LEGENDplex™ · measurement of the adipokines is critical for abetter understanding of disease progression and immune responses. The LEGENDplexTM NHP Adipokine Panel is a bead-based

®

LEGENDplex™Mul�-Analyte Flow Assay Kit

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

Cat. No. 740233

Non-human Primate (NHP) Adipokine Panel

(13-plex)

For Cell Culture and Urine Samples

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

LEGENDplex™ NHP Adipokine Panel

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……………….........

Performing the Assay Using Microtubes, or a 96-Well V- orU-bottomMicroplate…..................................................

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

Setup Procedure for FACSCaliburTM with Dual Laser..........

Setup Procedure for FACSCaliburTM with a Single Laser.....

Setup Procedure for BD FACSAriaTM, FACSCantoTM and LSR Series..........................................................................

Setup Procedure for Other Flow Cytometers ...................

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

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

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

3

3

3

4

6

6

7

8

9

9

9

9

11

12

12

15

18

19

23

30

34

35

35

36

Tel: 858-768-5800


2

Chapter 6: FREQUENTLY ASKED QUESTIONS...........................

Chapter 7: ASSAY CHARACTERIZATION..........................................

StandardCurve.………………………………………………………........

37

41

41

41

42

42

43

44

45

46

49

52

53

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

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

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

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

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

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

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

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

PLATE MAP...............……………………………………………………...............

RACK MAP....................................................................................

biolegend.com 3


Chapter 1: KIT DESCRIPTION

Introduction

Adiposetissue(AT)playsanactiveroleinregulatingmetabolismandenergyhomeostasis. AT expresses and secretes numerous soluble factors, including cytokines,chemokinesandhormones,collectivelyreferredtoasadipokinesherein. Altered levels of these proteins have been associated with a variety of metabolicdiseasesandinflammatory-autoimmuneconditions.Theaccuratemeasurementoftheadipokinesiscriticalforabetterunderstanding of disease progression and immune responses.

The LEGENDplexTM NHP Adipokine Panel is a bead-based multiplexassay, uing fluorescence–encodedbeadssuitableforuseonvariousflowcytometers.Thispanelallowssimultaneousquantificationof13adipokinesfromRhesus or Cyno-molgus monkey,includingadiponectin,adipsin,RBP4,MCP-1,IL-1β,IP-10,IL-10,IL-8,leptin,IL-6,IFN-γ,resistinandTNF-α.Thispanelprovideshighsensitiv-ity and broad dynamic range. The panel has been validated for use on urine and cell culture supernatant samples. The panel is not suitable for serum or plasma samplesduetodifferentsampledilutionrequirementsfordifferentanalytes.

The NHP Adipokine Panel isdesignedtoallowflexiblecustomization.Formixandmatch within the panel, please visit www.biolegend.com/legendplex.

This assay is for research use only.

Principle of the Assay

BioLegend’s LEGENDplexTM assays are bead-based immunoassays using the same basic principle as sandwich immunoassays.

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

Sincethebeadsaredifferentiatedbysizeandinternalfluorescenceintensityonaflowcytometer,analyte-specificpopulationscanbesegregatedandquanti-fiedbythePEfluorescentsignal.Theconcentrationofaparticularanalyteisdetemined by a standard curve generated in the same assay.

Tel: 858-768-5800


4

Beads Usage

The NHP Adipokine Panelincludestwosetsofbeads.Eachsethasauniquesizethatcanbeidentifiedonflowcytometerbasedontheirforwardscatter(FSC)andsidescatter(SSC)profiles(BeadsAandBeadsB,Figure1).Eachbeadsetcanbefurtherresolvedbasedontheirinternalfluorescenceintensities.TheinternaldyecanbedetectedusingFL3,FL4,orAPCchannel,dependingonthetypeofflowcytometerused.ThesmallerBeadsAconsistsof6beadpopula-tionsandthelargerBeadsBconsistsof7beadpopulations(Figure2-3).

Usingatotalof13beadpopulationsdistinguishedbysizeandinternalfluo-rescent dye, the NHP Adipokine Panelallowssimultaneousdetectionof13adipokinesinonesampletest.Eachanalyteisassociatedwithaparticularbeadset as indicated in Table 1.

Figure1.BeadsDifferentiatedbySize

Beads A = smaller beads

Beads B = larger beads

Figure2.BeadsAClassificationbyFL4

A5 A7 A8

A4

A6

A10

biolegend.com 5


Figure3.BeadsBClassificationbyFL4

For Beads usage in the panel, please refer to Table 1 below:

Table1.BeadsIDandTargetInformation

Target Bead ID Top Standard Concentrations(ng/mL)

NHPAdiponectin A4 200

NHP Adipsin A5 50

NHPRBP4 A6 50

NHP MCP-1 A7 10

NHPIL-1β A8 10

NHP IP-10 A10 10

NHP IL-10 B2 10

NHP IL-8 B3 10

NHPLeptin B4 10

NHP IL-6 B5 10

NHPIFN-γ B6 10

NHPResistin B7 10

NHPTNF-α B9 10

*BeadIDisusedtoassociateabeadpopulationtoaparticularanalyteintheLEGENDplexTMDataAnalysisSoftware.TheassociationofanalyteandbeadIDwillbedefinedduringthegatingstepofthedataanalysis.

WhenenteringanalyteandbeadIDinfomationduringthegatingstep,alwaysenterinthesequentialorderofthebeadID(e.g,A4,A5,A6...B2,B3,B4...).Please refer to the LEGENDplexTMDataAnalysisSoftwareUserGuideandOnlineHelpfordetails(www.biolegend.com/legendplex).

B4 B5

B6 B7 B3

B9

B2

Tel: 858-768-5800


6

StorageInformation

Recommended storage for all original kit components is between 2°C and 8°C. DONOTFREEZEPre-mixedBeads,DetectionAntibodiesorSA-PE.

• Oncethestandardshavebeenreconstituted,immediatelytransfercon-tents into polypropylene vials. DO NOT STORE RECONSTITUTED STAN-DARDS IN GLASS VIALS.

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

Materials Supplied

The LEGENDplexTM kit contains reagents for 100 tests listed in the table below. Whenassayedinduplicate,thisisenoughforan8-pointstandardcurveand40samples.

Kit Components Quantity Volume Part #

Setup Beads 1: FITC Beads 1 vial 1 mL 77840

Setup Beads 2: PE Beads 1 vial 1 mL 77842Setup Beads 3: Raw Beads 1 vial 2 mL 77844

NHP Adipokine Panel Premixed Beads 1bottle 3.5 mL 76601

NHPAdipokinePanelDetectionAntibodies 1bottle 3.5 mL 76604

NHP Adipokine Panel Standard Cock-tail,Lyophilized 1 vial lyophilized 76607

LEGENDplexTM SA-PE 1bottle 3.5 mL 77743LEGENDplexTMAssayBuffer 1bottle 25 mL 77562LEGENDplexTMWashBuffer,20X 1bottle 25 mL 77564Filter plate 1 plate 76187Plate Sealers 4sheets 78101DataAnalysisSoftwareDongle 1 21217NHP Adipokine Panel Manual 1 76599

biolegend.com 7


Materials to be Provided by the End-User

• Aflowcytometerequippedwithtwolasers(e.g.,a488nmbluelaseror532nmgreenlaseranda633-635nmredlaser)capableofdistinguishing575 nm and 660 nm oraflowcytometerequippedwithonelaser(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.

Forsettinguptheaboveflowcytometers,pleasefollowtheFlow Cytom-eter Setup guide in this manual or visit: www.biolegend.com/legendplex.

Forflowcytometersnotlistedhere,theend-userneedstosetupthemachine following similar guidelines. Please refer to Setup Procedure for Other Flow CytometerssectioninChapter4.

• Multichannelpipettescapableofdispensing5μLto200μL

• Reagentreservoirsformultichannelpipette

• Polypropylenemicrofugetubes(1.5mL)

• Laboratory vortex mixer

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

• Aluminum foil

• Absorbent pads or paper towels

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

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

Tel: 858-768-5800


8

Iftheassayisrunusingthefilterplateprovided(recommended),

• Avacuumfiltrationunit(MilliporeMultiScreen®HTSVacuumManifold,catalog#MSVMHTS00orequivalent).Instructionsonhowtousethevacuum manifold can be found at the supplier’s website.

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

Iftheassayisruninmicrotubes,V-orU-bottom96-wellplate(optional),

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

• Centrifugewithaswingingbucketadaptorformicrotiterplatesormicro-tuberacks(e.g.,BeckmanCoulterAllegraTM 6R Centrifuge with MICROPLUS CARRIERadaptorforGH3.8andJS4.3Rotors).

• 96-well,V-orU-bottom,clearpolypropylenemicroplate(lowproteinbind-ing,e.g.,grenierbio-one,Catalog#651201orequivalent).

• Precautions

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

• Donotmixorsubstitutereagentsfromdifferentkitsorlots.Reagentsfromdifferentmanufacturersshouldnotbeusedwiththiskit.

• Donotusethiskitbeyonditsexpirationdate.

• SA-PEandBeadsarelight-sensitive.Minimizelightexposure.

biolegend.com 9

LEGENDplex™ NHP Adipokine PanelChapter 2: ASSAY PREPARATION

SampleCollectionandHandling

PreparationofTissueCultureSupernatantorUrineSamples:

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

ReagentPreparation

PreparationofAntibody-ImmobilizedBeads

SonicatethePre-mixedBeadsbottlefor1minuteinasonicatorbathandthen vortex for 30 seconds prior to use. If no sonicator bath is available, in-creasethevortexingtimeto1minutetocompletelyresuspendthebeads.

PreparationofWashBuffer

• Bringthe20XWashBuffertoroomtemperatureandmixtobringallsaltsintosolution.

• Dilute25mLof20XWashBufferwith475mLdeionizedwater.Storeun-usedportionsbetween2°Cand8°Cforuptoonemonth.

StandardPreparation

1. Priortouse,reconstitutethelyophilizedNHPAdipokinePanelStandardCocktailwith250µLAssayBuffer.

2. Mix and allow the vial to sit at room temperature for 10 minutes, and then transfer the standard to an appropriately labeled polypropylene microfuge tube. This will be used as the top standard C7.

3. Label6polypropylenemicrofugetubesasC6,C5,C4,C3,C2andC1,re-spectively.

4.Add75µLofAssayBuffertoeachofthesixtubes.Prepare1:4dilutionofthe top standard by transferring 25 µL of the top standard C7 to the C6 tube and mix well. This will be the C6 standard.

5. Inthesamemanner,performserial1:4dilutionstoobtainC5,C4,C3,C2andC1standards(see the tables below).AssayBufferwillbeusedasthe0pg/mLstandard(C0).

Tel: 858-768-5800


10

Tube Serial Dilution

Assay Buffertoadd(µL)

Standard to add

Final Conc.

(pg/mL)*

Final Conc.

(pg/mL)**

Final Conc.

(pg/mL)***

C7 -- -- -- 10,000 50,000 200,000

C6 1:4 75 25 µL of C7 2,500 12,500 50,000

C5 1:16 75 25 µL of C6 625 3,125 12,500

C4 1:64 75 25 µL of C5 156.3 781.3 3,125

C3 1:256 75 25 µLofC4 39.1 195.3 781.3

C2 1:1024 75 25 µL of C3 9.8 48.8 195.3

C1 1:4096 75 25 µL of C2 2.4 12.2 48.8

C0 -- 75 -- 0 0 0

*TopstandardconcentrationofMCP-1,IL-1β,IP-10,IL-10,IL-8,Leptin,IL-6,IFN-γ,ResistinandTNF-αis10ng/mL.

**TopstandardconcentrationofAdipsinandRBP4is50ng/mL.

***TopstandardconcentrationofAdiponectinis200ng/mL.

biolegend.com 11


SampleDilution

• For cell culture supernatant samples, the levels of analyte can vary greatly from sample to sample. While the samples can be tested without dilu-tions,apreliminaryexperimentmayberequiredtodeterminetheappro-priatedilutionfactor.

Ifsampledilutionisdesired,dilutionshouldbedonewithcorrespondingfreshcellculturemediumorAssayBuffertoensureaccuratemeasure-ment.

• Urinesamplesshouldbedilutedtwo-foldwithAssayBufferbeforetesting(e.g.dilute50µLofsamplewith50µLofAssayBuffer).Furtherdilutionisneededifthesampleconcentrationisabovetheupperlimitofthestandard range.

Tel: 858-768-5800


12

Chapter 3: ASSAY PROCEDURE

The LEGENDplexTM assaycanbeperformedeitherinafilterplate,inmicrotubes,orinaV-orU-bottommicroplate.

• Thein-filterplateassayprocedureishighlyrecommendedduetoitsgoodsample to sample consistency, assay robustness and ease of handling. This procedurerequiresavacuumfiltrationunitforwashing(seeMaterials to beProvidedbytheend-user,Page7).IfyouhaveperformedaLuminex®-basedmultiplexassaybefore,yourlabshouldalreadyhavethevacuumfiltrationunitsetup.

• Ifthein-filterplateassayprocedureisnotpossible,orifyouprefer,theas-saycanbeperformedinmicrotubesorinaV-orU-bottommicroplate.Forin-tubeassay,werecommendusingmicroFACStubes(seeMaterials to be Providedbytheend-user,Page7).

Performing the Assay Using a Filter Plate

• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.

• Setthefilterplateonaninvertedplatecoveratalltimesduringassaysetupandincubationstepssothatthebottomoftheplatedoesnottouchanysurface. Touching a surface may cause leakage.

• Keeptheplateuprightduringtheentireassayprocedure,includingthewashing steps, to avoid losing beads.

• The plate should be placed in the dark or wrapped with aluminum foil for allincubationsteps.

• Standards and samples should be run in duplicate and arranged on the plateinaverticalconfigurationconvenientfordataacquisitionandanaly-sis(asshowninattachedPLATEMAP,Page52-53).Besuretoloadstan-dardsinthefirsttwocolumns.Ifanautomationdeviceisusedforread-ing,theorientationandreadingsequenceshouldbecarefullyplanned.

1. Pre-wettheplatebyadding100μLofLEGENDplexTM1XWashBuffertoeach well and let it sit for 1 minute at room temperature. To remove the excess volume, place the plate on the vacuum manifold and apply vacuum. Donotexceed10”Hgofvacuum.Vacuumuntilwellsaredrained(5-10seconds).BlotexcessWashBufferfromthebottomoftheplatebypress-ing the plate on a stack of clean paper towels. Place the plate on top of the inverted plate cover.

biolegend.com 13


For measuring cell culture supernatant and urine samples:

• Add25µLofAssayBuffertoallwells.• Add 25 µL of each standard to the standard wells. • Add25µLofeachsampletothesamplewells(SeeSampleDilution)

2. Vortexthepre-mixedbeadsbottlefor30seconds.Add25μLofthepre-mixedbeadstoeachwell.Thevolumeshouldbe75μLineachwellafterbeadsaddition.(Note:Duringadditionofthebeads,shakethepre-mixedbeadsbottleintermittentlytoavoidbeadsettling).

3. Seal the plate with a plate sealer. Toavoidplateleaking,donotapplypositivepressuretothesealerwhensealingtheplate.Wraptheentireplate, including the inverted plate cover, with aluminum foil. Place the plate on a plate shaker, secure it and shake at approximate 500 rpm 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 washing step once more.

5. Add25µLofDetectionAntibodiestoeachwell.

6. Sealtheplatewithafreshplatesealer.Wraptheentireplate,includingtheinverted plate cover, with aluminum foil. Place the plate on a plate shaker and shake at approximately 500 rpm for 1 hour at room temperature.

7. Do not vacuum! Add 25 µL of SA-PE to each well directly.

8. Sealtheplatewithafreshplatesealer.Wraptheentireplate,includingtheinverted plate cover, with aluminum foil. Place the plate on a plate shaker and shake at approximate 500 rpm for 30 minutes at room temperature.

9. Repeatstep4above.

10. Add200µLof1XWashBuffertoeachwell(or150µLifplateistobereadbyanautosampler.Highervolumemayresultinleakingofthefilterplateduring reading. Besuretosetthesamplevolumetobeanalyzedto70uLor less so that sample can be read again if needed).Resuspendthebeadson a plate shaker for 1 minute.

11. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay (Note:Prolongedsamplestoragecanleadtoreducedsignal)

Tel: 858-768-5800


14

Iftheflowcytometerisequippedwithanautosampler,readtheplatedirectly using the autosampler. The probe height may need to be adjusted when using an autosampler.

If an autosampler is not available, the samples need to be transferred from thefilterplatetoFACStubesandreadmanually.

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

For cell culture supernatant/urine samples, Add to the plate:25 μL Assay Bu�er to all wells25 μL diluted standard to standard wells25 μL sample to sample wells25 μL pre-mixed beads to all wells

Wash 2 times using vacuum �ltration unitAdd 200 μL (or 150µL) of 1x Wash Bu�er Read on a �ow cytometer

BA

C

A B C

A B C

biolegend.com 15


PerformingtheAssayUsingMicrotubesora96-WellV-orU-bot-tom Microplate

• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.

• Determine the number of assays to run. Standards and samples should be runinduplicateandarrangedintheorderconvenientfordataacquisitionand analysis (see below). Ifanautomationdeviceisusedforreading,theorientationandreadingsequenceshouldbecarefullyplanned.

Ifusingtubes,labelalltubesandarrangetubesonarack(asshowninat-tached RACK MAP, assuming using micro FACS tubes and a 96-microtube rack,e.g.,NationalScientificSupplyCo.,Catalog#:TN0946-01R).A96-mi-crotuberackisrecommendedbecauseitallowspipettingwithamultichan-nelpipette.

If using a microplate, arrange the standard and samples according to the PLATEMAPattached.

1. For measuring cell culture supernatant and urine samples:

• Add25µLofAssayBuffertoalltubes/wells.• Add25µLofeachstandardtothestandardtubes/wells.• Add25µLofeachsampletothesampletubes/wells(See Sample Dilu-tion).• Add25µLofthepre-mixedbeadstoalltubes/wells.• Add25µLDetectionAntibodiestoalltubes/wells.

Note: Thevolumeshouldbe100μLineachtube/well.Shakebeadsbottleintermittentlyduringtheadditiontoavoidbeadsettling.

2. Covertheentirerack/platewithaluminumfoiltoprotectthetubes/platefrom light. Shake(approximate1,000rpmforin-tubeassayand600rpmforin-plateassay) on a plate shaker for 2 hours at room temperature.

3. Withoutwashingthetubes/plate,add25µLSA-PEtoeachtube/wells.

4. Covertheentirerack/platewithaluminumfoiltoprotectthetubes/platefrom light. Shake(approximate1,000rpmforin-tubeassayand600rpmforin-plateassay)on a plate shaker for 30 minutes at room temperature.

5. Centrifugethetubes/plateat1,000xg for 5 minutes, using a swinging bucketrotorwithmicroplateadaptor(Please refer to Materials to be Pro-videdbytheend-user,Page7).

6. Removethesupernatantusingamicrochannelpipette.Becarefulnottoremove beads, but to remove as much liquid as possible.

Tel: 858-768-5800


16

7. Add200µLof1XWashBuffertoalltubes/well.Resuspendthebeadsbyvortexing(forin-tubeassay)orshakingonaplateshakerfor1minute(forin-plateassay).Centrifugethetubes/plateat1,000xg for 5 minutes, using a swinging bucket rotor with microplate adaptor. Remove the supernatant.

8. Add200µLof1XWashBuffertoalltube/well(or 150 µL if plate is to be readwithanautosampler.Highervolumemayresultinleakingofthefil-terplateduringreading.Besuretosetthesamplevolumetobeanalyzedto 70 uL or less so that sample can be read again if needed).Resuspendthebeadsbyvortexing(forin-tubeassay)orpipetting(forin-plateassay).

9. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay(Note:Prolongedsamplestoragecanleadtoreducedsignal).

Iftheflowcytometerisequippedwithanautosampler,thesamplescanbereadeitherdirectly(forin-plateassay)orafterbeingtransfferedtoa96-wellplate(forin-tubeassay).The probe height may need to be adjusted when using an autosampler.

If an autosampler is not available, the samples can be read directly in FACS tubesorafterbeingtransferredfromthemicroplatetomicroFACStubes.

biolegend.com 17


Assay Procedure Summary for Tubes (or V-Bottom Plate)

Arrange the number of tubes needed on a rack (or set up the plate)

Incubate 2 hours, RT, shaking

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

For cell culture supernatant/urine samples, Add to the tubes/wells:25 μL Assay Bu�er to all tubes/wells25 μL diluted standard to standard tubes/wells25 μL sample to sample tubes/wells25 μL pre-mixed beads to all tubes/wells25 μL Detection Antibodies to all tubes/wells

Spin down beadsWash one timeAdd 200 μL (or 150µL) of 1x Wash Bu�erRead on a �ow cytometer

A B

C

A

B

C

Capture beads

Detection Antibody

Analytes

Tel: 858-768-5800


18

Chapter 4: FLOW CYTOMETER SETUP

Inordertogeneratereliabledata,theflowcytometermustbesetupproperlybeforedataacquisition.Thefollowingsectionswilladdressmachinesetupfortheflowcytometerslistedbelow.

ListofFlowCytometersandPossibleConfigurations

Flow CytometerReporterChannel

Channel Emission

ClassificationChannel

ChannelEmission

Compensationneeded?

BD FACSCaliburTM (singlelaser) FL2 575 nm FL3 670 nm Yes

BD FACSCaliburTM (duallaser) FL2 575 nm FL4 660 nm No*

BD FACSCantoTM,BD FACSCantoTM II PE 575 nm APC 660 nm No*

BDTM LSR, LSRII,BD LSRFortessaTM PE

575-585 nm APC 660 nm No*

BD FACSAriaTM PE 575 nm APC 660 nm No*

*Compensationisnotrequiredforthespecifiedflowcytometerswhensetupproperly,butisrecommendedforconsistentresults.

Forflowcytometersnotlistedhere,theend-userneedstosetupthemachinefollowing similar guidelines. Please refer to Setup Procedure for Other Flow Cytometerssectioninthischapter.

The setup process typically includes the following steps. Please see the detailed setupprocedurethatfollows,regardingyourspecificinstrument.

1).Startuptheinstrumentfollowingthemanufacturer’srecommendations.

2).Createatemplatefordataacquisitionusingyourinstrument’sdata acquisitionsoftware.Atemplateisadocumentorworksheetwithdensityplotsthatallowstheusertoperformmachinesetupanddataacquisition.

3).SetupthePMTvoltagesofeachchanneltobeusedfordataacquisition using the Setup Beads provided in the kit.

4).DeterminewhethercompensationisneededbasedontheconfigurationofyoursystemasshowninTable1.Ifcompensationisneeded,perform compensationusingtheSetupBeadsprovidedinthekit.

biolegend.com 19


Setup Procedure for FACSCaliburTM with Dual Lasers

For a dual laser FACSCaliburTM,useFL2forreporterandFL4forbeadsclassifica-tion.Ingeneral,thereisnoneedforcompensationbetweenthesechannelsifthe machine is set up properly, following the setup procedure described below.

1. Start up the Instrument

Performinstrumentstartupandverificationcheckfollowingthemanufac-turer’srecommendations.

2. ObtainaTemplateforDataAcquisition

A template for FACSCaliburTM is a document with density plot that allows theusertoperformmachinesetupanddataacquisition.

Ifyouhavealreadycreatedatemplatefortheflowcytometer,openthattemplate and proceed to Step 3.

If a template is not yet available, create a new template by following the instructionsbelow:

2.1 FromtheBDCellQuestdataacquisitionsoftware,gotoFile→new document.

2.2 CreateadotplotwithFSC(forwardscatter)forX-axisandSSC(side scatter)forY-axis.Be sure to set FSC and SSC to linear mode. Create twogatesandlabelthemBeadsAandBeadsB(Figure4).

Figure 4.

2.3 Create4fluorescentdotplotsasshownbelow(Figure5)withFL2for X-axis,FL4orFL1forY-axis.Forthefluorescentdotplots,gateon BeadsA(dotplotsontheleftpanelbelow)andBeadsB(dotplotson therightpanelbelow).The dot plots should be in log mode.

02

00

40

06

00

80

01

00

0S

SC

-H

00 200 400 600 800 1000FSC-H

Acquisition Dot Plot

Beads A

Beads B

10

01

01

10

21

03

10

4F

L3-H

100 101 102 103 104

FL2-H


10

01

01

10

21

03

10

4F

L3-H

100 101 102 103 104

FL2-H


10

01

01

10

21

03

10

4F

L1-H

100 101 102 103 104

FL2-H


10

01

01

10

21

03

10

4F

L1-H

100 101 102 103 104

FL2-H


Page 1

Gated on Beads BGated on Beads A

Tel: 858-768-5800


20

2.4 Savethenewdocumentas“LEGENDplexTemplateforFACSCalibur Dual Laser” and proceed to the next step of the setup.

Figure 5.

3. Set up the PMT Voltages

The Setup Beads 3: Raw Beads are used to set up the PMT voltage of the classificationchannel(FL4/APC)andchannelFL1.TheSetupBeads2:PE

BeadsareusedtosetupthePMTvoltageofthereporterchannel(FL2/PE)The Setup Beads 1: FITC Beads are not needed for this setup.

FollowtheinstructionsbelowforsettingupthePMTsettings:

3.1 Vortex the vial of the Raw Beads for 30 seconds to resuspend the beads.

3.2 Transfer400μLoftheRawBeadstoafreshFACStube.

3.3 Settheflowcytometerflowratetolow.Insetupmode,runthe RawBeads.AdjustthesettingsforFSCandSSCsothatbothbead populationsarevisible(Figure6).

Pauseandrestartacquisitionfrequentlyduringthesetupprocedure torefreshthebeadpopulationsafteradjustingsettings.

biolegend.com 21

LEGENDplex™ NHP Adipokine Panel Figure 6.

3.4 ContinueadjustingthesettingssothatBeadsAandBeadsBarewell separatedandtheFSCandSSCreadingsare>200.

3.5 Move the gates for Beads A and Beads B so that the smaller beads fall into Beads A gate and the larger beads fall into Beads B gate

(Figure6).

3.6 AdjusttheFL1settingsothattheFL1signalsforallbeadsare between 1x100 and 1x101(Note:Thisstepisnotrequired,butis

recommended).

3.7 AdjusttheFL4settingsothattheFL4signalsforallbeadsarebe- tween 1x101 and 5x103(Figure7).

Figure 7.

3.8 Vortex the vial of Setup Beads 2: PE Beads for 30 seconds to resuspend the beads.

Tel: 858-768-5800


22

3.9 Transfer400μLofthePEBeadstoafreshFACStube.

3.10ReplacetheRawBeadstubefromtheflowcytometerwiththe PE beads tube.

3.11Settheflowcytometerflowratetolow.Insetupmode,runthePE Beads.Note:PEbeadsareonlyofsmallsize,fallingintheBeadsA

gate(Figure8).

3.12AdjusttheFL2settingsothatthemedianfluorescenceintensityof thePEbeadsfallsbetweenthelot-specificrangelabeledonthePE Beadsvial(Figure8,gateR3).

Figure 8.

3.13 Save the document again for future use.

3.14Theflowcytometerisnowreadyforsampleacquisition.

biolegend.com 23


Setup Procedure for FACSCaliburTM with a Single Laser

For a single laser FACSCaliburTM,useFL2forreporterandFL3forbeadsclassifi-cation.Compensationisneededtoproperlysetuptheinstrument.




A template for FACSCaliburTM is a document with density plot that allows theusertoperformmachinesetupanddataacquisition.

Ifyouhavealreadycreatedatemplatefortheflowcytometer,openthattemplate and proceed to Step 3.

If a template is not yet available, create a new template by following the instructionsbelow:

2.1 FromtheBDCellQuestdataacquisitionsoftware,gotoFile→new document.

2.2 CreateadotplotwithFSC(forwardscatter)forX-axisandSSC(side scatter)forY-axis.Be sure to set FSC and SSC to linear mode. Create twogatesandlabelthemBeadsAandBeadsB(Figure9).

Figure9.

0200

400

600

800

1000

SS

C-H

00 200 400 600 800 1000FSC-H


Beads A

Beads B

10

010

110

210

310

4F

L3-H

100 101 102 103 104

FL2-H


10

010

110

210

310

4F

L3-H

100 101 102 103 104

FL2-H


10

010

110

210

310

4F

L1-H

100 101 102 103 104

FL2-H


10

010

110

210

310

4F

L1-H

100 101 102 103 104

FL2-H


Page 1

Gated on Beads BGated on Beads A2.3 Create4fluorescentdotplotsasshownbelow(Figure10)withFL2 forX-axis,andFL3orFL1forY-axis.Forthefluorescentdotplots,gate onBeadsA(dotplotsontheleftpanelbelow)andBeadsB(dotplots ontherightpanelbelow).The dot plots should be in log mode.

Tel: 858-768-5800


24

Figure 10.

0200

400

600

800

1000

SS

C-H

00 200 400 600 800 1000FSC-H


Beads A

Beads B

10

010

110

210

310

4F

L3-H

100 101 102 103 104

FL2-H


10

010

110

210

310

4F

L3-H

100 101 102 103 104

FL2-H


10

010

110

210

310

4F

L1-H

100 101 102 103 104

FL2-H


10

010

110

210

310

4F

L1-H

100 101 102 103 104

FL2-H


Page 1


2.4 Setallcompensationstozero.

2.5 Savethenewdocumentas“LEGENDplexTemplateforFACSCalibur Single Laser” and proceed to the next step of the setup.

biolegend.com 25

LEGENDplex™ NHP Adipokine Panel3. SetupPMTVoltagesandCompensation

The Setup Beads 3: Raw Beads are used to set up the PMT voltage of the classificationchannelFL3andchannelFL1.TheSetupBeads2:PEBeadsare used to set up the PMT voltage of the reporter channel FL2. All three setupbeadsareneededforsettingupcompensation.

FollowtheinstructionsbelowforsettingupthePMTsettingsandcompen-sation:

3.1 Vortex the vial of the Raw Beads for 30 seconds to resuspend the beads.


3.3 Settheflowcytometerflowratetolow.Insetupmode,runtheRaw Beads.AdjustthesettingsforFSCandSSCsothatbothbeads populationsarevisible(Figure11).

Pauseandrestartacquisitionfrequentlyduringthesetupprocedure torefreshthebeadpopulations,afteradjustingsettings.

Figure 11.

3.4 ContinueadjustingthesettingssothatBeadsAandBeadsBarewell separatedandtheFSCandSSCreadingsare>200.

3.5 Move the gates for Beads A and Beads B so that the smaller beads fall into Beads A gate and the larger beads fall into Beads B gate (Figure11).

3.6 AdjusttheFL1settingsothattheFL1signalsforallbeadsarebe- tween 1x100 and 1x101 (Figure12).

Tel: 858-768-5800


26

Figure 12.

3.7 AdjusttheFL2settingssothattheFL2signalsforallbeadsarebe- tween 1x100 and 1x101(Figure12).

3.8 AdjusttheFL3settingsothattheFL3signalsforallbeadsarebe- tween 1x101 and 5 x 103(Figure12).

3.9 Vortex the vial of the Setup Beads 1: FITC Beads for 30 seconds to resuspend the beads.

3.10Transfer200μLoftheFITCBeadstoafreshFACStube.Add200μL ofRawBeadsandmixwell(thiswillgenerateFITC-positiveand FITC-negativepopulationsofbeadsandisneededforproper compensation).

3.11 In setup mode, run the mixed FITC and Raw Beads.

3.12OntheFL1vsFL2dotplot(Figure13),thebeadswilldisplayasFITC- negativeandFITC-positivepopulations(indicatedbyanarrow).

Note:FITCbeadsareonlyofsmallsize,fallingintheBeadsAgate.

10

010

110

210

310

4F

L3-H

100 101 102 103 104

FL2-H


10

010

110

210

310

4F

L3-H

100 101 102 103 104

FL2-H


10

010

110

210

310

4F

L1-H

100 101 102 103 104

FL2-H


10

010

110

210

310

4F

L1-H

100 101 102 103 104

FL2-H


0200

400

600

800

1000

SS

C-H

00 200 400 600 800 1000FSC-H


Beads A

Beads B

Page 1


biolegend.com 27


3.13AdjusttheFL2-%FL1compensationsetting(e.g.,FL2-FL1=20%)so thattheFITC-negativeandFITC-positivepopulationshavesimilar meanFL2fluorescenceintensities(Figure14).

Figure 14.

3.14VortexthevialofPEBeadsfor30secondstoresuspendthebeads.

3.15Transfer200μLofthePEBeadstoafreshFACStube.Add200μLof RawBeadsandmixwell(ThiswillgeneratePE-positiveand PE-negativepopulationsofbeadsandisneededforpropercompen- sation).

3.16 In setup mode, run the mixed PE and Raw Beads.

3.17OntheFL1vsFL2dotplot(Figure15),thebeadswilldisplayasPE- negativeandPE-positivepopulations(indicatedbyanarrow).Note: PEbeadsareonlyofsmallsize,fallingintheBeadsAgate.

Tel: 858-768-5800


28

Figure 15.

3.18AdjusttheFL2settingsothatthemedianfluorescenceintensityofthe PEbeadsfallsbetweenthelot-specificrangelabeledonthePEBeads vial(Figure16).

3.19AdjusttheFL1-%FL2compensationsetting(e.g.,FL1-FL2=1.5%)so thatthePE-negativeandPE-positivepopulationshavesimilarmean FL1fluorescenceintensities(Figure16).

Figure 16.

3.20OntheFL3vsFL2dotplot(Figure17),thebeadswilldisplayasPE- negativeandPE-positivepopulations(indicatedbyanarrow).

3.21 AdjusttheFL3-%FL2compensationsetting(e.g.,FL3-%FL2=40%)so thattheFL3-lowpopulationandthePE-positivepopulationhave similarmedianFL3signal(Figure18).

biolegend.com 29


Figure 18.


3.23Theflowcytometerisnowcompensatedandreadyforsample analysis.

Tel: 858-768-5800


30

Setup Procedure for BD FACSAriaTM, FACSCantoTM and LSR Series

ThispartoftheguideappliestoBDdigitalflowcytometersusingFACSDivaTM softwareversion6.0andabove.

For the BD FACS machines running FACSDivaTM, use the PE channel for reporter andtheAPCchannelforbeadsclassification.Ingeneral,thereisnoneedforcompensationbetweenthesechannelsifthemachineissetupproperlyfollow-ing the setup procedure described below.

Thissetupprocedureisrequiredunderthefollowingsituations:

• YouarerunningtheLEGENDplexkitforthefirsttime.

• It has been over a month since the procedure was last performed.

• Yourflowcytometerhasbeenservicedsinceyoulastperformedthis procedure.

This setup process is not needed if you have run this experiment before and haveaccesstoasavedexperimenttemplate(Thesettingswillbesavedinthefinalstepofthissetupprocedureandanysettingssavedcanbeimportedtoanewexperiment.PleaserefertoStep2andStep3.9below).




A template for FACSDivaTM is a worksheet with density plots that allows the usertoperformmachinesetupanddataacquisition.

If a template is not yet available, create a new template by following the instructions.Afteratemplateiscreated,savethefileinD:\BDExport\Tem-plates\Experiment.DonotchangethenameoftheTemplatesfolder.

Ifyouhavealreadycreatedatemplatefortheflowcytometer,openthattemplateandproceedtoStep3.Toopenanexistingtemplate,selectEx-periment>NewExperiment.AlistoftemplatessavedinD:\BDExport\Tem-plates\Experimentwillpopup.Selectthedesiredtemplatefromthelist.

Tocreateanewtemplate,followtheinstructionsbelow:

2.1 From the BD FACSDivaTMsoftware,gotoExperiment>NewExperi- ment.

biolegend.com 31

LEGENDplex™ NHP Adipokine Panel 2.2 In the global worksheet, open the worksheet. Create a dot plot with

FSC(forwardscatter)forX-axisandSSC(sidescatter)forY-axis.Be sure to set FSC and SSC to linear mode. Create two gates and label themBeadsAandBeadsB(Figure19).

Figure19.

2.3 CreatetwodotplotswithPEforX-axis,APCforY-axis(Figure20), gatedonBeadsA(leftpanelbelow)andBeadsB(rightpanelbelow), respectively.CreateonedotplotwithFITCforX-axis,APCforY-axis, gatedonBeadsAandBeadsB(graphnotshown).The plots should all be in log mode.

Figure 20.

2.4 Savethedocumentas“LEGENDplexTemplateforFACSDiva” inD:\BDExport\Templates\Experimentandproceedtothenextstep of setup.

Tel: 858-768-5800


32

3. Set up PMT Voltages

The Setup Beads 3: Raw Beads are used to set up the PMT voltage of the classificationchannelAPC,reporterchannelPE,andFITCchannel.TheSetup Beads 1: FITC Beads and 2: PE Beads are not needed for this setup becausenocompensationisrequiredifthesetupproceduredescribedhereis closely followed.

FollowtheinstructionsbelowforsettingupthePMTsettings:

3.1 Vortex the vial of Raw Beads for 30 seconds to resuspend the beads.


3.3 Settheflowcytometerflowratetolow.RuntheRawBeads.Adjust thesettingsforFSCandSSCsothatbothbeadpopulationsarevisible (Figure21).

Pauseandrestartacquisitionfrequentlyduringthesetupprocedure torefreshthebeadspopulationsafteradjustingsettings.

Figure 21.

3.4 ContinueadjustingthesettingssothatBeadsAandBeadsBarewell separatedandtheFSCandSSCreadingsare>50(x1000).

3.5 Move the gates for Beads A and Beads B so that the smaller beads fall into Beads A gate and the larger beads fall into Beads B gate (Figure21).

3.6 AdjusttheFITCsettingsothattheFITCsignalforthemajorityof beads is between 1x101 and 1x102 (Figure22).

biolegend.com 33


Beads Beads A+ Beads B

3.7 AdjustthePEsettingsothatthePEsignalforthemajorityofbeadsis between 1x101 and 1x102(Figure23).

Figure 23.

3.8 AdjusttheAPCsettingssothatthetheAPCfluorescenceintensitiesof allbeadpopulationsarebetween1x102 and 5 x 104(Figure23).


Tosaveyourassay-specificsettings,inthebrowser,right-click CytometerSettingsandselectSavetoCatalog.Namethefile,and thenclickOK.Toimportthesavedsettingforanewexperiment,right clickoncytometersettingsandselectimportsettings.

3.10Theflowcytometerisnowreadyforsampleacquisition.

Tel: 858-768-5800


34

Setup Procedure for Other Flow Cytometers

Forflowcytometersnotaddressedabove,thesetupprocedurewilldifferfromone to another. It is very important for the end-user to set up the machine following similar guidelines. Inthiscase,machinecompensationbetweenthereporterandbeadsclassificationchannelsisstronglyrecommended.

Theflowcytometersetupinstructionsarealsopostedonourwebsite:www.biolegend.com/legendplex. Newflowcytometersetupinstructionswillbeadded to on our website when they become available.

biolegend.com 35


Chapter 5: DATA ACQUISITION AND ANALYSIS

DataAcquisition

1. Beforereadingsamples,makesurethattheflowcytometerissetupprop-erly.Forflowcytometersetup,pleasefollowtheFlowCytometerSetupguide in this manual or visit: www.biolegend.com/legendplex.

2. Createanewtemplateoropenanexistingtemplate(fordetailsonhowtocreateacytometer-specifictemplate,pleaserefertotheFlowCytometerSetupGuide).

3. Vortex each sample for 5 seconds before analysis.

4.Settheflowratetolow.Setthenumberofbeadstobeacquiredto2000-2500 on Beads A gate or Beads B gate. Do not acquire too many events (e.g.>10,000totalevents).

Note: Do not acquire too few or too many beads. Too few beads acquired may result in high CVs and too many beads acquired may result in slow data analysis later.

Whenreadingsamples,settheflowcytometertosetupmodefirstandwaituntilbeadpopulationisstabilizedbeforeswitchingtoacquisitionmode.

To simplify data analysis using the LEGENDplexTMDataAnalysisSoftware,read samples in the same order as shown on the PLATE MAP or RACK MAP attachedattheendofthemanual.Foranin-plateassay,readcolumnbycolumn(A1,B1,C1...A2,B2,C2...).Foranin-tubeassay,readrowbyrow(A1,A2,A3,...B1,B2,B3...).

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, create a separate folder for each assay.

5. Proceed to data analysis using LEGENDplexTMDataAnalysisSoftwarewhendataacquisitioniscompleted.

Tel: 858-768-5800


36

Data Analysis

• TheFCSfilegeneratedonaflowcytometershouldbeanalyzedusingBioLegend’s LEGENDplexTMDataAnalysisSoftwareorothercompatibledataanalysissoftware.TheLEGENDplexTMDataAnalysisSoftwarecanbedownloaded here: www.biolegend.com/legendplex.

• Afterdownloading,installitonaPC(runningWindows7orWindows8)anduseitinconjunctionwiththeDataAnalysisSoftwareDongleincludedin this kit. The dongle has a license key stored in it and is needed to run the software.Tousethedongle,simplyplugitintheUSBportofthecomputeronwhichthedataanalysissoftwareisinstalled,priortolaunchingthesoftware.

• TheSoftwareDonglehasafixednumberofpoints.Eachanalysiswillconsume a certain number of points. The number of points consumed dependsonassayplexsizeandnumberofsamples.Afterthepointsona dongle are consumed, a new dongle will be needed to run more data analysis.Althoughthesoftwarewillcontinuetobefunctional,thedatawillnotbesaveduntilanewdongleisavailable.Anewdongleisprovidedineach LEGENDplexTMkit.Asavedanalysiscanalwaysbere-analyzedusingthesoftwareregardlessofdonglestatus.

• Follow the LEGENDplexTMDataAnalysisSoftwareUserGuideandOnlineHelptousethesoftware(www.bioLegend.com/legendplex; or press F1 for online help at any step of the data analysis).

biolegend.com 37


Chapter 6: FREQUENTLY ASKED QUESTIONS

Q. WhatisthedifferencebetweenLEGENDplexTM and Luminex®Assays?

BioLegend’s LEGENDplexTM assays and Luminex®-basedmultiplexassaysare both bead-based immunoassays using the same basic principle of sandwichimmunoassays.Bothsystemsusefluorescence-codedbeadstoachievemultiplexing.Themajordifferenceishowthedataisacquired.LEGENDplexTMassaysusecommonlabflowcytometersandtheirrespec-tivesoftwarefordataacquisition,whereasLuminex®-based assays use dedicatedmachinesandsoftwarefordataacquisition.Therefore,oneofthe advantages of LEGENDplexTM assays is that they can be run on common flowcytometersandnospecializedmachineisneeded.

Q. Can I use LEGENDplexTM kits on Luminex®machines?

No. LEGENDplexTMkitsshouldbeusedonaregularflowcytometerandthedatacanbeanalyzedusingtheuser-friendlydataanalysissoftwareavail-ablefordownloadatwww.biolegend.com/legendplex.

Q. WhatarethecompatibleflowcytometersfortheLEGENDplex™assays?

In general, LEGENDplexTMassayscanbeusedonmostcommonflowcytom-eters, such as:

BD FACSCalibur™ BD FACSCanto™ BD FACSCanto™ II BD TM LSR I BD TM LSR II BD LSRFortessaTM BD FACSAria™ BD FACSArrayTM

PleaserefertotheMATERIALSTOBEPROVIDEDBYEND-USERsectionfordetailsonthechannelconfigurationsofthecytometer.

Forflowcytometersnotlistedabove,theenduserneedstomakesure

thatthemachineissetupproperlybeforeuse(refertoSetup Procedure for Other Flow CytometerssectioninChapter4).Inthiscase,machinecompensationbetweenthereporterandbeadsclassificationchannelsisstrongly recommended.

TheFCSorListmodefilesfromthefollowinginstrumentsarecompatiblewith the LEGENDplex TMDataAnalysisSoftware.

Tel: 858-768-5800


38

Vendor Instrument Model VendorInstrument

Model

BD

FACScan™ Sony iCyt Eclipse™

FACSCalibur™ Life Technologies Attune®

FACSCanto™ II Miltenyi Biotec MACSQuant®

LSRFortessa™ Partec PAS

LSR II Stratedigm S1400

FACSAria™

Beckman Coulter

CyAn™ ADP

FACSAria™ II FC 500

Accuri™ C6 Gallios™

ORFLO Moxi Flow™ MoFlo®Astrios™

CyteK DxP10™ MoFlo®XDP

Q. Whatistherightprocedureforrunningtheassay?

1. Perform the assay as instructed in this kit manual.

2. Determinethetypeofflowcytometeryouhaveandperformmachinesetup as instructed on Flow Cytometer Setup guide in this manual or visit: www.biolegend.com/legendplex.Openanexistingorcreateamachine-specifictemplatefordataacquisition.

3. AnalyzethesamplesontheflowcytometerandsavetheFCSfilesina new folder.

4. Install the LEGENDplexTM DataAnalysisSoftwarealongwiththesoft-waredongleonaPCwithoperatingsystemWindows7orWindows8(32bitor64bit).Makesuretoinstallthecorrectversion(32bitor64bitversion),dependingonyourcomputer’soperatingsystem.

5. TransferthefoldercontainingtheFCSfilesofyourexperimenttothecomputerwherethedataanalysissoftwareisinstalled.

6. PerformdataanalysisasinstructedintheDataAnalysisSoftwareUserGuide.

Q. WhendoIneedtodomachinecompensation?

Ifaflowcytometerequippedwithasinglelaserisusedforbothreporter(FL2/PE)andclassification(FL3),thencompensationisabsolutelyrequiredto compensate the signal spill over between the two channels, especially from FL2 to FL3. Please use the Setup Beads provided and follow the FLOW CYTOMETER SETUP guide in this manual.

biolegend.com 39

LEGENDplex™ NHP Adipokine Panel Ifaflowcytometerislistedinthecompatibleflowcytometerlistinthis

manualandisequippedwithtwolasers,oneforreporter(FL2/PE)andtheotherforclassification(FL4/APC),thencompensationisnotrequirediftheFLOW CYTOMETER SETUP guide in this manual is closely followed. How-ever,compensationisrecommendedforconsistentresults.

Forotherflowcytometersnotlistedinthecompatibleflowcytometerlist, the end-user needs to set up the machine following similar guidelines (refertomanufacturer’smanualforproperinstrumentsetup).Inthiscase,machinecompensationbetweenthereporterandbeadsclassificationchannels is strongly recommended.

Q. Isthereaspecialsoftwarerequiredfordataanalysis?

Theflowcytometerrawdatafiles(FCS2.0,3.0)canbeanalyzedusingtheLEGENDplexTMDataAnalysisSoftwareorotherequivalentanalysissoft-ware. Download the LEGENDplexTM DataAnalysisSoftwareforfreehere:www.biolegend.com/legendplex.Asoftwaredonglewithlicensekeyisprovided in the kit.

Fordataacquisition,nospecialsoftwareisneeded.Justusethedataac-quisitionsoftwarethatcomeswiththeflowcytometer,aslongasthedatageneratedisinFCSformat,whichmeetsFCSconvention2.0,3.0and3.1.

Q. CanIselecttomeasureonlysomeanalyteswithinapanel?

Yes.Thetargetswithinapanelarefullycustomizable.Thecustomercanselectanycombinationoftargetswithinapanelandorderacustomizedproduct.Pleaseuseourwebsitetargetselectiontoolfororderingcustom-izedproduct(www.biolegend.com/legendplex).

Q. CanIselecttomeasureanalytesacrosspanels?

Cross-panelcustomizationisalsopossible,aslongasthetotalplexsizeisno more than 13 and there is no overlapping beads region among targets selected.Forcross-panelcustomization,[email protected].

Q. I have run out of a component from the kit. Can I use a similar compo-nentfromadifferentkit?

TheLEGENDplex™Beads,Standards,DetectionAntibodiesandSA-PEarelot-specificandmustbeusedincombinationwitheachother.Donotmixthesecomponentsfromdifferentkitsorlots.

Othercomponents,suchasAssayBuffer,WashBuffer,Plate,andPlateSealerarenotlot-specificandcanthereforebeexchangedbetweendiffer-ent kits and lots.

Tel: 858-768-5800


40

Q. My standard curve is not linear; can I use the non-linear part of the stan-dardcurveduringanalysis?

Yes. It is possible to use the non-linear part of the standard curve for calculatingtheresults.TheLEGENDplexTMDataAnalysisSoftwareusesafive-parametercurvefittingalgorithm,whichdeterminestheminimumandmaximumdetectionconcentrationsofeachtargetandreportsthem.Ifsampleconcentrationsfallabovethemaximumdetectableconcentration,thesamplewillhavetobedilutedandreanalyzed.Ifsampleconcentra-tionsfallbelowtheminimumdetectionconcentration,itisconsideredNotDetectablebytheparticularassay.

Q. Duringdataacquisition,whydothebeadpopulations(definedbyFSCandSSC)sometimesappeartobedispersedorshifted?

Thisisusuallycausedbyafastflowrateorasuddenchangeinflowrate.Therearethreeflow-throughsettings:low,medium,andhigh.Ifyouareusingalowflowrateandthenchangetomediumandhighflowrate,thesuddenchangeofflowratemaysometimesresultinadispersedorshiftedbeadpopulation.Therefore,itisnotrecommendedtochangeflowrateduringdataacquisition.Thebestwayistorunthesampleinsetupmodeusinganidealflowrate,andoncethepopulationisstable,thenchangeittoacquisitionmode.

Q. DoestheLEGENDplex™DataAnalysisSoftwarerunonAppleMacintosh?

Notyet.ThecurrentversionofthesoftwarehastorunonaPCwithoperatingsystemWindows7orWindows8(32bitor64bit).IfyourflowcytometerisconnectedtoaMac,afterdataacquisition,theentirefoldercontainingthedata(FCSfiles)shouldbetransferredtoaPCandanalyzed.

Q. Whydoeseachkitincludeasoftwaredongle?

The dongle allows you to use our LEGENDplexTMsoftwarefordataanalysisand each dongle contains 1 million points.

Q. Iranoutofpointsonmydongle.CanIgetanewoneseparately?Or,canIuseadonglefromonekitforanotherkit?

Dongle is not sold separately. Contact BioLegend if you need a new dongle. The dongle can be used across kits.

biolegend.com 41


Chapter 7: ASSAY CHARACTERIZATION

Standard Curve

This standard curve was generated using the LEGENDplexTM NHP Adipokine Panelfordemonstrationpurposeonly.Astandardcurvemustberunwitheach assay.

AssaySensitivity

Theassaysensitivityorminimumdetectableconcentration(MDC)isthetheoreticallimitofdetectioncalculatedusingtheLEGENDplexTM Data AnalysisSoftwarebyapplyinga5-parametercurvefittingalgorithm.

Analyte MDC (pg/mL)NHPAdiponectin 39.0

NHP Adipsin 4.3

NHPRBP4 3.4

NHP MCP-1 1.3

NHPIL-1β 1.3

NHP IP-10 0.7

NHP IL-10 2.2

NHP IL-8 1.2

NHPLeptin 2.4

NHP IL-6 2.4

1

10

100

1000

10000

1 100 10000

MFI

Concentration (pg/mL)

Adiponectin Adipsin RBP4 MCP-1 IL-1b IP-10 IL-10 IL-8 Leptin IL-6 IFN-r Resistin TNF-a

Tel: 858-768-5800


42

NHPIFN-γ 1.9

NHPResistin 2.2

NHPTNF-α 0.6

Cross-Reactivity

Therewasnoornegligiblecross-reactivityfoundfortheanalytesinthispanel.

Accuracy (Spike Recovery)

Forspikerecoveryincellculturemedium(CCM),RPMIorDMEMwith10%FCSwasfirstdilutedtwo-foldwithAssayBufferandspikedwithtargetpro-teinsatthreedifferentlevelswithintheassayrange.Thespikedsampleswerethenassayed,andthemeasuredconcentrationswerecomparedwiththe expected values.

For spike recovery in urine, Rhesus and Cynomolgus monkey urine samples (n=16)werefirstdilutedtwo-foldwithAssayBufferandspikedwithtargetproteinsatthreedifferentlevelswithintheassayrange.Thespikedsam-pleswerethenassayed,andthemeasuredconcentrationswerecomparedwith the expected values.

Analyte Recovery in CCM Recovery in Urine NHPAdiponectin 96% 96%NHP Adipsin 94% 69%NHPRBP4 70% NANHP MCP-1 92% 109%NHPIL-1β 91% 62%NHP IP-10 89% 91%NHP IL-10 78% 106%NHP IL-8 108% 116%NHPLeptin 94% 94%NHP IL-6 89% 115%

biolegend.com 43


NHPIFN-γ 110% 131%NHPResistin 84% 103%NHPTNF-α 87% 105%

NA=NotApplicableduetohighendogenouslevels

LinearityofDilution

ForspikelinearityinCCM,RPMIorDMEMwith10%FCSwasfirstdilutedtwo-foldwithAssayBufferandspikedwithaknownconcentrationoftargetproteins.Thespikedsampleswereseriallydiluted1:2,1:4,1:8withAssaybufferandassayed.Themeasuredconcentrationsofseriallydilutedsamples were compared with that of the spiked samples.

For sprike linearity in urine, Rhesus and Cynomolgus monkey urine samples (n=16)weredilutedtwo-foldwithAssayBufferandspikedwithaknownconcentrationoftargetproteins.Thespikedsampleswereseriallydiluted1:2,1:4,1:8withAssayBufferandassayed.Themeasuredconcentrationsof serially diluted samples were compared with that of the spiked samples.

Analyte Linearity in CCMLinearity in

Urine

NHPAdiponectin 95% 102%

NHP Adipsin 115% 113%NHPRBP4 117% 100%NHP MCP-1 114% 110%NHPIL-1β 114% 105%NHP IP-10 111% 114%NHP IL-10 121% 103%NHP IL-8 119% 108%NHPLeptin 113% 109%NHP IL-6 111% 103%NHPIFN-γ 120% 96%NHPResistin 125% 110%NHPTNF-α 114% 103%

Tel: 858-768-5800


44

Intra-Assay Precision

Twosampleswithdifferentconcentrationsoftargetproteinswereanalyzedin one assay with 16 replicates for each sample. The intra-assay precision was calculated as below.

Analyte Sample Mean (pg/mL) STDEV %CV

NHP Adiponectin

Sample 1 923.9 108.8 12%Sample 2 3639.8 313.3 9%

NHP Adipsin

Sample 1 204.8 8.5 4%Sample 2 787.0 29.0 4%

NHPRBP4Sample 1 256.5 13.9 5%Sample 2 1024.5 41.9 4%

NHP MCP-1Sample 1 48.0 2.5 5%Sample 2 199.3 9.1 5%

NHPIL-1βSample 1 50.4 3.2 6%Sample 2 202.1 11.2 6%

NHP IP-10Sample 1 65.8 3.7 6%Sample 2 265.7 12.1 5%

NHP IL-10Sample 1 40.4 4.2 10%Sample 2 143.6 7.8 5%


NHPLeptinSample 1 52.8 3.0 6%Sample 2 201.7 8.8 4%


NHPIFN-γSample 1 47.5 4.7 10%Sample 2 182.5 9.9 5%

NHPResistinSample 1 52.2 2.4 5%Sample 2 194.7 7.1 4%

NHPTNF-αSample 1 54.2 3.9 7%Sample 2 206.9 10.4 5%

biolegend.com 45


Inter-Assay Precision

Twosampleswithdifferentconcentrationsoftargetproteinswereanalyzedin three independent assays with 3 replicates for each sample. The inter-assay precision was calculated as below.

Analyte Sample Mean (pg/mL) STDEV %CV

NHP Adiponectin

Sample 1 987.4 131.7 13%Sample 2 3554.8 336.5 9%

NHP Adipsin

Sample 1 214.9 17.5 8%Sample 2 775.2 45.8 6%

NHPRBP4Sample 1 274.3 30.7 11%Sample 2 1020.1 80.5 8%

NHP MCP-1Sample 1 50.9 4.5 9%Sample 2 195.6 14.5 7%

NHPIL-1βSample 1 52.6 4.9 9%Sample 2 205.2 16.4 8%

NHP IP-10Sample 1 76.0 10.6 14%Sample 2 290.3 34.0 12%



NHPLeptinSample 1 59.5 7.6 13%Sample 2 212.4 18.2 9%


NHPIFN-γSample 1 53.4 7.8 15%Sample 2 185.1 17.6 10%

NHPResistinSample 1 55.3 5.0 9%Sample 2 193.9 10.6 5%

NHPTNF-αSample 1 58.6 7.0 12%Sample 2 202.5 16.1 8%

Tel: 858-768-5800


46

Biological Samples

Urine

NormalRhesusmonkeyurinesamples(n=8)weretestedforendogenouslevelsofadipokines.Theconcentrationsmeasuredareshownbelow:

Analyte Range (pg/mL)% of

Detectable

Mean of detectable

(pg/mL)RhesusAdiponectin 3,244-714,352 100% 187,980

Rhesus Adipsin 104.8-1,159.6 100% 564.0

RhesusRBP4 665.0 - 201,520 100% 93,739

Rhesus MCP-1 3.3-13,484 100% 3,594

RhesusIL-1β 7.4-19.1 100% 11.4

Rhesus IP-10 3.5 - 226 100% 72.7

Rhesus IL-10 2.5 - 325.6 100% 60.9

Rhesus IL-8 ND - 923.9 75% 167.9

RhesusLeptin ND - 12.8 25% 10.8

Rhesus IL-6 ND - 190.1 38% 68.9

RhesusIFN-γ 2.8-31.7 100% 8.5

RhesusResistin 5.6-24,524 100% 7,185

RhesusTNF-α ND -7.7 25% 5.4 ND=NotDetectable

NormalCynomolgusmonkeyurinesamples(n=8)weretestedforendogenouslevelsofadipokines.Theconcentrationsmeasuredareshownbelow:

Analyte Range (pg/mL)% of

Detectable

Mean of detectable

(pg/mL)Cynomolgus Adiponectin 3,996-979,288 100% 302,385

Cynomolgus Adipsin 125.4-6,091 100% 1,767

CynomolgusRBP4 6,013-166,038 100% 106,457

Cynomolgus MCP-1 228.1-11,386 100% 2,396CynomolgusIL-1β 1.8-9.7 100% 5.6

biolegend.com 47


Cynomolgus IP-10 2.3 - 189.6 100% 40.5

Cynomolgus IL-10 19.7 - 270.5 100% 120.4

Cynomolgus IL-8 ND - 18.0 25% 7.7

CynomolgusLeptin ND - 31.0 38% 16.5

Cynomolgus IL-6 ND 0% ND

CynomolgusIFN-γ ND - 72.0 75% 18.4

CynomolgusResistin 228.8-13,629 100% 6,818

CynomolgusTNF-α ND - 9.9 38% 4.9 ND=NotDetectable

Cell Culture Supernatant

RhesusPBMC(1x106cells/mL)wereculturedundervariousconditions(PHA,5µg/mL;PMA,20ng/mL;LPS, 1 µg/mL;Ionomycin(I),500ng/mL;IFN-γ,100ng/mL).Supernatantswerecollectedafter3days.Allsamplesweredilutedtwo-foldwithAssayBufferandassayedwiththeLEGEND-plexTMNHPAdipokinePanel.Theresults(allinpg/mL)aresummarizedbelow.

Analyte (-) PHA PMA+I LPS IFN-γ + LPS

Rhesus Adiponectin 26.6 ND 24.6 18.8 47.2

Rhesus Adipsin 4,673 897.8 455.3 935.6 1,045

RhesusRBP4 21.2 13.1 20.6 19.0 19.8Rhesus MCP-1 1,964 10,259 1,375 12,832 11,026

RhesusIL-1β ND 54.9 24.7 72.2 91.8Rhesus IP-10 917.9 5,624 1,951 600.3 5,015Rhesus IL-10 7.3 79.2 247.8 75.0 38.9Rhesus IL-8 3,249 12,492 12,907 12,907 12,907RhesusLeptin ND ND ND ND ND

Rhesus IL-6 ND 2146 54.8 2,225 6,191RhesusIFN-γ ND 1,037 8,374 3.3 >13,977RhesusResistin 106.7 89.6 89.0 103.6 107.4RhesusTNF-α 7.2 70.3 454.9 62.4 888.4ND=NotDetectable

Tel: 858-768-5800


48

CynomolgusPBMC(1x106cells/mL)wereculturedundervariouscondi-tions(PHA,5µg/mL;PMA,20ng/mL;LPS, 1 µg/mL;Ionomycin(I),500ng/mL;IFN-γ,100ng/mL).Supernatantswerecollectedafter3days.Allsamplesweredilutedtwo-foldwithAssayBufferandassayedwiththeLEGENDplexTMNHPAdipokinePanel.Theresults(allinpg/mL)aresumma-rizedbelow.

Analyte (-) PHA PMA+I LPS IFN-γ + LPS

Cynomolgus Adiponectin 47.2 15.4 13.8 31.2 17.0

Cynomolgus Adipsin 7,914 1,506 608.8 1,349 1,648

Cynomolgus RBP4 18.6 20.6 26.3 21.8 22.5

Cynomolgus MCP-1 663.4 11,880 2,465 13,350 11,442

Cynomolgus IL-1β ND 106.7 21.0 100.0 175.9

Cynomolgus IP-10 2,338 6,778 2,247 522.4 6,548

Cynomolgus IL-10 10.2 135.9 458.5 170.4 31.7

Cynomolgus IL-8 1,776 12,907 12,907 12,907 12,907

Cynomolgus Leptin ND ND ND ND ND

Cynomolgus IL-6 2.3 3,080 53.1 2,397 6,191

Cynomolgus IFN-γ ND 126.0 8,909 3.0 >13,977

Cynomolgus Resistin 129.2 126.3 112.3 125.4 134.1

Cynomolgus TNF-α 6.3 151.9 728.1 140.9 3,401

ND=NotDetectable

biolegend.com 49


TROUBLESHOOTING

Problem Possible Cause Solution

Bead popula-tionshiftingupward or downward dur-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.

OptimizeinstrumentsettingsusingKitSetup Beads, and make appropriate com-pensationbetweenchannels.

Filter plate will not vacuum or some wells clogged

Vacuum pressure is insufficientorvacuummanifold does not seal properly.

Increase vacuum pressure such that 0.2mLbuffercanbesuctionedin3-5seconds. Clean the vacuum manifold and make sure no debris on the manifold. Press down the plate on the manifold to make a good seal.

Samples have insoluble particlesorsampleistooviscous(e.g.,serumandplasmasamples)

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

Ifsomewellsarestillcloggedduringwashing, try the following:

1).Addbuffertoallthewells,pipetteupand down the clogged wells and vacuum again.

2).Useapieceofcleanwipe,wipetheun-der side of the clogged wells and vacuum again.

3).Takeathinneedle(e.g.,insulinnee-dle),whileholdingtheplateupward,pokethelittleholeundereachofthecloggedwells and vacuum again. Do not poke too hard or too deep as it may damage the filterandcauseleaking.

Filter plate was used without pre-wet.

Pre-wetplatewithwashbufferbeforerunning the assay.

Tel: 858-768-5800


50

Insufficientbead count or slow reading

Beads inappropriately prepared

Sonicate bead vials and vortex just prior toaddition.Agitatethepre-mixedbeadsintermittentlyinreservoirwhilepipettingthis into the plate.

Samples cause beads aggregationduetoparticulatematterorviscosity


Beads were lost during washing for in-tube assay

Make sure beads are spun down by visu-allycheckthepellet(beadsareinlightblueorbluecolor).Beverycarefulwhenremoving supernatant during washing.

Probemaybepartiallyclogged

Sample probe may need to be cleaned, or if needed, probe should be removed and sonicated.

Plate leaked

Vacuum pressure set too high

Adjust vacuum pressure such that 0.2 mL buffercanbesuctionedin3-5seconds.Do not exceed 10” Hg of vacuum.

Plate set directly on table or absorbent tow-elsduringincubationsorreagentadditions

Set plate on plate holder or raised edge sobottomoffilterisnottouchinganysurface.

Liquid present on the under side of the plate aftervacuum

Afterwashing,pressdownplatefirmlyona stack of clean paper towels to dry the underside of the plate.

Pipettetouchinganddamagedplatefilterduringadditions

Pipettetothesideofwells.

High back-ground

Background wells were contaminated

Avoidcross-wellcontaminationbychang-ingtipsbetweenpipettingwhenperform-ingtheassayusingamultichannelpipette.

InsufficientwashesThe background may be due to non-spe-cificbindingofSA-PE.Increasenumberofwashes.

Debris(FSC/SSC)duringsample acquisi-tion

Debris or platelet may existinsamplesolution

Centrifugesamplesbeforeanalyzingsamples. Remove platelet as much as possible.

biolegend.com 51


Variationbe-tweenduplicate samples

Beadsaggregation Sonicate and vortex the Beads prior to use.

Multichannelpipettemay not be calibrated or inconsistent pipet-ting

CalibratePipette.Ensuregoodpipettingpractice.Primepipettebeforeusemayhelp.

Plate washing was not uniform

Make sure all reagents are vacuumed out completely in all wash steps.

Samples may contain particulatematters.


Low or poor standard curve signal

The standard was in-correctlyreconstituted,stored or diluted

Followtheprotocoltoreconstitute,storeand dilute standard. Double check your calculation.

Wrong or short incuba-tiontime

Ensurethetimeofallincubationswasappropriate.

Signals too high, standard curves satu-rated

PMTvalueforFL2/PEset too high

MakesurethePMTsettingforthere-porter channel is appropriate

Plateincubationtimewas too long Useshorterincubationtime.

Sample read-ings are out of range

Samples contain no or below detectable levels of analyte

Make sure the experiment to generate thesamplesworked.Useproperpositivecontrols.

Samplesconcentrationshigher than highest standard point.

Dilutesamplesandanalyzeagain.

Standard curve was saturated at higher end of curve.

MakesurethePMTsettingforthere-porter channel is appropriate. Use shorter incubationtimeifincubationtimewastoo long

Missed beads populationsduring reading, ordistributionis unequal

Sample may cause some beads to ag-gregate.


Beadspopulationsarenot mixed properly

Makesureallbeadpopulationsaremixed.and in similar numbers.

Tel: 858-768-5800


52

PLA

TE M

AP

(for

in-p

late

ass

ay)

1 2

3 4

5 6

7 8

9 10

11

12

A

C0

C4

Sa

mpl

e1

Sa

mpl

e5

Sa

mpl

e9

Sa

mpl

e 13

Sa

mpl

e17

Sa

mpl

e21

Sa

mpl

e25

Sa

mpl

e29

Sa

mpl

e33

Sa

mpl

e 37

B

C0

C4

Sa

mpl

e1

Sa

mpl

e5

Sa

mpl

e9

Sa

mpl

e 13

Sa

mpl

e17

Sa

mpl

e21

Sa

mpl

e25

Sa

mpl

e29

Sa

mpl

e33

Sa

mpl

e37

C

C1

C5

Sa

mpl

e2

Sa

mpl

e6

Sa

mpl

e10

Sa

mpl

e 14

Sa

mpl

e18

Sa

mpl

e22

Sa

mpl

e26

Sa

mpl

e30

Sa

mpl

e34

Sa

mpl

e38

D

C1

C5

Sa

mpl

e2

Sa

mpl

e6

Sa

mpl

e10

Sa

mpl

e 14

Sa

mpl

e18

Sa

mpl

e22

Sa

mpl

e26

Sa

mpl

e30

Sa

mpl

e34

Sa

mpl

e38

E

C2

C6

Sa

mpl

e3

Sa

mpl

e7

Sa

mpl

e11

Sa

mpl

e 15

Sa

mpl

e19

Sa

mpl

e23

Sa

mpl

e27

Sa

mpl

e31

Sa

mpl

e35

Sa

mpl

e39

F

C2

C6

Sa

mpl

e3

Sa

mpl

e7

Sa

mpl

e11

Sa

mpl

e 15

Sa

mpl

e19

Sa

mpl

e23

Sa

mpl

e27

Sa

mpl

e31

Sa

mpl

e35

Sa

mpl

e39

G

C3

C7

Sa

mpl

e4

Sa

mpl

e8

Sa

mpl

e12

Sa

mpl

e 16

Sa

mpl

e20

Sa

mpl

e24

Sa

mpl

e28

Sa

mpl

e32

Sa

mpl

e36

Sa

mpl

e40

H

C3

C7

Sa

mpl

e4

Sa

mpl

e8

Sa

mpl

e12

Sa

mpl

e 16

Sa

mpl

e20

Sa

mpl

e24

Sa

mpl

e28

Sa

mpl

e32

Sa

mpl

e36

Sa

mpl

e40

biolegend.com 53


RA

CK

MA

P (

for

in-t

ub

e as

say)

1

2 3

4 5

6 7

8 9

10

11

12

A

C0

C0

C1

C1

C2

C2

C3

C3

C4

C4

C5

C5

B

C6

C6

C7

C7

Sam

ple

1 Sa

mp

le

1 Sa

mp

le

2 Sa

mp

le

2 Sa

mp

le

3 Sa

mp

le

3 Sa

mp

le

4 Sa

mp

le4

C

Sam

ple

5

Sam

ple

5

Sam

ple

6

Sam

ple

6

Sam

ple

7

Sam

ple

7

Sam

ple

8

Sam

ple

8

Sam

ple

9

Sam

ple

9

Sam

ple

10

Sa

mp

le

10

D

Sam

ple

11

Sa

mp

le

11

Sam

ple

12

Sa

mp

le

12

Sam

ple

13

Sa

mp

le

13

Sam

ple

14

Sa

mp

le

14

Sam

ple

15

Sa

mp

le

15

Sam

ple

16

Sa

mp

le

16

E Sa

mp

le

17

Sam

ple

17

Sa

mp

le

18

Sam

ple

18

Sam

ple

19

Sa

mp

le

19

Sam

ple

20

Sa

mp

le

20

Sam

ple

21

Sa

mp

le

21

Sam

ple

22

Sa

mp

le

22

F Sa

mp

le

23

Sam

ple

23

Sa

mp

le

24

Sam

ple

24

Sa

mp

le

25

Sam

ple

25

Sa

mp

le

26

Sam

ple

26

Sa

mp

le

27

Sam

ple

27

Sa

mp

le

28

Sam

ple

28

G

Sam

ple

29

Sa

mp

le

29

Sam

ple

30

Sa

mp

le

30

Sam

ple

31

Sa

mp

le

31

Sam

ple

32

Sa

mp

le

32

Sam

ple

33

Sa

mp

le

33

Sam

ple

34

Sa

mp

le

34

H

Sam

ple

35

Sa

mp

le

35

Sam

ple

36

Sa

mp

le

36

Sam

ple

37

Sa

mp

le

37

Sam

ple

38

Sa

mp

le

38

Sam

ple

39

Sa

mp

le

39

Sam

ple

40

Sa

mp

le

40

LEGENDplex™ Kits are manufactured by BioLegend Inc. 9727 Pacific Heights Blvd.San 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

®

76599_V01

Documents

LEGENDplex™ · measurement of the adipokines is critical for abetter understanding of disease progression and immune responses. The LEGENDplexTM NHP Adipokine Panel is a bead-based