legendplex™ · please read the entire manual before running the assay ... • sonicator bath...

®

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. 740001, Human Th Cytokine Panel (13-plex ) Cat. No. 740009, Human Th1 Panel (5-plex) Cat. No. 740011, Human Th2 Panel (6-plex)Cat. No. 740013, Human Th1/Th2 Panel (8-plex) Cat. No. 740015, Human Th17 Panel (8-plex) Cat. No. 740019, Human Th9 Panel (3-plex)Cat. No. 740021, Human Th22 Panel (4-plex)Cat. No. 740023, Human Tfh Panel (4-plex)

Please read the entire manual before running the assay.

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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.

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LEGENDplex™ Human Th Cytokine Panel

Table of Contents Page

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

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

IntendedUse……………………....…………………........................

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

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

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

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

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

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

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

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

ReagentsPreparation………………………………………...............

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

DilutionofSamples....................................……...........…….

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

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

PerformingtheAssayUsingMicrotubes……………….............

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

Setup Procedure for FACSCalibur with Dual Laser..............

Setup Procedure for FACSCalibur 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.....................................................................

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

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

TypicalData……………….……………………………………………........

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

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

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

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

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

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

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

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

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

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

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Chapter 1: KIT DESCRIPTION

Introduction

Thelper(Th)cellsplayimportantrolesinregulatingimmuneresponses.Theysecretecytokinestostimulatevariouseffectorcells,suchascytotoxicTcells,B cells and macrophages. Accurate measurement of Th cytokine expression is criticaltoidentifythecorrespondingThcellsandforindepthunderstandingofthe immune responses.

TheHumanThCytokinePanelisamultiplexbead-basedassaypanel,usingfluorescence–encodedbeadssuitableforuseonvariousflowcytometers.Thispanelallowssimultaneousquantificationof13humancytokines,includingIL-2,4, 5, 6, 9, 10, 13, 17A, 17F, 21, 22, IFN-γandTNF-α,whicharecollectivelyse-creted by Th1, Th2, Th9, Th17, Th22 and T follicular cells. This assay panel pro-videshigherdetectionsensitivitiesandbroaderdynamicrangesthantraditionalELISAmethods.Thepanelhasbeenvalidatedbydetectingexpectedchangesinbiological samples.

TheHumanThCytokinePanelisdesignedtoallowflexiblecustomizationwithinthepanel.Itcanalsobedividedintocelltype-specificsubpanels.Pleaserefertothetargetselectiontableforpanel-specifictargetinformation(Table1).Formixand match within the panel, please visit www.biolegend.com/legendplex.

Intended Use

BioLegend’s LEGENDplexTMHumanThCytokineanditssubpanelsarespecificallydesignedfortheaccuratequantificationofmultiplehumanThcytokinesfromcell culture supernatant, serum, plasma and other biological samples.

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.Eachbeadsetisconjugatedwithaspecificantibodyonitssurfaceandservesasthecap-turebeadsforthatparticularanalyte.Whenaselectedpanelofcapturebeadsismixedandincubatedwithasamplecontainingtargetanalytesspecifictothecaptureantibodies,eachanalytewillbindtoitsspecificcapturebeads.Afterwashing,abiotinylateddetectionantibodycocktailisadded,andeachdetec-tionantibodyinthecocktailwillbindtoitsspecificanalyteboundonthecap-turebeads,thusformingcapturebead-analyte-detectionantibodysandwiches.Streptavidin-phycoerythrin (SA-PE) is subsequently added, which will bind to thebiotinylateddetectionantibodies,providingfluorescentsignalintensitiesin

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proportiontotheamountofboundanalytes.

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

Beads Usage

TheHumanThCytokinePanelusestwosetsofbeads.Eachsethasauniquesizethatcanbeidentifiedbasedontheirforwardscatter(FSC)andsidescatter(SSC)profiles(BeadsAandBeadsB,Figure1).Eachbeadsetcanbefurtherresolvedbasedontheirinternalfluorescenceintensities.Theinternaldyecanbede-tectedusingFL3,FL4,orAPCchannel,dependingonthetypeofflowcytometerused.ThesmallerBeadsAconsistsof6beadpopulationsandthelargerBeadsBconsistsof7beadpopulations(Figure2-3).

Usingatotalof13beadpopulationsdistinguishedbysizeandinternalfluores-centdye,theHumanThCytokinePanelallowssimultaneousdetectionof13cytokinesinasinglesample.Eachanalyteisassociatedwithaparticularbeadset as indicated (Figures 2-3 and Table 1)

Figure 1. Beads Differentiated by Size

Figure 2. Beads A Classification by FL4

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Figure 3. Beads B Classification by FL4

For Beads usage in various panels, please refer to Table 1 below:

Table 1. Beads ID* and Panel-Specific Target Selection

TargetBead

IDTh

Cat. No.740001

Th1Cat. No.740009

Th2Cat. No.740011

Th17Cat. No.740015

Th1/Th2Cat. No.740013

Th9Cat. No.740019

Th22Cat. No.740021

TfhCat. No.740023

IL-5 A4 √ √ √

IL-13 A5 √ √ √

IL-2 A6 √ √ √

IL-6 A7 √ √ √ √ √ √ √

IL-9 A8 √ √

IL-10 A10 √ √ √ √ √ √ √ √

IFN-γ B2 √ √ √ √

TNF-α B3 √ √ √ √ √ √ √ √

IL-17A B4 √ √

IL-17F B5 √ √

IL-4 B6 √ √ √

IL-21 B7 √ √ √

IL-22 B9 √ √ √

*BeadIDisusedtoassociateabeadpopulationtoaparticularanalyteintheLEGENDplexTMDataAnalysisSoftware.TheassociationofanalyteandbeadIDwillbedefinedduringthegatingstepofthedataanalysis.

When entering analyte and bead ID infomation during the gating step, always enter in the sequential order of the bead ID (e.g, A4, A5, A6...B2, B3, B4...). Please refer to the LEGENDplexTMDataAnalysisSoftwareUserGuideandOnlineHelp for details (www.biolegend.com/legendplex).

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Storage Information

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,leftoverstandardandMatrixBshouldbestoredat≤-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 77842

Setup Beads 3: Raw Beads 1 vial 2 mL 77844

Capture Beads* (see tables below for more information)

varies varies varies

HumanThCytokinePanelDetectionAntibodies

1bottle 3.5 mL 77707

Human Th Cytokine Panel StandardCocktail,Lyophilized

1 vial lyophilized 77709

LEGENDplexTM SA-PE 1bottle 3.5 mL 77743

LEGENDplexTMMatrixB,Lyophilized 1 vial lyophilized 77549

LEGENDplexTMAssayBuffer 1bottle 25 mL 77562

LEGENDplexTMWashBuffer,20X 1bottle 25 mL 77564

Filter plate 1 plate 21209

Plate Sealers 4 sheets 78101

DataAnalysisSoftwareDongle 1 21217

Human Th Cytokine Panel Manual 1 77550

* For Human Th Cytokine Panel, premixed beads are provided ready-to-use.

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ForHumanThCytokinesubpanels,individualbeadsareprovidedat13Xcon-centration(seetablesbelow):

Capture beads for Human Th Cytokine Panel:


Human Th Cytokine Panel Premixed Beads 1bottle 3.5 mL 77679

Capture beads for Human Th Cytokine subpanels*:


Human IL-5 beads, 13X 1 vial 270 µL 77685

Human IL-13 Beads, 13X 1 vial 270 µL 77693





Human IFN-γ Beads, 13X 1 vial 270 µL 77703

Human TNF-α Beads, 13X 1 vial 270 µLL 77705

Human IL-17A Beads, 13X 1 vial 270 µL 77695

Human IL-17F Beads, 13X 1 vial 270 µL 77697




* Please refer to Beads ID* and Panel-Specific Target Selection table (Table 1), to see which capture beads are included in each panel.

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Materials to be Provided by the End-User

• Aflowcytometerequippedwithtwolasers(e.g.,a488nmbluelaseror532nmgreenlaseranda633-635nmredlaser)capableofdistinguishing575 nm and 660 nm oraflowcytometerequippedwithonelaser(e.g.,488nmbluelaser)capableofdistinguishing575nmand670nm.

Partial list of compatible flow cytometers:

Flow Cytometer

Reporter Channel

ChannelEmission

Classification Channel

Channel Emission

Compensa-tion needed?

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*

BDTM LSR, LSR IIBD LSRFortessaTM PE 575 nm APC 660 nm No*

BD FACSAriaTM PE 575 nm APC 660 nm No*

*Compensation is not required for the specified flow cytometers when set up properly, but is recommended for consistent results.

Forsettingupoftheaboveflowcytometers,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

• Polypropylene microfuge tubes (1.5 mL)

• Laboratory vortex mixer

• Sonicator bath (e.g., Branson Ultrasonic Cleaner model #B200, or equiva-lent)

• Aluminum foil

• Absorbent pads or paper towels

• Plate shaker (e.g., Lab-Line Instruments model #4625, or equivalent)

• Tabletop centrifuges (e.g., Eppendorf centrifuge 5415 C, or equivalent)

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LEGENDplex™ Human Th Cytokine Panel If the assay is run using the filter plate provided (recommended),

• Avacuumfiltrationunit(MilliporeMultiScreen®HTSVacuumManifold,catalog#MSVMHTS00orequivalent).Instructionsonhowtousethevacuum manifold can be found at www.biolegend.com/legendplex.

• A vacuum source (mini vacuum pump or line vacuum, e.g., Millipore VacuumPump,catalog#WP6111560,orequivalent)

If the assay is run in microtubes (optional),

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

• Centrifugewithaswingingbucketadaptorformicrotiterplatesormicro-tube racks (e.g., Beckman Coulter AllegraTM 6R Centrifuge with MICROPLUS CARRIER adaptor for GH3.8 and JS4.3 Rotors) .

Precautions

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

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

• Matrix B for LEGENDplexTM kits contains components of human origin and shouldbehandledaspotentiallyhazardous.TherawmaterialhasbeenscreenedforinfectiousdiseasesandisnegativeforHIV,HBVandHCVusingFDA-approved test methods.

• Donotmixorsubstitutereagentsfromdifferentkitsorlots.Reagentsfromdifferentmanufacturersshouldnotbeusedwiththiskit.

• Donotusethiskitbeyonditsexpirationdate.

• SA-PEandPremixedBeadsarelight-sensitive.Minimizelightexposure.

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Chapter 2: ASSAY PREPARATION

Sample Collection and Handling

Preparation of Serum Samples:

• Allow the blood to clot for at least 30 minutes and centrifuge for 10 min-utes at 1,000 x g.

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

• Whenusingfrozensamples,itisrecommendedthatsamplesarethawedcompletely,mixedandcentrifugedtoremoveparticulatespriortouse.

Preparation of Plasma Samples:

• PlasmacollectionusingEDTAasananti-coagulantisrecommended.Centri-fuge for 10 minutes at 1,000 x gwithin30minutesofbloodcollection.

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

• Whenusingfrozensamples,itisrecommendedthatsamplesarethawedcompletely,mixedwellandcentrifugedtoremoveparticulates.

Preparation of Tissue Culture Supernatant:

• Centrifuge the sample to remove debris and assay immediately, or aliquot andstoresamplesat≤-20°C.Avoidmultiple(>2)freeze/thawcycles.

Reagents Preparation

Preparation of Antibody-Immobilized Beads

• If pre-mixed beads are provided in the kit:

SonicatePre-mixedBeadsbottlefor1minuteinasonicatorbathandthen vortex for 30 seconds prior to use. If no sonicator bath is available, increasethevortexingtimeto1minutetocompletelyresuspendthebeads.

• If individual beads (13X) are provided in the kit:

Theindividualbeads(13X)shouldbemixedanddilutedto1XwithAssayBufferpriortouse.Tomixthebeads,followthestepsbelow(the5-plexTh1 Panel is used as an example):

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

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vortex for 30 seconds prior to use.

2. Calculate the amount of mixed and diluted beads needed for the assay. Prepareextratocompensateforpipettingloss.Eachreactionneeds 25µLofmixedanddilutedbeads.For50reactions,prepare1.5mLof mixedbeads.For96reactions,prepare3mLofmixedbeads.

3.Tomake1.5mlof5-plex(e.g.,Th1Panel)1Xdilutedbeads,transfer 115µLofeachofthe5individualbeads(13X)toafreshtube(total beadvolume=575µL)andadd925µLofAssayBuffertomakethe finalvolumeof1.5mL.

Preparation of Wash Buffer

• Bringthe20XWashBuffertoroomtemperatureandmixtobringallsaltsintosolution.

• Dilute25mLof20XWashBufferwith475mLdeionizedwater.Storeun-usedportionsbetween2°Cand8°Cforuptoonemonth.

Preparation of Matrix B (for Serum or Plasma Samples Only)

• Add 5.0 mL LEGENDplexTMAssayBuffertothebottlecontaininglyophilized MatrixB.Allowatleast15minutesforcompletereconstitution.Vortexto

mixwell.LeftoverreconstitutedMatrixBshouldbestoredat≤-70°Cforup to one month.

Standard Preparation

1. Priortouse,reconstitutethelyophilizedHumanThCytokineStandardCocktailwith250µLAssayBuffer.

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.

Note: Each analyte in this panel has a top standard concentration of 10,000 pg/mL.

3. Label 6 polypropylene microfuge tubes as C6, C5, C4, C3, C2 and C1, respectively.

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,C2and C1 standards (see the table below).AssayBufferwillbeusedasthe0pg/mLstandard(C0).

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Tube/Standard ID

Serial Dilution

Assay Buffer to add (µL)

Standard to add

Final Conc. (pg/mL)

C7 -- -- -- 10,000

C6 1:4 75 25 µL of C7 2,500

C5 1:16 75 25 µL of C6 625

C4 1:64 75 25 µL of C5 156.3

C3 1:256 75 25 µL of C4 39.1

C2 1:1024 75 25 µL of C3 9.8

C1 1:4096 75 25 µL of C2 2.4

C0 -- 75 -- 0

Dilution of Samples

• Serumorplasmasamplesmustbediluted2-foldwithAssayBufferbeforebeingtested(e.g.dilute50µLofsamplewith50µLofAssayBuffer).

Iffurthersampledilutionisdesired,dilutionshouldbedonewithMatrixBto ensure accurate measurement.

Adding serum or plasma samples without dilution will result in low assay accuracy and possibly, clogging of the filter plate.

• For cell culture supernatant samples, the levels of analyte can vary greatly fromsampletosample.Whilethesamplecanbetestedwithoutdilutions,a preliminary experiment may be required to determine the appropriate dilutionfactorforsamples.

Ifsampledilutionisdesired,dilutionshouldbedonewithcorrespondingfreshcellculturemediumorAssayBuffertoensureaccuratemeasurement.

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Chapter 3: ASSAY PROCEDURE

The LEGENDplexTM assaycanbeperformedeitherinafilterplate(in-plateas-say) or in microtubes (in-tube assay).

• The in-plate assay procedure is highly recommended due to its good sample to sample consistency, assay robustness and ease of handling. This procedurerequiresavacuumfiltrationunitforwashing(seeMaterials to be Provided by the End-User).IfyouhaveperformedaLuminex®-basedmultiplexassaybefore,yourlabshouldalreadyhavethevacuumfiltrationunit set up.

• If the in-plate assay procedure is not possible or if you prefer, the in-tube assay can be performed. In this case, we recommend using micro FACS tubes for the assay (see Materials to be Provided by the End-User).

Performing the Assay Using a Filter Plate

• Allow all reagents to warm to room temperature (20-25°C) before use.

• Setthefilterplateonaninvertedplatecoveratalltimesduringassaysetupandincubationsteps,sothatthebottomoftheplatedoesnottouch any surface. 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 plate in a vertical configuration convenient for data acquisition and analysis (as shown in attached PLATE MAP). Be sure to load standards in the first two columns.

1. Pre-wettheplatebyadding100μLofLEGENDplexTM1XWashBufferto each 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).BlotexcessWashBufferfromthebottomoftheplatebypressing the plate on a stack of clean paper towels. Place the plate on top of the inverted plate cover.

For measuring cell culture supernatant samples:

• Add25µLofAssayBuffertoallwells.• Add 25 µL of each standard to the standard wells. • Add 25 µL of each sample to the sample wells (See Dilution of

Samples)

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For measuring serum or plasma samples:

• Add 25 µL of Matrix B to the standard wells.• Add 25 µLofAssayBuffertothesamplewells.• Add 25 µL of each standard to the standard wells. • Add 25 µL of each diluted serum or plasma sample to the sample

wells (See Dilution of Samples).

2. Vortexmixedbeadsfor30seconds.Add25μLofmixedbeadstoeachwell.Thevolumeshouldbe75μLineachwellafterreadsaddition.(Note:Duringadditionofthebeads,shakemixedbeadsbottleintermittentlytoavoidbeadsettling).

3. Seal the plate with a plate sealer. To avoid plate leaking, do not apply posi-tive pressure to the sealer when sealing the plate. 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. Repeat step 4 above.

10. Add200µLof1XWashBuffertoeachwell.Resuspendthebeadsonaplate shaker for 1-5 minutes.

11. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay (Note: Prolonged sample storage can lead to reduced signal and sensitivity).

Iftheflowcytometerisequippedwithanautosampler,readtheplatedi-rectly using the autosampler. The probe height might need to be adjusted

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when using an autosampler.

If an autosampler is not available, the samples need to be transferred from thefilterplatetoFACStubesandreadmanually.Inthiscase,thesamplevolume may need to be increased from 200 µL to 300 µL by adding extra 100µLof1XWashBuffertoeachtubetoavoidsamplerunningdrywhenreadonaflowcytometer.

In-Plate Assay Procedure SummaryAdd 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 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 mixed beads to all wells

Wash 2 times using vacuum �ltration unitAdd 200 μL 1x Wash Bu�er Read on a �ow cytometer

For serum and plasma samples,Add to the plate:25 μL Matrix B to standard wells25 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

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Performing the Assay Using Microtubes

1. Allow all reagents to warm to room temperature (20-25°C) before use.

2. Determine the number of tubes needed, arrange tubes on a rack, and label all tubes.

Standards and samples should be run in deplicate and arranged on a rack in the order convenient for data acquisition and analysis (as shown in attachedRACKMAP,assumingusingmicroFACStubesanda96-microtuberack,e.g.,NationalScientificSupplyCo,catalog#:TN0946-01R).A96-mi-crotuberackisrecommendedbecauseitallowspipettingwithamultichan-nelpipette.

3. For measuring cell culture supernatant samples:

• Add25µLofAssayBuffertoalltubes.• Add 25 µL of each standard to the standard tubes.• Add 25 µL of each sample to the sample tubes (See Dilution of

Samples).• Add 25 µL of mixed beads to all tubes.• Add25µLDetectionAntibodiestoalltubes.

For measuring serum or plasma samples:

• Add 25 µL of Matrix B to the standard tubes.• Add25µLofAssayBuffertosampletubes.• Add 25 µL of each standard to standard tubes.• Add25µLofeachdilutedserumorplasmasampletosampletubes

(See Dilution of Samples).• Add 25 µL of mixed beads to all tubes.• Add25µLDetectionAntibodiestoalltubes.

Note: Thevolumeshouldbe100μLineachwell.Shakebeadsbottleintermittentlyduringtheadditiontoavoidbeadsettling.

4. Covertheentirerackwithaluminumfoiltoprotectthetubesfromlight.Shake vigorously (approximate 1,000 rpm) on a plate shaker for 2 hours at room temperature.

5. Withoutwashingthetubes,add25µLSA-PEtoeachtube.

6. Covertheentirerackwithaluminumfoiltoprotectthetubesfromlight.Shake vigorously (approximate 1,000 rpm) on a plate shaker for 30 minutes at room temperature.

7. Centrifuge the tubes at 1,000 x g for 5 minutes, using a swinging bucket rotor with microplate adaptor (Please refer to Materials to be Provided by the End-User).

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8. Removethesupernatantusingamicropipette.Becarefulnottoremovebeads, but to remove as much liquid as possible.

9. Add200µLof1XWashBuffertoalltubes.Resuspendthebeadsbyvortex-ingbriefly.Centrifuge the tubes at 1,000 x g for 5 minutes, using a swinging bucket rotor with microplate adaptor. Remove the supernatant.

10. Add200µLof1XWashBuffertoalltubes.Resuspendthebeadsbyvortex-ingbriefly.

11. Readsampleonaflowcytometer,preferablywithinthesamedayoftheassay (Note: Prolonged sample storage can lead to reduced signal and sensitivity).

Iftheflowcytometerisequippedwithanautosampler,thesamplesinthetubescanbetransferredtoa96-wellplateandanalyzed.The probe height might need to be adjusted when using an autosampler.

If an autosampler is not available, the samples can be read manually. In this case, the sample volume may need to be increased from 200 µL to 300 µLbyaddingextra100µLof1XWashBuffertoeachtube,toavoidsamplerunningdrywhenanalyzedonaflowcytometer.

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In-Tube Assay Procedure Summary

Arrange the number of tubes needed on a rack

Incubate 2 hours, RT, shaking

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

For cell culture supernatant samples, Add to the tube:25 μL Assay Bu�er to all tubes25 μL diluted standard to standard tubes25 μL sample to sample tubes25 μL mixed beads to all tubes25 μL Detection Antibodies to all tubes

Spin down beadsWash one timeAdd 200 μL 1x Wash Bu�erRead on a �ow cytometer

For serum and plasma samples, Add to the tube:25 μL Matrix B to standard tubes25 μL Assay Bu�er to sample tubes25 μL diluted standard to standard tubes25 μL sample to sample tubes25 μL mixed beads to all tubes25 μL Detection Antibodies to all tubes

A B

C

A

B

C

Capture beads

Detection Antibody

Analytes

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Chapter 4: FLOW CYTOMETER SETUP

Inordertogeneratereliabledata,theflowcytometermustbesetupproperlybeforedataacquisition.Thefollowingsectionswilladdressmachinesetupfortheflowcytometerslistedbelow.

List of Flow Cytometers and Possible Configurations

Flow CytometerReporterChannel

Channel Emission

Classification Channel

ChannelEmission

Compensationneeded?

BD FACSCaliburTM

(single laser)FL2 575 nm FL3 670 nm Yes

BD FACSCaliburTM

(dual laser)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*

*Compensation is not required for the specified flow cytometers when set up properly, but is recommended for consistent results.

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.

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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. Obtain a Template for Data Acquisition

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 two gates and label them Beads A and Beads B (Figure 4).

Figure 4.

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

02

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Gated on Beads BGated on Beads A

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2.4 Save the new document as “LEGENDplex Template for FACSCalibur 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.

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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

(Figure 6).

3.6 AdjusttheFL1settingsothattheFL1signalsforallbeadsare between 1x100 and 1x101 (Note: This step is not required, but is

recommended).

3.7 AdjusttheFL4settingsothattheFL4signalsforallbeadsarebe- tween 1x101 and 5x103 (Figure 7).

Figure 7.

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

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3.9 Transfer400μLofthePEBeadstoafreshFACStube.

3.10ReplacetheRawBeadstubefromtheflowcytometerwiththe PE beads tube.

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

gate (Figure 8).

3.12AdjusttheFL2settingsothatthemedianfluorescenceintensityof thePEbeadsfallsbetweenthelot-specificrangelabeledonthePE Beads vial (Figure 8, gate R3).

Figure 8.

3.13 Save the document again for future use.

3.14Theflowcytometerisnowreadyforsampleanalysis.

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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 two gates and label them Beads A and Beads B (Figure 9).

Figure 9.

0200

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

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Figure 10.

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2.4 Setallcompensationstozero.

2.5 Save the new document as “LEGENDplex Template for FACSCalibur Single Laser” and proceed to the next step of the setup.

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3. Set up PMT Voltages and Compensation

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 (Figure 11).

3.6 AdjusttheFL1settingsothattheFL1signalsforallbeadsarebe tween 1x100 and 1x101 (Figure 12).

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Figure 12.

3.7 AdjusttheFL2settingssothattheFL2signalsforallbeadsarebe- tween 1x100 and 1x101 (Figure 12).

3.8 AdjusttheFL3settingsothattheFL3signalsforallbeadsarebe- tween 1x101 and 5 x 103 (Figure 12).

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.

3.12 On the FL1 vs FL2 dot plot (Figure 13), the beads will display as FITC- negativeandFITC-positivepopulations(indicatedbyanarrow). Note:FITCbeadsareonlyofsmallsize,fallingintheBeadsAgate.

10

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Figure 13.

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

Figure 14.

3.14 Vortex the vial of PE Beads for 30 seconds to resuspend the beads.

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

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

3.17 On the FL1 vs FL2 dot plot (Figure 15), the beads will display as PE- negativeandPE-positivepopulations(indicatedbyanarrow).Note: PEbeadsareonlyofsmallsize,fallingintheBeadsAgate.

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Figure 15.

3.18AdjusttheFL2settingsothatthemedianfluorescenceintensityofthe PEbeadsfallsbetweenthelot-specificrangelabeledonthePEBeads vial (Figure 16).

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

Figure 16.

3.20 On the FL3 vs FL2 dot plot (Figure 17), the beads will display as PE- negativeandPE-positivepopulations(indicatedbyanarrow).

3.21 AdjusttheFL3-%FL2compensationsetting(e.g.,FL3-%FL2=40%)so thattheFL3-lowpopulationandthePE-positivepopulationhave similar median FL3 signal (Figure 18).

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Figure 17.

Figure 18.


3.23Theflowcytometerisnowcompensatedandreadyforsample analysis.

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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(Thesettingswillbesavedinthefinalstepofthissetupprocedureandanysettingssavedcanbeimportedtoanew experiment. Please refer to Step 2 and Step 3.9 below).




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 instructionsinstep2.Afteratemplateiscreated,savethefileinD:\BDEx-port\Templates\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.

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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 them Beads A and Beads B (Figure 19).

Figure 19.

2.3CreatetwodotplotswithPEforX-axis,APCforY-axis(Figure20), gatedonBeadsA(leftpanelbelow)andBeadsB(rightpanel below),respectively.CreateonedotplotwithFITCforX-axis,APCfor Y-axis, gated on Beads A and Beads B (graph not shown). The plots should all be in log mode.

Figure 20.

2.4 Save the document as “LEGENDplex Template for FACS Diva” inD:\BDExport\Templates\Experimentandproceedtothenextstep of setup.

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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 (Figure 21).

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 (Figure 21).

3.6 AdjusttheFITCsettingsothattheFITCsignalforthemajorityof beads is between 1x101 and 1x102 (Figure 22).

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Figure 22.

Beads Beads A+ Beads B

3.7 AdjustthePEsettingsothatthePEsignalforthemajorityofbeadsis between 1x101 and 1x102 (Figure 23).

Figure 23.

3.8 AdjusttheAPCsettingssothatthetheAPCfluorescenceintensitiesof allbeadpopulationsarebetween1x102 and 5 x 104 (Figure 23).


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

3.10Theflowcytometerisnowreadyforsampleanalysis.

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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. In this case, machine compensation between the reporter and beads classification channels is strongly recommended.

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

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Chapter 5: DATA ACQUISITION AND ANALYSIS

Data Acquisition

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

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

3. Vortex each sample for 5 seconds before analysis.

4. Settheflowratetolow.Setthenumberofbeadstobeacquiredto2000-2500 on Beads A gate or Beads B gate.

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.

5. Read samples.

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...). For an in-tube assay, read row by row (A1, A2, A3,...B1, B2, B3...).

Whennamingdatafiles,trytousesimplenameswithaconsecutivenum-bering for easy data analysis (e.g. for standards, C0.001, C0.002, C1.003, C1.004, C2.005, C2.006, C3.007, C3.008, ... C7.015, C7.016; for samples, S1.017,S1.018,S2.019,S2.020,S3.021,S3.022…)

StoreallFCSfilesinthesamefolderforeachassay.Ifrunningmultipleas-says, create a separate folder for each assay.

6. Proceed to data analysis using LEGENDplexTMDataAnalysisSoftwarewhendataacquisitioniscompleted.

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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.

• TheSoftwareDonglehasafixednumberofpoints.Eachanalysiswillcon-sume a certain number of points from the dongle. The number of points consumeddependsonassayplexsizeandnumberofsamples.Afterthepoints on a dongle are consumed, a new dongle will be needed to run moredataanalysis.Althoughthesoftwarewillcontinuetobefunctional,thedatawillnotbesaveduntilanewdongleisavailable.Anewdongleisprovided in each LEGENDplexTM kit. A saved analysis can always be re-ana-lyzedusingthesoftwareregardlessofdonglestatus.

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

`

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Chapter 6: FREQUENTLY ASKED QUESTIONS

Q. What is the difference between LEGENDplexTM 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. What are the compatible flow cytometers for the LEGENDplex™ 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

that the machine is set up properly before use (refer to Setup Procedure for Other Flow CytometerssectioninChapter4).In this case, machine compensation between the reporter and beads classification channels is strongly recommended.

TheFCSorListmodefilesfromthefollowinginstrumentsarecompatiblewith the LEGENDplex TMDataAnalysisSoftware.

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VendorInstrument

ModelVendor

Instrument 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. What is the right procedure for running the assay?

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(32 bit or 64 bit). Make sure to install the correct version (32 bit or 64 bitversion),dependingonyourcomputer’soperatingsystem.

5. TransferthefoldercontainingtheFCSfilesofyourexperimenttothecomputerwherethedataanalysissoftwareisinstalled.

6. PerformdataanalysisasinstructedintheDataAnalysisSoftwareUserGuide.

Q. When do I need to do machine compensation?

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.

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Ifaflowcytometerislistedinthecompatibleflowcytometerlistinthismanualandisequippedwithtwolasers,oneforreporter(FL2/PE)andtheotherforclassification(FL4/APC),thencompensationisnotrequirediftheFLOW CYTOMETER SETUP guide in this manual is closely followed. How-ever, compensation is recommended for consistent results.

Forotherflowcytometersnotlistedinthecompatibleflowcytometerlist, the end-user needs to set up the machine following similar guidelines (refer to manufacturer’s manual for proper instrument setup). In this case, machine compensation between the reporter and beads classification channels is strongly recommended.

Q. Is there a special software required for data analysis?

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. Can I select to measure only some analytes within a panel?

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

Q. Can I select to measure analytes across panels?

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-nent from a different kit?

TheLEGENDplex™Beads,Standards,DetectionAntibodies,MatrixB,andSA-PEarelot-specificandmustbeusedincombinationwitheachother.Donotmixthesecomponentsfromdifferentkitsorlots.

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

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

Yes. It is possible to use the non-linear part of the standard curve for calculatingtheresults.TheLEGENDplexTMDataAnalysisSoftwareusesafive-parametercurvefittingalgorithm,whichdeterminestheminimumandmaximumdetectionconcentrationsofeachtargetandreportsthem.Ifsampleconcentrationsfallabovethemaximumdetectableconcentration,thesamplewillhavetobedilutedandreanalyzed.Ifsampleconcentrationsfallbelowtheminimumdetectionconcentration,itisconsiderednon-detectablebytheparticularassay.

Q. During data acquisition, why do the bead populations (defined by FSC and SSC) sometimes appear to be dispersed or shifted?

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

Q. Does the LEGENDplex™ Data Analysis Software run on Apple Macintosh?

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

Q. Why does each kit include a software dongle?

Thedongleallowsyoutousethedataanalysissoftware.However,thereisalimitednumberofpointswitheachdongle.Whenthetotalnumberofpoints is consumed for a dongle, the dongle needs to be replaced with a new one. Each kit includes a dongle to ensure the data analysis will not be impactedbythelimitedusageofadongle.Eachdongleissufficientforatleast 192 tests or two full 13-plex assay panels, equivalent to 2600 points

Q. I ran out of points on my dongle. Can I get a new one separately? Or, can I use a dongle from one kit for another kit?

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

Tel: 858-768-5800


42

Chapter 7: ASSAY CHARACTERIZATION

Representative Standard Curve

This standard curve was generated using the LEGENDplexTM Human Th CytokinePanelfordemonstrationpurposeonly.Astandardcurvemustberun with each assay.

1

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Concentration (pg/mL)

IL-5

IL-13

IL-2

IL-6

IL-9

IL-10

IFN-r

TNF-a

IL-17A

IL-17F

IL-4

IL-21

IL-22

Assay Sensitivity

Theassaysensitivityorminimumdetectableconcentration(MDC)isthetheoreticallimitofdetectioncalculatedusingtheLEGENDplexTM Data AnalysisSoftwarebyapplyinga5-parametercurvefittingalgorithm.

AnalyteMDC in Cell Culture

Medium (pg/mL)MDC in Serum

(pg/mL)

Human IL-5 1.1 1.3

Human IL-13 0.8 1.1

Human IL-2 1.0 1.3

Human IL-6 1.1 1.1

Human IL-9 1.0 1.2

Human IL-10 1.1 0.9

HumanIFN-γ 1.0 1.4

HumanTNF-α 1.0 0.9

Human IL-17A 1.5 1.8

Human IL-17F 1.1 1.3

biolegend.com 43


Human IL-4 0.7 1.1

Human IL-21 1.4 2.3

Human IL-22 2.0 2.2

Cross-Reactivity

Thefollowingrecombinantproteinsweretestedat50ng/mLusingtheLEGENDplexTM HumanThCytokinePanel.Noornegligiblecross-reactivitywas found.

ENA-78 Eotaxin GROα IFN-α2 IFN-γ IL-1α IL-1β

IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-8

IL-9 IL-10 IL-12p70 IL-13 IL-15 IL-17A IL-17F

IL-18 IL-21 IL-22 IL-23 IL-27 IL-31 IL-33

IP-10 I-TAC MCP-1 MIG MIP-1α MIP-1β MIP-3α

PARC RANTES TARC TNF-α TNF-β

Accuracy (Spike Recovery)

For spike recovery in cell culture medium, target proteins with known concentrationswerespikedintocellculturemedium(RPMIandDMEMwith10%FCS)atthreedifferentlevelswithintheassayrange.Thespikedsampleswerethenassayed,andthemeasuredconcentrationswerecom-pared with expected values.

Forspikerecoveryinserum,asamplewithknownhighconcentrationsoftarget proteins was spiked into unknown serum samples. The spiked sam-pleswerethenassayed,andthemeasuredconcentrationswerecomparedwith expected values.

Analyte% of Recovery in Cell

Culture Medium% of Recovery in

Serum

Human IL-5 91% 125%

Human IL-13 55% 88%

Human IL-2 93% 99%

Human IL-6 94% 116%

Human IL-9 100% 91%

Human IL-10 93% 100%

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44

HumanIFN-γ 100% 82%

HumanTNF-α 96% 83%

Human IL-17A 82% 72%

Human IL-17F 80% 86%

Human IL-4 77% 86%

Human IL-21 114% 89%

Human IL-22 56% 89%

Linearity of Dilution

Fortestinglinearityofdilution,serumsampleswerefirstdilutedtwo-foldwithAssayBuffer,thenseriallydiluted1:2,1:4,1:8withMatrixBandas-sayed.Themeasuredconcentrationsofseriallydilutedsampleswerethencompared with that of the two-fold diluted samples.

AnalyteLinearity of

DilutionAnalyte

Linearity of Dilution

Human IL-5 111% HumanTNF-α 118%

Human IL-13 113% Human IL-17A 115%

Human IL-2 113% Human IL-17F 113%

Human IL-6 117% Human IL-4 120%



HumanIFN-γ 127%

biolegend.com 45


Intra-Assay Precision

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

Analyte SampleMean

(pg/mL)STDEV %CV

Human IL-5Sample 1 34.8 3.1 9%

Sample 2 613.2 42.0 7%


Sample 2 585.3 52.8 9%


Sample 2 660.0 62.7 9%


Sample 2 604.7 70.1 12%


Sample 2 581.7 44.8 8%


Sample 2 658.4 46.9 7%

HumanIFN-γSample 1 28.1 4.6 16%

Sample 2 608.7 62.1 10%

HumanTNF-αSample 1 36.2 2.9 8%

Sample 2 623.4 55.1 9%

Human IL-17ASample 1 35.1 2.2 6%

Sample 2 583.9 27.9 5%

Human IL-17FSample 1 33.8 3.3 10%

Sample 2 661.7 81.4 12%


Sample 2 588.2 34.1 6%


Sample 2 569.5 42.7 8%


Sample 2 601.5 42.7 7%

Tel: 858-768-5800


46

Inter-Assay Precision

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

Analyte SampleMean

(pg/mL)STDEV %CV


Sample 2 603.3 39.0 6%


Sample 2 567.9 34.4 6%


Sample 2 627.2 63.0 10%


Sample 2 633.5 78.7 12%


Sample 2 611.6 73.1 12%


Sample 2 635.3 41.6 7%

HumanIFN-γSample 1 37.5 7.6 20%

Sample 2 631.7 75.2 12%

HumanTNF-αSample 1 37.1 2.9 8%

Sample 2 647.3 73.6 11%

Human IL-17ASample 1 37.7 3.5 9%

Sample 2 621.4 42.4 7%

Human IL-17FSample 1 35.1 2.9 8%

Sample 2 652.6 59.3 9%


Sample 2 610.0 61.3 10%


Sample 2 596.6 69.0 12%


Sample 2 610.4 30.6 5%

biolegend.com 47


Biological Samples

Serum and Plasma (Samples are not paired)

Normal human serum samples (n=30) were tested for endogenous levels oftheThcytokines.Theconcentrationsmeasuredareshownbelow:

AnalyteRange

(pg/ml)No. of

Detectable% of

DetectableMean

(pg/mL)

Human IL-5 ND 0 0% ND


Human IL-2 ND - 33.1 7 23% 3.1

Human IL-6 ND -85.0 18 60% 12.9

Human IL-9 ND -9.7 1 3% 0.4


HumanIFN-γ ND - 4.6 1 3% 0.2

HumanTNF-α ND - 35.4 11 37% 2.3

Human IL-17A ND 0 0% ND

Human IL-17F ND - 35.8 5 17% 2.6


Human IL-21 ND - 43.4 3 10% 2.7

Human IL-22 ND - 27.8 7 23% 3.6

ND = Non-detectable

Normal human plasma samples (n=18) were tested for endogenous levels ofThcytokines.Theconcentrationsmeasuredareshownbelow:

AnalyteRange

(pg/mL)No. of

Detectable% of

DetectableMean

(pg/mL)

Human IL-5 ND -29.6 4 21% 2.5

Human IL-13 ND - 10.9 4 21% 1.3

Human IL-2 ND -24.6 7 37% 2.4

Human IL-6 ND - 39.9 8 42% 4.3

Human IL-9 ND - 15.5 2 11% 1.1

Human IL-10 ND -8.7 1 5% 0.5

HumanIFN-γ ND - 97.7 7 37% 8.7

HumanTNF-α ND - 176.8 11 58% 12.8

Human IL-17A ND - 29.0 6 32% 4.8

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48

Human IL-17F ND - 154.5 8 42% 15.1

Human IL-4 ND - 68.4 2 11% 3.8

Human IL-21 ND - 329.8 9 47% 36.3

Human IL-22 ND - 236.1 14 74% 34.0

ND = Non-detectable

Cell Culture Supernatant

Human PBMC (1 x 106cells/mL)wereculturedundervariousconditions(LPS,100ng/mL;CD3,1µg/mLplate-coated;CD28,1µg/mLsoluble;PMA,20ng/mL;Ionomycin,500ng/mL).Supernatantswerecollectedafter48hours and assayed with the LEGENDplexTM Human Th Cytokine Panel kit. Theresults(allinpg/mL)aresummarizedbelow.

Analyte Control LPS CD3 + CD28 PMA +

Ionomycin

Human IL-5 8.3 53.8 5020.2 6174.1

Human IL-13 13.6 134.6 4713.6 4671.4

Human IL-2 21.3 378.8 1412.5 >10,000

Human IL-6 12.0 6982.0 3088.6 >10,000

Human IL-9 18.3 131.0 >10,000 >10,000

Human IL-10 12.6 399.8 6634.2 1362.6

HumanIFN-γ 9.5 578.1 >10,000 >10,000

HumanTNF-α 4.1 11.2 9337.6 >10,000

Human IL-17A 7.8 30.0 766.1 484.5

Human IL-17F 16.7 198.2 9008.0 1399.7

Human IL-4 40.0 38.9 1302.4 1201.3

Human IL-21 17.2 16.7 606.1 606.1

Human IL-22 32.5 14.3 1012.5 489.0

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 0.2 mLbuffercanbesuctionedin3-5seconds.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 particlesorsampleistoo viscous (e.g., serum and plasma samples)

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

Ifsomewellsarestillcloggedduringwash-ing, try the following:

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

2). Use a piece of clean wipe, wipe the un-der side of the clogged wells and vacuum again.

3). Take a thin needle (e.g., insulin needle), while holding the plate upward, poke the littleholeundereachofthecloggedwellsand vacuum again. Do not poke too hard ortoodeepasitmaydamagethefilterand cause leaking.

Filter plate was used without pre-wet.

Pre-wetplatewithwashbufferbeforerun-ning the assay.

Tel: 858-768-5800


50

Insufficientbead count or slow reading

Beads inappropriately prepared

Sonicate bead vials and vortex just prior toaddition.Agitatemixedbeadsintermit-tentlyinreservoirwhilepipettingthisintothe plate.

Samples cause beads aggregationduetoparticulatematterorviscosity.


Beads were lost during washing for in-tube assay

Make sure beads are spun down by visu-ally check the pellet (beads are in light blue or blue color). Be very careful when removing supernatant during washing.

Probe might be par-tiallyclogged.

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.Donot 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-specificbindingofSA-PE.Increasenumberof washes.

Debris(FSC/SSC) during sample acquisi-tion

Debris or platelet may exist in sample solu-tion.

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.

Wrongorshortincuba-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 incubationtimeifincubationtimewastoolong

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

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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

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740001_V01

legendplex™ · please read the entire manual before running the assay ... • sonicator bath...

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