended that this manual be read in its entirety before using this product. Do not use this kit beyond the expiration date.

biolegend.com 1

NHP Th Cytokine Mix and Match Subpanel

Table of Contents Page

Chapter 1: KIT DESCRIPTION..................................................

Introduction……………………………………………..........................

PrincipleoftheAssay……………………....……………....….…......

BeadsUsage...........................................………..……………...

StorageInformation…………………………………….......…..........

MaterialsSupplied………………….....……………….................…

MaterialstobeProvidedbytheEnd-User……...........……...

Precautions.................................……………………................

Chapter 2: ASSAY PREPARATION.............................................

SampleCollectionandHandling…………………………............

ReagentPreparation…………………………………………...............

StandardPreparation.........................................................

SampleDilution...........................................……...........…….

Chapter 3: ASSAY PROCEDURE..................................................

PerformingtheAssayUsingaFilterPlate……………….........

PerformingtheAssayUsingaV-bottomPlate………………..

Chapter 4: FLOW CYTOMETER SETUP......................................

Chapter 5: DATA ACQUISITION AND ANALYSIS.........................

DataAcquisition..................................................................

Data Analysis.....................................................................

Chapter 6: ASSAY CHARACTERIZATION.......................................

RepresentativeStandardCurve……...………......…………........

AssaySensitivity...……………………………………………………..…..

Cross-Reactivity……………………………………………………..........

Accuracy.............................................................................

LinearityofDilution………………………………………………..........

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Intra-AssayPrecision……………………………………...................

Inter-AssayPrecision……………………………………...................

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BiologicalSamples…………………………………………….………....

TROUBLESHOOTING...........................………………….………………....

PLATE MAP....................................................................................

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Chapter 1: KIT DESCRIPTION

Introduction

Thelper(Th)cellsplayimportantrolesinregulatingimmuneresponses.Theysecretecytokinestostimulatevariouseffectorcells,suchascytotoxicTcells,Bcellsandmacrophages.AccuratemeasurementofThcytokineexpressioniscriticaltoidentifythecorrespondingThcellsandforin-depthunderstandingofthe immune responses.

The LEGENDplexTM Non-HumanPrimate(NHP) Th Cytokine Panel is a bead-basedmultiplexassay,usingfluorescence–encodedbeadssuitableforuseonvariousflowcytometers.Thispanelallowssimultaneousquantificationof10NHPcytokines,includingIL-2,4,5,6,10,13,17A,21,IFN-γandTNF-α,whicharecollectivelysecretedbyTh1,Th2,Th17,andTfollicularcells.Thisassaypro-videshighsensitivitiesandbroaddynamicrange.The panel has been validated foruseonserum,plasmaandcellculturesupernatantsamplesinbothRhesusand Cynomolgus species.

TheNHPThCytokinePanelisdesignedtoallowflexiblecustomizationwithinthepanel.Itcanalsobedividedintocelltype-specificsubpanels.Pleaserefertothetargetselectiontableforpanel-specifictargetinformation(Table1).Formixandmatchwithinthepanel,please visit www.biolegend.com/legendplex.

This assay is for research use only.

Principle of the Assay

BioLegend’sLEGENDplexTM assays are bead-based immunoassays using the samebasicprincipleassandwichimmunoassays.

Beadsaredifferentiatedbysizeandinternalfluorescenceintensities.Eachbeadsetisconjugatedwithaspecificantibodyonitssurfaceandservesasthecap-turebeadsforthatparticularanalyte.Whenaselectedpanelofcapturebeadsismixedandincubatedwithasamplecontainingtargetanalytesspecifictothecaptureantibodies,eachanalytewillbindtoitsspecificcapturebeads.Afterwashing,abiotinylateddetectionantibodycocktailisadded,andeachdetec-tionantibodyinthecocktailwillbindtoitsspecificanalyteboundonthecap-turebeads,thusformingcapturebead-analyte-detectionantibodysandwiches.Streptavidin-phycoerythrin(SA-PE)issubsequentlyadded,whichwillbindtothebiotinylateddetectionantibodies,providingfluorescentsignalintensitiesinproportiontotheamountofboundanalytes.

Sincethebeadsaredifferentiatedbysizeandinternalfluorescenceintensityonaflowcytometer,analyte-specificpopulationscanbesegregatedandPEfluorescentsignalquantified.Theconcentrationofaparticularanalyteisdeter-mined using a standard curve generated in the same assay.

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Beads Usage

TheNHPThCytokinePanelusestwosetsofbeads.Eachsethasauniquesizethatcanbeidentifiedbasedontheirforwardscatter(FSC)andsidescatter(SSC)profiles(BeadsAandBeadsB,Figure1).Eachbeadsetcanbefurtherresolvedbasedontheirinternalfluorescenceintensities.Theinternaldyecanbede-tectedusingFL3,FL4,orAPCchannel,dependingonthetypeofflowcytometerused.ThesmallerBeadsAconsistsof5beadpopulationsandthelargerBeadsBconsistsof5beadpopulations(Figure2-3).

Usingatotalof10beadpopulationsdistinguishedbysizeandinternalfluo-rescentdye,theNHPThCytokinePanelallowssimultaneousdetectionof10cytokinesinasinglesample.Eachanalyteisassociatedwithaparticularbeadset as indicated (Figures 2-3 and Table 1).

Figure1.BeadsDifferentiatedbySize

Beads A = smaller beads

Beads B = larger beads

Figure2.BeadsAClassificationbyFL4

A5 A7 A8

A4

A6

A10

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Figure3.BeadsBClassificationbyFL4

ForBeadsusageinvariouspanels,pleaserefertoTable1below:

Table1.BeadsIDandPanel-SpecificTargetSelection

Target Bead ID

Th1Cat. No.740381*

&740391**

Th2Cat. No.740382*

&740392**

Th1/Th2Cat. No.740383*

&740393**

Th17Cat. No.740384*

&740394**

TfhCat. No.740385*

&740395**

Top Standard concentration

(ng/mL)

IL-2 A4 √ v The top standard

concentrationof each target may vary and may be sub-

ject to change from lot to lot. Please refer to thelot-specificCertificateofAnalysis for

this informa-tion

IL-5 A5 √ √

IL-6 A6 √ √ √ √ √

IL-10 A7 √ √ √ √ √

IL-13 A10 √ √

TNF-α B2 √ √ √ √ √

IFN-γ B3 √ √ √ √

IL-4 B4 √ √ √

IL-17A B5 √

IL-21 B6 √ √

*Cat#forkitswithfilterplates.**Cat#forkitswithV-bottomplates

BeadIDisusedtoassociateabeadpopulationtoaparticularanalyteintheLEGENDplexTMDataAnalysisSoftware.TheassociationofanalyteandbeadIDwillbedefinedduringthegatingstepofthedataanalysis.

WhenenteringanalyteandbeadIDinfomationduringthegatingstep,alwaysenterinthesequentialorderofthebeadID(e.g,A4,A5,A6...B2,B3,B4...). PleaserefertotheLEGENDplexTMDataAnalysisSoftwareUserGuideandOnlineHelpfordetails(www.biolegend.com/legendplex).

B4 B5

B6 B7

B3

B9

B2

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StorageInformation

Recommendedstorageforalloriginalkitcomponentsisbetween2°Cand8°C.DONOTFREEZEBeads,DetectionAntibodiesorSA-PE.

• Oncethestandardshavebeenreconstituted,immediatelytransfercon-tentsintopolypropylenevials.DONOTSTORERECONSTITUTEDSTAN-DARDS IN GLASS VIALS.

• Uponreconstitution,leftoverstandardandMatrixBshouldbestoredat≤-70°Cforusewithinonemonth.Avoidmultiple(>2)freeze-thawcycles.Discardanyleftoverdilutedstandards.

Materials Supplied

TheLEGENDplexTMkitcontainsreagentsfor100tests,listedinthetablebelow.Whenassayedinduplicate,thisisenoughforan8-pointstandardcurveand40samples.

FortheMixandMatchSubpanels,individualbeadsareprovidedat13Xconcen-tration.TheBufferSetcontainsSetupBeads,allBuffers,PlateSealers,Matrix,SA-PEandDataAnalysisSoftwareDongle.AmanualisprovidedforeachMixand Match subpanel.

Kit Components Quantity Volume Cat #

Capture Beads* (see tables below for moreinformation) varies 270µL varies

LEGENDplex™NHPThCytokineDe-tectionAntibodies 1bottle 3.5mL 740315

LEGENDplex™NHPThCytokineStandard 1 vial lyophilized 740314

LEGENDplex™BufferSetA 1 740368Filter Plate* or V-bottomPlate** 1 plate 740377*or

740379**NHPThCytokineMixandMatchCustom Subpanel Manual 1 76761

*Forkitwithfilterplate.**ForkitwithV-bottomplate.

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Capture beads for Mix and Match Subpanels:

Bead Name Quan-tity

Vol-ume Cat#

LEGENDplex™NHPIL-2CaptureBeadA4,13X 1 vial 270µL 740304LEGENDplex™NHPIL-5CaptureBeadA5,13X 1 vial 270µL 740305LEGENDplex™NHPIL-6CaptureBeadA6,13X 1 vial 270µL 740306LEGENDplex™NHPIL-10CaptureBeadA7,13X 1 vial 270µL 740307LEGENDplex™NHPIL-13CaptureBeadA10,13X 1 vial 270µL 740308LEGENDplex™NHPTNF-αCaptureBeadB2,13X 1 vial 270µL 740309LEGENDplex™NHPIFN-γCaptureBeadB3,13X 1 vial 270µL 740310LEGENDplex™NHPIL-4CaptureBeadB4,13X 1 vial 270µL 740311LEGENDplex™NHPIL-17ACaptureBeadB5,13X 1 vial 270µL 740312LEGENDplex™NHPIL-21CaptureBeadB6,13X 1 vial 270µL 740313

Please refer to BeadsIDandPanel-SpecificTargetSelectiontable(Table1),to see which capture beads are included in each panel.

LEGENDplex™BufferSetA(Cat#:740368)

Component Quantity Volume Part #SetupBeads1:FITCBeads 1 vial 1 mL 77840SetupBeads2:PEBeads 1 vial 1 mL 77842SetupBeads3:RawBeads 1 vial 2 mL 77844LEGENDplexTM SA-PE 1bottle 3.5mL 77743LEGENDplexTMMatrixB,Lyophilized 1 vial lyophilized 77549LEGENDplexTMAssayBuffer 1bottle 25mL 77562LEGENDplexTMWashBuffer,20X 1bottle 25mL 77564DataAnalysisSoftwareDongle 1 21217Plate Sealers 4sheets 78101

NoplateisincludedinBufferSetA.Plateneedtobeorderedseparately.Please order the correct type of plate based on the preferred assay protocol (Cat#740377forFilterPlateandCat#740379forV-bottomPlate)

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Materials to be Provided by the End-User

• Aflowcytometerequippedwithtwolasers(e.g.,a488nmbluelaseror532nmgreenlaseranda633-635nmredlaser)capableofdistinguishing575nmand660nmoraflowcytometerequippedwithonelaser(e.g.,488nmbluelaser)capableofdistinguishing575nmand670nm.

Partiallistofcompatibleflowcytometers:

Flow Cytometer

Reporter Channel

ChannelEmission

ClassificationChannel

Channel Emission

Compensa-tionneeded?

BD FACSCaliburTM

(single laser)FL2 575 nm FL3 670 nm Yes

BD FACSCaliburTM

(dual laser)FL2 575 nm FL4 660 nm No*

BD FACSArrayTM Yellow 575 nm Red 660 nm No*

BD FACSCantoTM

BD FACSCantoTM IIPE 575 nm APC 660 nm No*

BDTMLSR,LSRIIBD LSRFortessaTM PE 575-585

nm APC 660 nm No*

BD FACSAriaTM PE 575 nm APC 660 nm No*

*Compensationisnotrequiredforthespecifiedflowcytometerswhensetupproperly,butisrecommendedforconsistentresults.

Forsettingupvariousflowcytometers,pleasevisit:www.biolegend.com/legendplex and click on the Instrument Setup tab.

• Multichannelpipettescapableofdispensing5μLto200μL

• Reagentreservoirsformultichannelpipette

• Polypropylenemicrofugetubes(1.5mL)

• Laboratoryvortexmixer

• Sonicatorbath(e.g.,BransonUltrasonicCleanermodel#B200,orequiva-lent)

• Aluminum foil

• Absorbentpadsorpapertowels

• Plateshaker(e.g.,Lab-LineInstrumentsmodel#4625,orequivalent)

• Tabletopcentrifuges(e.g.,Eppendorfcentrifuge5415C,orequivalent)

• 1.1mLpolypropylenemicroFACStubes,in96-tuberack(e.g.,NationalScientificSupplyCo,catalog#TN0946-01R,orequivalent).

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NHP Th Cytokine Mix and Match SubpanelIftheassayisperformedinafilterplate;

• Avacuumfiltrationunit(MilliporeMultiScreen®HTSVacuumManifold,cat#MSVMHTS00orequivalent).Instructionsonhowtousethevacuummanifoldcanbefoundatthesupplier’swebsite.

• Avacuumsource(minivacuumpumporlinevacuum,e.g.,MilliporeVacuumPump,catalog#WP6111560,orequivalent).

• Ifneeded,additionalFilterplatecanbeorderedfromBioLegend(Cat#740377or740378).

IftheassayisperformedinaV-bottomplate;

• Centrifugewithaswingingbucketadaptorformicrotiterplates(e.g.,Beck-man Coulter AllegraTM6RCentrifugewithMICROPLUSCARRIERadaptorforGH3.8andJS4.3Rotors).

• Ifneeded,additionalV-bottomplatecanbeorderedfromBioLegend(Cat#740379).

Precautions

• All blood components and biological materials should be handled as poten-tiallyhazardous.FollowuniversalprecautionsasestablishedbytheCenterforDiseaseControlandPreventionandbytheOccupationalSafetyandHealthAdministrationwhenhandlinganddisposingofinfectiousagents.

• Sodiumazidehasbeenaddedtosomereagentsasapreservative.Al-thoughtheconcentrationsarelow,sodiumazidemayreactwithleadandcopperplumbingtoformhighlyexplosivemetalazides.Ondisposal,flushwithalargevolumeofwatertopreventazidebuild-up.

• MatrixBforLEGENDplexTM kits contains components of human origin and shouldbehandledaspotentiallyhazardous.TherawmaterialhasbeenscreenedforinfectiousdiseasesandisnegativeforHIV,HBVandHCVusingFDA-approved test methods.

• Donotmixorsubstitutereagentsfromdifferentkitsorlots.Reagentsfromdifferentmanufacturersshouldnotbeusedwiththiskit.

• Donotusethiskitbeyonditsexpirationdate.

• SA-PEandBeadsarelight-sensitive.Minimizelightexposure.

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Chapter 2: ASSAY PREPARATION

SampleCollectionandHandling

PreparationofSerumSamples:

• Allowthebloodtoclotforatleast30minutesandcentrifugefor10min-utesat1,000xg.

• Remove serum and assay immediately or aliquot and store samples at ≤-20°C.Avoidmultiple(>2)freeze/thawcycles.

• Whenusingfrozensamples,itisrecommendedthatsamplesarethawedcompletely,mixedandcentrifugedtoremoveparticulatespriortouse.

PreparationofPlasmaSamples:

• PlasmacollectionusingEDTAasananti-coagulantisrecommended.Centri-fugefor10minutesat1,000xgwithin30minutesofbloodcollection.

• Removeplasmaandassayimmediately,oraliquotandstoresamplesat≤-20°C.Avoidmultiple(>2)freeze/thawcycles.

• Whenusingfrozensamples,itisrecommendedthatsamplesarethawedcompletely,mixedwellandcentrifugedtoremoveparticulates.

PreparationofTissueCultureSupernatant:

• Centrifuge the sample to remove debris and assay immediately. If not pos-sible,aliquotandstoresamplesat≤-20°C.Avoidmultiple(>2)freeze/thawcycles.

ReagentPreparation

PreparationofAntibody-ImmobilizedBeads

Theindividualbeads(13X)shouldbemixedanddilutedto1XwithAssayBufferpriortouse.Tomixthebeads,followthestepsbelow(a5-plexsub-panelisusedasanexample):

1. Sonicate the beads vials for 1 minute in a sonicator bath and then vortexfor30secondspriortouse.

2.Calculatetheamountofmixedanddilutedbeadsneededfortheassay. Prepareextratocompensateforpipettingloss.Eachreactionneeds 25µLofmixedanddilutedbeads.For50reactions,prepare1.5mLof mixedbeads.For96reactions,prepare3mLofmixedbeads.

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NHP Th Cytokine Mix and Match Subpanel3.Tomake1.5mlof5-plex1Xdilutedbeads,transfer115µLofeachof

the5individualbeads(13X)toafreshtube(totalbeadvolume=575µL)andadd925µLofAssayBuffertomakethefinalvolumeof1.5mL.

PreparationofWashBuffer

• Bringthe20XWashBuffertoroomtemperatureandmixtobringallsaltsintosolution.

• Dilute25mLof20XWashBufferwith475mLdeionizedwater.Storeun-usedportionsbetween2°Cand8°Cforuptoonemonth.

PreparationofMatrixB(forSerumorPlasmaSamplesOnly)

• Add10.0mLLEGENDplexTMAssayBuffertothebottlecontaininglyophi-lizedMatrixB.Allowatleast15minutesforcompletereconstitution.Vortextomixwell.LeftoverreconstitutedMatrixBshouldbestoredat≤-70°Cforuptoonemonth.

StandardPreparation

1. Priortouse,reconstitutethelyophilizedNHPThCytokineStandardwith250µLAssayBuffer.

2. Mixandallowthevialtositatroomtemperaturefor10minutes,andthen transfer the standard to an appropriately labeled polypropylene microcentrifugetube.ThiswillbeusedasthetopstandardC7.

3. Label6polypropylenemicrocentrifugetubesasC6,C5,C4,C3,C2andC1,respectively.

4.Add75µLofAssayBuffertoeachofthesixtubes.Prepare1:4dilutionofthetopstandardbytransferring25µLofthetopstandardC7totheC6tubeandmixwell.ThiswillbetheC6standard.

5.Inthesamemanner,performserial1:4dilutionstoobtainC5,C4,C3,C2and C1 standards (see the table below using 10ng/mL of top standard concentrationasanexample).AssayBufferwillbeusedasthe0pg/mLstandard(C0).

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Tube/Standard

ID

Serial Dilution

Assay Buffertoadd (µL)

Standard to add

Final Conc. (pg/mL)*

C7 -- -- -- 10,000

C6 1:4 75 25µLofC7 2,500

C5 1:16 75 25µLofC6 625

C4 1:64 75 25µLofC5 156.3

C3 1:256 75 25µLofC4 39.1

C2 1:1024 75 25µL of C3 9.8

C1 1:4096 75 25µL of C2 2.4

C0 -- 75 -- 0

SampleDilution

• Serumorplasmasamplesmustbediluted4-foldwithAssayBufferbeforebeingtested(e.g.dilute50µLofsamplewith150µLofAssayBuffer).

Iffurthersampledilutionisdesired,dilutionshouldbedonewithMatrixBto ensure accurate measurement.

Addingserumorplasmasampleswithoutdilutionwillresultinlowassayaccuracyandpossibly,cloggingofthefilterplate.

• Forcellculturesupernatantsamples,thelevelsofanalytecanvarygreatlyfromsampletosample.Whilethesamplescanbetestedwithoutdilutions,apreliminaryexperimentmayberequiredtodeterminetheappropriatedilutionfactor.

Ifsampledilutionisdesired,dilutionshouldbedonewithcorrespondingfreshcellculturemediumorAssayBuffertoensureaccuratemeasurement.

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Chapter 3: ASSAY PROCEDURE

TheLEGENDplexTM assaycanbeperformedinafilterplate,orinaV-bottomplate.

• Thein-filterplateassayprocedurerequiresavacuumfiltrationunitforwashing(seeMaterialstobeProvidedbytheEnd-User,page8). If you haveperformedbead-basedmultiplexassaysbefore,yourlabmayalreadyhavethevacuumfiltrationunitsetup.

• Ifthein-filterplateassayprocedureisnotpossibleorifyouprefer,theas-saycanbeperformedinaV-bottomplate.

Performing the Assay Using a Filter Plate

• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.

• Setthefilterplateonaninvertedplatecoveratalltimesduringassaysetupandincubationsteps,sothatthebottomoftheplatedoesnottouchany surface. Touching a surface may cause leakage.

• Keeptheplateuprightduringtheentireassayprocedure,includingthewashingsteps,toavoidlosingbeads.

• Theplateshouldbeplacedinthedarkorwrappedwithaluminumfoilforallincubationsteps.

• Standards and samples should be run in duplicate and arranged on the plateinaverticalconfigurationconvenientfordataacquisitionandanalysis(asshowninattachedPLATEMAP,page32).Besuretoloadstan-dardsinthefirsttwocolumns.Ifanautomationdeviceisusedforread-ing,theorientationandreadingsequenceshouldbecarefullyplanned.

1. Pre-wettheplatebyadding100μLofLEGENDplexTM1XWashBuffertoeachwellandletitsitfor1minuteatroomtemperature.Toremovetheexcessvolume,placetheplateonthevacuummanifoldandapplyvacuum.Donotexceed10”Hgofvacuum.Vacuumuntilwellsaredrained(5-10seconds).BlotexcessWashBufferfromthebottomoftheplatebypressingtheplateonastackofcleanpapertowels.Placetheplateontopoftheinverted plate cover.

Formeasuringcellculturesupernatantsamples,loadtheplateasshowninthetablebelow(intheorderfromlefttoright):

AssayBuffer MatrixB Standard Sample*

StandardWells 25µL --- 25µL ---

Samplewells 25µL --- --- 25µL

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Formeasuringserumsamples,loadtheplateasshowninthetablebelow(intheorderfromlefttoright):


StandardWells --- 25µL 25µL ---


*See SampleDilution

2. Vortexmixedbeadsbottlefor30seconds.Add25μLofmixedbeadstoeachwell.Thevolumeshouldbe75μLineachwellafterbeadsaddition.(Note:Duringadditionofthebeads,shakemixedbeadsbottleintermit-tentlytoavoidbeadsettling).

3. Sealtheplatewithaplatesealer.Toavoidplateleaking,donotapplyposi-tivepressuretothesealerwhensealingtheplate.Wraptheentireplate,includingtheinvertedplatecover,withaluminumfoil.Placetheplateonaplateshaker,secureitwitharubberbandandshakeatapproximate500rpm for 2 hours at room temperature.

4. Do not invert the plate! Place the plate on the vacuum manifold and apply vacuumasbeforeinStep1.Add200µLof1XWashBuffertoeachwell.RemoveWashBufferbyvacuumfiltration.BlotexcessWashBufferfromthebottomoftheplatewithanabsorbentpadorpapertowels. Repeat this washingsteponcemore.

5. Add25µLofDetectionAntibodiestoeachwell.

6. Sealtheplatewithafreshplatesealer.Wraptheentireplate,includingtheinvertedplatecover,withaluminumfoil.Placetheplateonaplateshakerandshakeatapproximately500rpmfor1houratroomtemperature.

7. Do not vacuum!Add25µLofSA-PEtoeachwelldirectly.

8. Sealtheplatewithafreshplatesealer.Wraptheentireplate,includingtheinvertedplatecover,withaluminumfoil.Placetheplateonaplateshakerandshakeatapproximate500rpmfor30minutesatroomtemperature.

9. Repeatstep4above.

10. Add150µLof1XWashBuffertoeachwell.Resuspendthebeadsonaplateshaker for 1 minute.

11. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay(Note:Prolongedsamplestoragecanleadtoreducedsignal).

Iftheflowcytometerisequippedwithanautosampler,readtheplatedirectly using the autosampler. Please be sure to program the autosampler

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to resuspend beads in the well immediately before taking samples. The probe height may need to be adjusted when using an autosampler.

Ifanautosamplerisnotavailable,thesamplescanbetransferredfromthefilterplateto micro FACS (or FACS) tubes and read manually.

Assay Procedure Summary for Filter PlateAdd 100 μL 1X Wash Bu�er to �lter plate wells

Vacuum to remove excess bu�er

Incubate 2 hours, RT, shaking

Capture beads

Biotinylated Detection Antibody

Analytes

Without washing, add 25 μL SA-PEIncubate 30 min, RT, shaking

Wash 2 times using vacuum �ltration unitAdd 25 μL Detection AntibodiesIncubate 1 hr, RT, shaking

Wash 2 times using vacuum �ltration unitAdd 150 µL of 1x Wash Bu�er Read on a �ow cytometer

BA

C

A B C

A B C

Add to the plate:25 μL Assay Bu�er or Matrix to standard wells (Refer to Assay Procedure) 25 μL Assay Bu�er to sample wells25 μL diluted standard to standard wells25 μL sample to sample wells25 μL mixed beads to all wells

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PerformingtheAssayUsingaV-bottomPlate

• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.

• Keeptheplateuprightduringtheentireassayprocedure,includingthewashingsteps,toavoidlosingbeads.

• Theplateshouldbeplacedinthedarkorwrappedwithaluminumfoilforallincubationsteps.

• Standards and samples should be run in duplicate and arranged on the plateinaverticalconfigurationconvenientfordataacquisitionandanalysis(asshowninattachedPLATEMAP,page32).Besuretoloadstandardsinthefirsttwocolumns.Ifanautomationdeviceisusedforreading,theori-entationandreadingsequenceshouldbecarefullyplanned.

1. For measuring cell culture supernatant samples,loadtheplateasshowninthetablebelow(intheorderfromlefttoright):


StandardWells 25µL --- 25µL ---

Samplewells 25µL --- --- 25µL Formeasuringserumsamples,loadtheplateasshown inthetablebe-

low(intheorderfromlefttoright):AssayBuffer MatrixB Standard Sample*

StandardWells --- 25µL 25µL ---


*See SampleDilution

2. Vortexmixedbeadsfor30seconds.Add25μLofmixedbeadstoeachwell.Thetotalvolumeshouldbe75μLineachwellafterbeadsaddition.(Note:Duringbeadsaddition,shakemixedbeadsbottleintermittentlytoavoidbeadsettling).

3. Sealtheplatewithaplatesealer.Covertheentireplatewithaluminumfoil to protect the plate from light. Shakeat800rpmon a plate shaker for 2 hours at room temperature (Dependingontheshaker,thespeedmayneedtobeadjusted.Theoptimalspeedisonethatishighenoughtokeepbeadsinsuspensionduringincubation,butnottoohighsoitcausesspillfrom the wells).

4. Centrifugetheplateat1050rpm(~250g)for5minutes,usingaswingingbucketrotor(G.H3.8)withmicroplateadaptor(Please refer to Materials to beProvidedbytheEnd-User,page8).Donotuseexcessivecentrifugationspeed as it may make it harder to resuspend beads in later steps. Make

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surethetimerofthecentrifugeworksproperlyandstandbytomakesurethe centrifuge reaches preset speed.

5. Immediatelyaftercentrifugation,dumpthesupernatantintoasinkbyquicklyinvertingandflickingtheplateinonecontinuousandforcefulmo-tion.Donotworryaboutlosingbeadsevenifthepelletisnotvisible.Thebeadswillstayinthetipofthewellnicely.Blottheplateonastackofcleanpapertowelanddraintheremainingliquidfromthewellasmuchaspos-sible. Be careful not to disturb the bead pellet.

Alternatively,removalofthesupernatantmaybecompletedusingamultichannelpipettesetat75µL. Try to remove as much liquid as possible withoutremovinganybeads. Be sure to changepipettetipsbetweeneachroworcolumn.

6. Washtheplatebydispensing200μLof1XWashBufferintoeachwellandincubate for one minute. Repeatstep4and5above.Asecondwashisoptional,butmayhelpreducebackground.

7. Add25µLofDetectionAntibodiestoeachwell.

8. Sealtheplatewithanewplatesealer.Covertheentireplatewithalumi-num foil to protect the plate from light. Shakeat800rpmon a plate shaker for 1 hour at room temperature.

9. Do not wash the plate!Add25µLofSA-PEtoeachwelldirectly.

10. Sealtheplatewithanewplatesealer.Wraptheentireplatewithaluminumfoilandshaketheplateonaplateshakeratapproximate800rpmfor30minutes at room temperature.

11. Repeatstep4,and5.

12. Washtheplatebydispensing200μLof1XWashBufferintoeachwellandincubate for one minute. Repeatstep4and5above.Thiswashingstepisoptionalbuthelpstoreducethebackground.

13. Add150µLof1XWashBuffertoeachwell.Resuspendthebeadsbypipet-ting.

14. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay(Note:Prolongedsamplestoragecanleadtoreducedsignal).

Iftheflowcytometerisequippedwithanautosampler,thesamplescanberead directly. Please be sure to program the autosampler to resuspend beads in the well immediately before taking samples. The probe height may need to be adjusted when using an autosampler.

Ifanautosamplerisnotavailable,thesamplescanbetransferredfromtheplate to micro FACS (or FACS) tubes and read manually.

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18

Assay Procedure Summary for V-bottom Plate

Incubate 2 hours, RT, shaking

Capture beads

Biotinylated Detection Antibody

Analytes

Without washing, add 25 μL SA-PEIncubate 30 min, RT, shaking

Spin down beads, remove supernatant Wash 1 timeAdd 25 μL Detection AntibodiesIncubate 1 hr, RT, shaking

Spin down beads, remove supernatant Wash 1 time (optional)Add 150 µL of 1x Wash Bu�er Read on a �ow cytometer

Add to the plate:25 μL Assay Bu�er or Matrix to standard wells (Refer to Assay Procedure) 25 μL Assay Bu�er to sample wells25 μL diluted standard to standard wells25 μL sample to sample wells25 μL mixed beads to all wells

BA

C

A B C

A B C

biolegend.com 19


Chapter 4: FLOW CYTOMETER SETUP

Inordertogeneratereliabledata,theflowcytometermustbesetupproperlybeforedataacquisition.

Thesetupinstructionshavebeenremovedfromthismanualanduploadedontoourwebsitetosavepaper.

Toaccessthesetupinstructions,pleasevisit:www.biolegend.com/legendplex and click on the Instrument Setup tab.

Chapter 5: DATA ACQUISITION AND ANALYSIS

DataAcquisition

1. Beforereadingsamples,makesurethattheflowcytometerissetupprop-erly.

2. Createanewtemplateoropenanexistingtemplate(fordetailsonhowtocreateacytometer-specifictemplate,pleaserefertotheFlowCytometerSetup Guide).

3. Vortexeachsamplefor5secondsbeforeanalysis.

4.Settheflowratetolow.Setthenumberofbeadstobeacquiredtoabout300peranalyte(e.g.,acquire2,400beadsfora8-plexassayor4000beadsfora13-plexassay).Donotsettoacquiretotaleventsassamplesmaycontainlargeamountsofdebris.Instead,createalargegatetoincludebothBeads A and Beads B (gate A+B) and set to acquire the number of events in gateA+B.Thiswillexludemajorityofthedebris.

Note:Donotacquiretoofewortoomanybeads.ToofewbeadsacquiredmayresultinhighCVsandtoomanybeadsacquiredmayresultinslowdata analysis later.

5.Readsamples.

Whenreadingsamples,settheflowcytometertosetupmodefirstandwaituntilbeadpopulationisstabilizedbeforerecordingorswitchingtoacquisi-tionmode.

TosimplifydataanalysisusingtheLEGENDplexTMDataAnalysisSoftware,readsamplesinthesameorderasshownonthePLATEMAPattachedattheendofthemanual.Foranin-plateassay,readcolumnbycolumn(A1,B1,C1...A2,B2,C2...).

Tel: 858-768-5800


20

Whennamingdatafiles,trytousesimplenameswithaconsecutivenum-beringforeasydataanalysis(e.g.forstandards,C0.001,C0.002,C1.003,C1.004,C2.005,C2.006,C3.007,C3.008,...C7.015,C7.016;forsamples,S1.017,S1.018,S2.019,S2.020,S3.021,S3.022…)

StoreallFCSfilesinthesamefolderforeachassay.Ifrunningmultipleas-says,createaseparatefolderforeachassay.

6.ProceedtodataanalysisusingLEGENDplexTMDataAnalysisSoftwarewhendataacquisitioniscompleted.

Data Analysis

• TheFCSfilegeneratedonaflowcytometershouldbeanalyzedusingBioLegend’sLEGENDplexTMDataAnalysisSoftware.TheLEGENDplexTM Data AnalysisSoftwarecanbedownloadedforfreehere:www.biolegend.com/legendplex.

• ForPCusers,installthesoftwareonaPCrunningWindows7orWindows8anduseitinconjunctionwiththeDataAnalysisSoftwareDongleincludedin this kit. The dongle has a license key stored in it and is needed to run the software.Tousethedongle,simplyplugitintheUSBportofthecomputeronwhichthedataanalysissoftwareisinstalled,priortolaunchingthesoftware.

• ForMacusers,installonaMacrunningMacOSXversion10.7(Lion)orlaterandyouwillbepromotedtorequestasoftwarelicensekeyafterthesoftwareinstallation.

• FollowtheLEGENDplexTMDataAnalysisSoftwareUserGuideandOnlineHelptousethesoftware(www.bioLegend.com/legendplex;orpress F1 for

online help at any step of the data analysis).

biolegend.com 21


Chapter 6: ASSAY CHARACTERIZATION

RepresentativeStandardCurve

ThisstandardcurvewasgeneratedusingtheLEGENDplexTMNHPThCyto-kinePanelfordemonstrationpurposeonly.Astandardcurvemustberunwitheachassay.

1

10

100

1000

10000

1 10 100 1000 10000 100000

MFI

Concentration (pg/mL)

IL-2 IL-5 IL-6 IL-10 IL-13 TNF-α IFN-γ IL-4 IL-17A IL-21

AssaySensitivity

Theassaysensitivityorminimumdetectableconcentration(MDC)isthetheoreticallimitofdetectioncalculatedusingtheLEGENDplexTM Data AnalysisSoftwarebyapplyinga5-parametercurvefittingalgorithm.

Analyte MDC in Cell Culture Medium (pg/mL)

MDC in Serum (pg/mL)

NHPIL-2 1.7 0.8

NHPIL-5 1.0 0.7

NHPIL-6 0.8 1.0

NHPIL-10 8.1 4.9

NHPIL-13 0.9 0.8

NHPTNF-α 0.8 0.9

NHPIFN-γ 0.9 0.8

NHPIL-4 0.8 0.7

NHPIL-17A 2.0 1.5

NHPIL-21 1.9 1.8

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22

Cross-Reactivity

Therewasnoornegligiblecross-reactivityfoundfortheanalytesinthispanel.

Accuracy (Spike Recovery)

Forspikerecoveryincellculturemedium,RPMIorDMEMwith10%FCSwasspikedwithtargetproteinsatthreedifferentlevelswithintheassayrange.Thespikedsampleswerethenassayed,andthemeasuredconcen-trationswerecomparedwiththeexpectedvalues.

Forspikerecoveryinserumandplasma,bothRhesusandCynomolgussamplesweretested.Serum(n=12)orplasma(n=8)werefirstdilutedfour-foldwithAssayBufferandspikedwithtargetproteinsatthreedifferentlevelswithintheassayrange.Thespikedsampleswerethenassayed,andthemeasuredconcentrationswerecomparedwiththeexpectedvalues.

Analyte% of Recovery in Cell Culture

Medium

% of Recovery in Serum

% of Recovery in Plasma

NHPIL-2 106% 76% 79%NHPIL-5 98% 97% 90%NHPIL-6 102% 78% 87%NHPIL-10 85% 71% 68%NHPIL-13 87% 66% 70%NHPTNF-α 92% 62% 70%NHPIFN-γ 85% 82% 68%NHPIL-4 88% 80% 64%NHPIL-17A 88% 78% 82%NHPIL-21 97% 134% 127%

LinearityofDilution

Forspikelinearityincellculturemedium,RPMIorDMEMwith10%FCSwasfirstspikedwithaknownconcentrationoftargetproteins.Thespikedsampleswereseriallydiluted1:2,1:4,1:8withassaybufferandassayed.Themeasuredconcentrationsofseriallydilutedsampleswerecomparedwiththatofthespikedsamples.

biolegend.com 23


Forspikelinearityinserumandplasma,bothRhesusandCynomolgussamplesweretested.Serum(n=12)orplasma(n=8)werefirstdilutedfour-foldwithAssayBufferandspikedwithaknownconcentrationoftargetproteins.Thespikedsampleswereseriallydiluted1:2,1:4,1:8withMatrixBandassayed.Themeasuredconcentrationsofseriallydilutedsampleswerecomparedwiththatofthespikedsamples.

AnalyteLinearity of

DilutioninCellCulture Medium

Linearity of Dilutionin

Serum

Linearity of Dilutionin

PlasmaNHPIL-2 109% 110% 131%NHPIL-5 95% 109% 94%NHPIL-6 96% 105% 100%NHPIL-10 100% 124% 117%NHPIL-13 118% 127% 129%NHPTNF-α 107% 138% 126%NHPIFN-γ 105% 139% 121%NHPIL-4 105% 132% 119%NHPIL-17A 102% 135% 126%NHPIL-21 97% 127% 109%

Intra-Assay Precision

Twosampleswithdifferentconcentrationsoftargetproteinswereanalyzedinoneassaywith16replicatesforeachsample.Theintra-assayprecisionwascalculatedasbelow.

Analyte Sample Mean (pg/mL) STDEV %CV

NHPIL-2Sample 1 33.3 2.6 8%Sample 2 443.8 32.9 7%



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24



NHPTNF-αSample 1 41.2 3.2 8%Sample 2 572.4 64.5 11%

NHPIFN-γSample 1 40.3 2.9 7%Sample 2 521.0 44.6 9%


NHPIL-17ASample 1 39.2 2.8 7%Sample 2 526.1 37.4 7%


Inter-Assay Precision

Twosampleswithdifferentconcentrationsoftargetproteinswereanalyzedinthreeindependentassayswith3replicatesforeachsample.Theinter-assayprecisionwascalculatedasbelow.

Analyte Sample Mean (pg/mL) STDEV %CV






biolegend.com 25


NHPTNF-αSample 1 41.5 3.1 8%Sample 2 595.6 64.3 11%

NHPIFN-γSample 1 40.2 3.0 7%Sample 2 570.0 67.9 12%


NHPIL-17ASample 1 39.1 2.7 7%Sample 2 553.2 56.4 10%


Biological Samples

Serum and Plasma (Samples are not paired)

NormalRhesus(n=16)andCynomolgus(n=16)serumsamplesweretestedforendogenouslevelsoftheThcytokines.Theconcentrationsmeasuredareshownbelow:

Analyte Range (pg/ml)

No. of Detectable

% of Detectable

Mean (pg/mL)

NHPIL-2 5.2-63.3 32 100% 13.5

NHPIL-5 ND-32.4 21 66% 7.4

NHPIL-6 ND-439.0 23 72% 25.7

NHPIL-10 ND-48.4 6 19% 28.3

NHPIL-13 ND-9.5 4 13% 7.1

NHPTNF-α ND-8.7 8 25% 3.7

NHPIFN-γ ND-20.7 29 91% 4.7

NHPIL-4 ND-16.5 3 9% 8.6

NHPIL-17A ND-17.0 5 16% 10.9

NHPIL-21 ND-125.8 24 75% 26.1

ND=Non-detectable

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26

NormalRhesus(n=4)andCynomolgus(n=4)plasmasamplesweretestedforendogenouslevelsofThcytokines.Theconcentrationsmeasuredareshownbelow:

Analyte Range (pg/mL)

No. of Detectable

% of Detectable

Mean (pg/mL)

NHPIL-2 6.1-26.4 8 100% 13.3

NHPIL-5 ND-18.0 6 75% 6.5

NHPIL-6 ND-14.5 3 38% 7.9

NHPIL-10 ND 0 0% ND

NHPIL-13 ND 0 0% ND

NHPTNF-α ND 0 0% ND

NHPIFN-γ ND-7.0 7 88% 4.1

NHPIL-4 ND 0 0% ND

NHPIL-17A ND 0 0% ND

NHPIL-21 ND-129.9 6 75% 35.0

ND=Non-detectable

Cell Culture Supernatant

RhesusorCynomolgusPBMCs(1x106cells/mL)wereculturedundervariousconditions(PHA,5µg/mL;PMA,20ng/mL;Ionomycin(I),500ng/mL;LPS,1µg/mL;IFN-γ,100ng/mL).Supernatantswerecollectedaf-ter72hoursandassayedwiththeLEGENDplexTMNHPThCytokinePanel.Theresults(allinpg/mL)aresummarizedbelow.

Analyte Control PHA PMA + I LPS IFN-γ+LPS

Rhesus IL-2 18.2 1.8 7173.4 4.3 8.1

RhesusIL-5 1.7 23.9 57.2 ND 0.9

RhesusIL-6 5.2 4282.0 118.0 3774.4 14444.2

RhesusIL-10 3.3 139.4 568.3 197.1 29.1

Rhesus IL-13 3.1 112.3 347.9 6.9 8.3

RhesusTNF-α 7.6 84.6 846.4 94.8 2816.9

RhesusIFN-γ ND 20.7 2351.0 ND 9841.0

RhesusIL-4 ND 1.9 4.2 ND 0.6

RhesusIL-17A 1.7 33.6 68.4 3.6 5.8

Rhesus IL-21 4.3 36.3 22.5 33.4 61.7

biolegend.com 27


Analyte Control PHA PMA + I LPS IFN-γ+LPS

Cynomolgus IL-2 16.2 15.0 5261.0 8.9 19.3

CynomolgusIL-5 3.0 106.7 132.1 1.3 2.4

CynomolgusIL-6 2.4 3833.3 100.3 3497.4 9289.3

CynomolgusIL-10 ND 86.6 233.2 75.3 37.6

Cynomolgus IL-13 1.2 212.8 270.8 2.1 3.0

CynomolgusTNF-α 8.6 53.3 358.2 53.3 621.7

CynomolgusIFN-γ ND 165.2 1607.1 ND 5315.0

CynomolgusIL-4 ND 16.6 11.6 ND 1.0

CynomolgusIL-17A ND 91.6 91.0 2.5 4.8

Cynomolgus IL-21 ND 47.6 27.5 39.3 79.6ND=Non-detectable

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28

TROUBLESHOOTING

Problem Possible Cause Solution

Bead popula-tionshiftingupwardordownwarddur-ingacquisition

The strong PE signal from high concentra-tionsamplesorstan-dards may spill over to classificationChannel(e.g.,FL3/FL4/APC)and mess up the bead separation.

OptimizeinstrumentsettingsusingKitSetupBeads,andmakeappropriatecom-pensationbetweenchannels.

Filterplatewillnot vacuum orsomewellsclogged

Vacuum pressure is insufficientorvacuummanifold does not seal properly.

Increasevacuumpressuresuchthat0.2mLbuffercanbesuctionedin3-5seconds.Clean the vacuum manifold and make sure nodebrisonthemanifold.Pressdowntheplate on the manifold to make a good seal.

Samples have insoluble particlesorsampleistooviscous(e.g.,serumand plasma samples)

Centrifuge samples just prior to assay setup and use supernatant. If high lipid contentispresent,removelipidlayeraftercentrifugation.Samplemayneeddilutionif too viscous.

Ifsomewellsarestillcloggedduringwash-ing,trythefollowing:

1).Addbuffertoallthewells,pipetteupanddownthecloggedwellsandvacuumagain.

2).Useapieceofcleanwipe,wipetheun-dersideofthecloggedwellsandvacuumagain.

3).Takeathinneedle(e.g.,insulinneedle),whileholdingtheplateupward,pokethelittleholeundereachofthecloggedwellsand vacuum again. Do not poke too hard ortoodeepasitmaydamagethefilterand cause leaking.

Filterplatewasusedwithoutpre-wet.

Pre-wetplatewithwashbufferbeforerun-ning the assay.

biolegend.com 29


Insufficientbead count or slowreading

Beads inappropriately prepared

Sonicatebeadvialsandvortexjustpriortoaddition.Agitatemixedbeadsintermit-tentlyinreservoirwhilepipettingthisintothe plate.

Samples cause beads aggregationduetoparticulatematterorviscosity.


Beadswerelostduringwashingforin-tubeassay

Makesurebeadsarespundownbyvisu-ally check the pellet (beads are in light blueorbluecolor).Beverycarefulwhenremovingsupernatantduringwashing.

Probe might be par-tiallyclogged.

Sampleprobemayneedtobecleaned,orifneeded,probeshouldberemovedandsonicated.

Plate leaked

Vacuum pressure set too high

Adjustvacuumpressuresuchthat0.2mLbuffercanbesuctionedin3-5seconds.Donotexceed10”Hgofvacuum.

Plate set directly on tableorabsorbenttow-elsduringincubationsorreagentadditions

Set plate on plate holder or raised edge sobottomoffilterisnottouchinganysurface.

Liquid present on the under side of the plate aftervacuum

Afterwashing,pressdownplatefirmlyonastackofcleanpapertowelstodrytheunderside of the plate.

Pipettetouchinganddamagedplatefilterduringadditions.

Pipettetothesideofwells.

HighBack-ground

Backgroundwellswerecontaminated

Avoidcross-wellcontaminationbychang-ingtipsbetweenpipettingwhenperform-ingtheassayusingamultichannelpipette.

InsufficientwashesThe background may be due to non-specificbindingofSA-PE.Increasenumberofwashes.

Debris(FSC/SSC) during sample acquisi-tion

Debris or platelet may existinsamplesolu-tion.

Centrifugesamplesbeforeanalyzingsamples. Remove platelet as much as possible.

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30

Variationbe-tweenduplicate samples

Beadsaggregation SonicateandvortextheBeadspriortouse.

Multichannelpipettemay not be calibrated or inconsistent pipet-ting

CalibratePipette.Ensuregoodpipettingpractice.Primepipettebeforeusemayhelp.

Platewashingwasnotuniform

Make sure all reagents are vacuumed out completelyinallwashsteps.

Samples may contain particulatematters.


Loworpoorstandard curve signal

Thestandardwasin-correctlyreconstituted,stored or diluted

Followtheprotocoltoreconstitute,storeand dilute standard. Double check your calculation.

Wrongorshortincuba-tiontime

Ensurethetimeofallincubationswasappropriate.

Signals too high,standardcurves satu-rated

PMTvalueforFL2/PEset too high

MakesurethePMTsettingforthere-porter channel is appropriate

Plateincubationtimewastoolong Useshorterincubationtime.

Sample read-ings are out of range

Samples contain no or belowdetectablelevelsof analyte

Makesuretheexperimenttogeneratethesamplesworked.Useproperpositivecontrols.

Samplesconcentrationshigher than highest standard point.

Dilutesamplesandanalyzeagain.

Standardcurvewassaturated at higher end of curve.

MakesurethePMTsettingforthere-porter channel is appropriate. Use shorter incubationtimeifincubationtimewastoolong

Missed beads populationsduringreading,ordistributionis unequal

Sample may cause some beads to ag-gregate.


Beadspopulationsarenotmixedproperly

Makesureallbeadpopulationsaremixed.and in similar numbers.

biolegend.com 31


Tel: 858-768-5800


32

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biolegend.com 33


76761_V03

LEGENDplex™ Kits are manufactured by BioLegend 8999 BioLegend WaySan Diego, CA 92121Tel: 1.858.768.5800Tel: US & Canada Toll-Free: 1.877.Bio-Legend (1.877.246.5343)Fax: 1.877.455.9587Email: [email protected]

For a complete list of world-wide BioLegend offices and distributors, please visit our website at: biolegend.com


Documents

LEGENDplex™...NP Th Cytokine Mi and Match Subpanel 6 Storage Information Recommended storage for all original kit components is between 2 C and 8 C. DO NOT FREEZE Beads, Detection