legendplex™ · legendplextm assay buffer 1 bottle 25 ml 77562 legendplextm wash buffer, 20x 1...

LEGENDplex™Mul�-Analyte Flow Assay Kit

Enabling Legendary Discovery™

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

Serum, Plasma and Other Biological Samples

Please read the entire manual before running the assay

BioLegend.com

LEGENDplex™Mul�-Analyte Flow Assay Kit


Cat. No. 740102

Human Cytokine Panel 2(13-plex)

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.

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LEGENDplex™ Human Cytokine Panel 2

Table of Contents Page

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

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

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

Cytokines, which are soluble extracelluar proteins or glycoproteins, are key in-tercellular regulators and mobilizers of cells. They are secreted by immune cells and act on other cells to coordinate appropriate immune responses. They are crucialtoinnateandadaptivehostdefenses,cellgrowth,celldeath,differentia-tion,aswellasdevelopmentandrepairprocesses.

The Human cytokine panel 2 isamultiplexbead-basedassaypanel,usingfluorescence–encodedbeadssuitableforuseonvariousflowcytometers.Thispanelallowssimultaneousquantificationof13humancytokines,including TSLP, IL-1α,IL-1β,GM-CSF, IFN-α2,IL-23,IL-12p40,IL-12p70,IL-15,IL-18,IL-11,IL-27,and IL-33.ThisassaypanelprovideshigherdetectionsensitivitiesandbroaderdynamicrangesthantraditionalELISAmethods.Thepanelhasbeenvalidatedbydetectingexpectedchangesinbiologicalsamples.

The Human Cytokine Panel2isdesignedtoallowflexiblecustomizationwithinthe panel. Please visit www.biolegend.com/legendplex formoreinformationonhow to mix and match within the panel.

The Human Cytokine Panel2anditscustomsubpanelsarespecificallydesignedfortheaccuratequantificationofmultiplehumancytokines from cell 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,providingfluorescentsignalintensitiesinproportiontotheamountofboundanalytes.

Sincethebeadsaredifferentiatedbysizeandinternalfluorescenceintensity

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

Beads Usage

The Human Cytokine Panel 2 uses two sets of beads. Each set has a unique size thatcanbeidentifiedbasedontheirforwardscatter(FSC)andsidescatter(SSC)profiles(BeadsAandBeadsB,Figure1).Eachbeadsetcanbefurtherresolvedbasedontheirinternalfluorescenceintensities.Theinternaldyecanbede-tectedusingFL3,FL4,orAPCchannel,dependingonthetypeofflowcytometerused.ThesmallerBeadsAconsistsof6beadpopulationsandthelargerBeadsBconsistsof7beadpopulations(Figure2-3).

Usingatotalof13beadpopulationsdistinguishedbysizeandinternalfluo-rescent dye, the Human CytokinePanel2allowssimultaneousdetectionof13cytokinesinasinglesample.Eachanalyteisassociatedwithaparticularbeadset as indicated (Figures 2-3 and Table 1).

Figure1.BeadsDifferentiatedbySize

Beads A = Smaller beads

Beads B = larger beads

Figure2.BeadsAClassificationbyFL4

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Figure3.BeadsBClassificationbyFL4

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

Table1.PanelTargets,BeadIDandTopStandardConcentration

Target Bead ID*Top Standard Concentration

(ng/mL)

TSLP A4 10

IL-1α A5 10

IL-1β A6 10

GM-CSF A7 10

IFN-α2 A8 10

IL-23 A10 10

IL-12p40 B2 50

IL-12p70 B3 10

IL-15 B4 50

IL-18 B5 10

IL-11 B6 50

IL-27 B7 10

IL-33 B9 50

*BeadIDisusedtoassociateabeadpopulationtoaparticularanalyteintheLEGENDplexTMDataAnalysisSoftware.TheassociationofanalyteandbeadIDwillbedefinedduringthegatingstepofthedataanalysis.

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

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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,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 77840Setup Beads 2: PE Beads 1 vial 1 mL 77842Setup Beads 3: Raw Beads 1 vial 2 mL 77844Human Cytokine Panel 2 Premixed Beads 1bottle 3.5 mL 76101

Human Cytokine Panel 2 DetectionAntibodies 1bottle 3.5 mL 76115

Human Cytokine Panel 2 Standard Cocktail, Lyophilized 1 vial lyophilized 76859

LEGENDplexTM SA-PE 1bottle 3.5 mL 77743LEGENDplexTM Matrix B, Lyophilized 1 vial lyophilized 77549LEGENDplexTMAssayBuffer 1bottle 25 mL 77562LEGENDplexTMWashBuffer,20X 1bottle 25 mL 77564Filter plate 1 plate 76187Plate Sealers 4sheets 78101DataAnalysisSoftwareDongle 1 21217Human Cytokine Panel 2 Manual 1 76119

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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.

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*

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

BD FACSAriaTM PE 575 nm APC 660 nm No*

*Compensationisnotrequiredforthespecifiedflowcytometerswhenset 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

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

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

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Iftheassayisrunusingthefilterplateprovided(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)

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

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

• Centrifugewithaswingingbucketadaptorformicrotiterplatesormicro-tube racks (e.g., Beckman Coulter AllegraTM 6R Centrifuge with MICROPLUS CARRIERadaptorforGH3.8andJS4.3Rotors).

• 96-well,V-bottom,clearpolypropylenemicroplate(lowproteinbinding,e.g., greiner bio-one, Catalog # 651201 or equivalent).

Precautions

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

• Sodiumazidehasbeenaddedtosomereagentsasapreservative.Al-thoughtheconcentrationsarelow,sodiumazidemayreactwithleadandcopperplumbingtoformhighlyexplosivemetalazides.Ondisposal,flushwith a large volume of water to prevent azide build-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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LEGENDplex™ Human Cytokine Panel 2Chapter 2: ASSAY PREPARATION

SampleCollectionandHandling

PreparationofSerumSamples:

• 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.

PreparationofPlasmaSamples:

• 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.

PreparationofTissueCultureSupernatant:

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

ReagentsPreparation

PreparationofAntibody-ImmobilizedBeads

SonicatePre-mixedBeadsbottlefor1minuteinasonicatorbathandthenvortex for 30 seconds prior to use. If no sonicator bath is available, increase thevortexingtimeto1minutetocompletelyresuspendthebeads.

PreparationofWashBuffer

• Bringthe20XWashBuffertoroomtemperatureandmixtobringallsaltsintosolution.

• Dilute25mLof20XWashBufferwith475mLdeionizedwater.Storeun-usedportionsbetween2°Cand8°Cforuptoonemonth.

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PreparationofMatrixB(forSerumorPlasmaSamplesOnly)

•Add 5.0 mL LEGENDplexTMAssayBuffertothebottlecontaininglyophilized MatrixB.Allowatleast15minutesforcompletereconstitution.Vortexto

mixwell.LeftoverreconstitutedMatrixBshouldbestoredat≤-70°Cforupto one month.

StandardPreparation

1. Priortouse,reconstitutethelyophilizedHumanCytokine Panel 2 Standard Cocktailwith250µLAssayBuffer.

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:Analytesinthispanelmayhavedifferenttopstandardconcentra-tions.(SeeTable1fordetails)

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

Tube/Stan-dard ID

Serial Dilution

Assay Buffertoadd (µL)

Standard to add

Final Conc. (pg/mL)*

Final Conc. (pg/mL)**

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

C6 1:4 75 25 µL of C7 2,500 12,500

C5 1:16 75 25 µL of C6 625 3,125

C4 1:64 75 25 µL of C5 156.3 781.3

C3 1:256 75 25 µLofC4 39.1 195.3

C2 1:1024 75 25 µL of C3 9.8 48.8

C1 1:4096 75 25 µL of C2 2.4 12.2

C0 -- 75 -- 0 0

*TopstandardconcentrationofTSLP, IL-1α,IL-1β,GM-CSF,IFN-α2,IL-23,IL-12p70,IL-18andIL-27is10ng/mL;**TopstandardconcentrationofIL-12p40,IL-15, IL-11 and IL-33 is 50 ng/mL.

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DilutionofSamples

• In general, serum or plasma samples need to be diluted 2-fold with Assay Bufferbeforetesting(e.g.dilute50µLofsamplewith50µLofAssayBuf-fer).

Iffurthersampledilutionisdesired,dilutionshouldbedonewithMatrixBto eusure accurate measurement.

Addingserumorplasmasampleswithoutdilutionwillresultinlowas-sayaccuracyandpossibly,cloggingofthefilterplate.

• 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,dilutionshouldbedonewithcorrespondingfreshcellculturemediumorAssayBuffertoensureaccuratemeasure-ment.

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Chapter 3: ASSAY PROCEDURE

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

• Thein-filterplateassayprocedureisrecommendedduetoitsgoodsampleto sample consistency, assay robustness and ease of handling. This pro-cedurerequiresavacuumfiltrationunitforwashing(seeMaterials to be Provided by the End-User).IfyouhaveperformedaLuminex®-basedmultiplexassaybefore,yourlabshouldalreadyhavethevacuumfiltrationunit set up.

• Ifthein-filterplateassayprocedureisnotpossibleorifyouprefer,theas-saycanbeperformedinmicrotubesorinaU-orV-bottommicroplate.Forin-tube assay, we recommend using micro FACS tubes (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,sothatthebottomoftheplatedoesnottouchanysurface. 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 plateinaverticalconfigurationconvenientfordataacquisitionandanalysis(asshowninattachedPLATEMAP).Besuretoloadstandardsinthefirsttwocolumns.Ifanautomationdeviceisusedforreading,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.

For measuring cell culture supernatant samples:

• Add25µLofAssayBuffertoallwells.• Add 25 µL of each standard to the standard wells.

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LEGENDplex™ Human Cytokine Panel 2• Add 25 µL of each sample to the sample wells (See Dilutionof

Samples)

For measuring serum or plasma samples:

• Add 25 µL of Matrix B to the standard wells.• Add25µLofAssayBuffertothesamplewells.• 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 DilutionofSamples).

2. Vortexmixedbeadsfor30seconds.Add25μLofmixedbeadstoeachwell.Thevolumeshouldbe75μLineachwellafterbeadsaddition.(Note:Dur-ingadditionofthebeads,shakemixedbeadsbottleintermittentlytoavoidbeadsettling).

3. Seal the plate with a plate sealer. To avoid plate leaking, do not apply posi-tivepressuretothesealerwhensealingtheplate.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(or 150 µL if plate is to be read withanautoampler.Highervolumemayresultinleakingofthefilterplateduringreading.Besuretosetthesamplevolumetobeanalyzedto70uLor less so that sample can be read again if needed). Resuspend the beads on a plate shaker for 1 minute.

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

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

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 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 (or 150µL) of 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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PerformingtheAssayUsingMicrotubesora96-WellU-orV-bottomMicroplate

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

2. Determine the number of assays to run. Standards and samples should be runinduplicateandarrangedintheorderconvenientfordataacquisitionandanalysis(seebelow).Ifanautomationdeviceisusedforreading,theorientationandreadingsequenceshouldbecarefullyplanned.

If using tubes, label all tubes and arrange tubes on a rack (as shown in at-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 thePLATEMAPattached.

3. For measuring cell culture supernatant samples:

• Add25µLofAssayBuffertoalltubes/wells.• Add25µLofeachstandardtothestandardtubes/wells.• Add25µLofeachsampletothesampletubes/wells(See Dilutionof

Samples).• Add25µLofmixedbeadstoalltubes/wells.• Add25µLDetectionAntibodiestoalltubes/wells.

For measuring serum or plasma samples:

• Add25µLofMatrixBtothestandardtubes/wells.• Add25µLofAssayBuffertosampletubes/wells.• Add25µLofeachstandardtostandardtubes/wells.• Add25µLofeachdilutedserumorplasmasampletosampletubes/

wells (See DilutionofSamples).• Add25µLofmixedbeadstoalltubes/wells.• Add25µLDetectionAntibodiestoalltubes/wells.

Note: Thevolumeshouldbe100μLineachtube/well.Shake beads bottleintermittentlyduringtheadditiontoavoidbeadsettling.

4. Covertheentirerack/platewithaluminumfoiltoprotectthetubes/platefrom light. Shake (approximate 1,000 rpm for in-tube assay and 600 rpm for in-plate assay) on a plate shaker for 2 hours at room temperature.

5. Withoutwashingthetubes/plate,add25µLSA-PEtoeachtube/well.

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6. Covertheentirerack/platewithaluminumfoiltoprotectthetubes/platefrom light. Shake (approximate 1,000 rpm for in-tube assay and 600 rpm for in-plate assay) on a plate shaker for 30 minutes at room temperature.

7. Centrifugethetubes/plateat1,000xg for 5 minutes, using a swinging bucket rotor with microplate adaptor (Please refer to Materials to be Pro-vided by the End-User).

8. Removethesupernatantusingamultichannelpipette.Becarefulnottoremove beads, but to remove as much liquid as possible.

9. Add200µLof1XWashBuffertoalltubes/wells.Resuspendthebeadsbyvortexing (for in-tube assay) or shaking on a plate shaker for 1 minute (for in-plate assay). Centrifugethetubes/plateat1,000xg for 5 minutes, using a swinging bucket rotor with microplate adaptor. Remove the supernatant.

10. Add200µLof1XWashBuffertoeachtube/well(or 150 µL if plate is to be readwithanautoampler.Highervolumemayresultinleakingofthefilterplateduringreading.Besuretosetthesamplevolumetobeanalyzedto70 uL or less so that sample can be read again if needed). Resuspend the beadsbyvortexing(forin-tubeassay)orpippeting(forin-plateassay).

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

Iftheflowcytometerisequippedwithanautosampler,thesamplescanbereadeitherdirectly(forin-plateassay)orafterbeingtransferredtoa96-well plate ((for in-tube assay). The probe height might need to be adjusted when using an autosampler.

If an autosampler is not available, the samples can be read directly in FACS tubesorafterbeingtransferredfrommicroplatetomicroFACStubes.Inthis case, the sample volume may need to be increased from 200 µL to 300 µLbyaddingextra100µLof1XWashBuffertoeachtube,toavoidsamplerunningdrywhenanalyzedonaflowcytometer.

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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 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 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

For serum and plasma samples, Add to the tubes/well:25 μL Matrix B to standard tubes/wells25 μL Assay Bu�er to sample tubes/wells25 μL diluted standard to standard tubes/wells25 μL sample to sample tubes/wells25 μL mixed beads to all tubes/wells25 μL Detection Antibodies to all tubes/wells

A B

C

A

B

C

Capture beads

Detection Antibody

Analytes

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Chapter 4: FLOW CYTOMETER SETUP

Inordertogeneratereliabledata,theflowcytometermustbesetupproperlybeforedataacquisition.Thefollowingsectionswilladdressmachinesetupfortheflowcytometerslistedbelow.

ListofFlowCytometersandPossibleConfigurations

Flow CytometerReporterChannel

Channel Emission

ClassificationChannel

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*

*Compensationisnotrequiredforthespecifiedflowcytometerswhensetupproperly, 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. 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 Create4dotplotsasshownbelow(Figure5)withFL2forX-axis,FL4 or FL1 for Y-axis. For these dot plots, gate on Beads A (dot plots on theleftpanelbelow)andBeadsB(dotplotsontherightpanelbelow). The dot plots should be in log mode.

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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.

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LEGENDplex™ Human Cytokine Panel 2 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: PE beads are only of small size, falling in the Beads A

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.


Performinstrumentstartupandverificationcheckfollowingthemanufac-turer’srecommendations.


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).

Figure9.

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Gated on Beads BGated on Beads A2.3 Create4dotplotsasshownbelow(Figure10)withFL2forX-axis,and FL3 or FL1 for Y-axis. For these dot plots, gate on Beads A (dot plots on theleftpanelbelow)andBeadsB(dotplotsontherightpanelbelow). The dot plots should be in log mode.

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Figure 10.

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2.4 Setallcompensationstozero.

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

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LEGENDplex™ Human Cytokine Panel 23. 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 (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).

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LEGENDplex™ Human Cytokine Panel 2Note: FITC beads are only of small size, falling in the Beads A gate.

Figure 13.

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.

3.17 On the FL1 vs FL2 dot plot (Figure 15), the beads will display as PE- negativeandPE-positivepopulations(indicatedbyanarrow).Note: PE beads are only of small size, falling in the Beads A gate.

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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 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).


Performinstrumentstartupandverificationcheckfollowingthemanufac-ture’srecommendations.


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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LEGENDplex™ Human Cytokine Panel 2 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).

Figure19.

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

Figure 20.

2.4 Savethedocumentas“LEGENDplexTemplateforFACSDiva” 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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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. Inthiscase,machinecompensationbetweenthereporterandbeadsclassificationchannelsisstronglyrecommended.

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

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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,pleaserefertotheFlowCytometerSetup Guide).

3. Vortex each sample for 5 seconds before analysis.

4.Settheflowratetolow.Setthenumberofbeadstobeacquiredtoabout300peranalyte(e.g.,acquire4,000beadsfora13-plexpanel).Donotacquiretoomanyevents(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.

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;forsamples,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,simplyplugitintheUSBportofthecom-puteronwhichthedataanalysissoftwareisinstalled,priortolaunchthesoftware.

• 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. 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

that the machine is set up properly before use (refer to Setup Procedure for Other Flow CytometerssectioninChapter4).In this case, machine compensationbetweenthereporterandbeadsclassificationchannelsisstrongly recommended.

TheFCSorListmodefilesfromthefollowinginstrumentsarecompatiblewith the LEGENDplex TMDataAnalysisSoftware.

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Vendor Instrument Model Vendor 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. 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.

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LEGENDplex™ Human Cytokine Panel 2 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 (refer to manufacturer’s manual for proper instrument setup). In this case, 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. The targets within a panel are fully customizable. The customer can selectanycombinationoftargetswithinapanelandorderacustomizedproduct.Pleaseuseourwebsitetargetselectiontoolfororderingcustom-ized product (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,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-dardcurveduringanalysis?

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. 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?

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. Iranoutofpointsonmydongle.CanIgetanewoneseparately?Or,canIuseadonglefromonekitforanotherkit?

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.

biolegend.com41


Chapter 7: ASSAY CHARACTERIZATION

RepresentativeStandardCurve

This standard curve was generated using the LEGENDplexTM Human Cyto-kinePanel2fordemonstrationpurposeonly.Astandardcurvemustberun with each assay.

1.0

10.0

100.0

1000.0

10000.0

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MFI

Concentration (pg/mL)

TSLPIL-1α IL-1β GM-CSFIFN-α2 IL-23IL-12p40IL-12p70IL-15IL-18IL-11IL-27IL-33

AssaySensitivity

Theassaysensitivityorminimumdetectableconcentration(MDC)isthetheoreticallimitofdetectioncalculatedusingtheLEGENDplexTM Data AnalysisSoftwarebyapplyinga5-parametercurvefittingalgorithm.

Analyte MDC in Cell Culture Medium (pg/mL)

MDC in Serum (pg/mL)

Human TSLP 1.2 1.1

HumanIL-1α 1.1 1.5

HumanIL-1β 0.8 0.8

Human GM-CSF 1.1 0.6

HumanIFN-α2 1.1 1.0

Human IL-23 0.7 1.4

HumanIL-12p40 6.0 5.1

Human IL-12p70 0.9 0.9

Human IL-15 7.4 5.7

Human IL-18 0.7 0.7

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Human IL-11 8.0 4.6

Human IL-27 1.3 1.0

Human IL-33 7.2 5.0

Cross-Reactivity

Thefollowinghumanrecombinantproteinsweretestedat50ng/mLusingthe LEGENDplexTM Human Cytokine Panel 2. IL-23 showed 5.0% cross-reactivitywithIL-12p40assay,IL-27showed0.3%cross-reactivitywithIL-11assay.Noornegligiblecross-reactivitywasfoundamongallotheranalytes.

IL-2 IL-4 IL-5 IL-6 IL-9 IL-10 IL-13

IL-17A IL-22 IFN-γ TNF-α IL-17F IL-21 IL-8

IP-10 CCL11 TARC MCP-1 RANTES MIP-1α MIG

ENA-78 MIP-3α GROα I-TAC MIP-1β IFN-α2 TSLP

IL-1α IL-1β IL-3 IL-7 IL-11 IL-12p40 IL-12p70

IL-15 IL-18 IL-23 IL-27 IL-33 GM-CSF

Accuracy (Spike Recovery)

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

Forspikerecoveryinserum,asamplewithknownhighconcentrationsoftargetproteinswasspikedintounknownserumsamples(n=10).Thespikedsampleswerethenassayed,andthemeasuredconcentrationswerecom-pared with the expected values.

Analyte % of Recovery in Cell Culture Medium

% of Recovery in Serum

Human TSLP 86% 153%HumanIL-1α 103% 132%HumanIL-1β 104% 88%Human GM-CSF 101% 119%HumanIFN-α2 106% 140%

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Human IL-23 100% 103%HumanIL-12p40 117% 163%Human IL-12p70 93% 90%Human IL-15 78% 80%Human IL-18 104% 97%Human IL-11 109% 117%Human IL-27 108% 83%Human IL-33 100% 79%

LinearityofDilution

Fortestinglinearityofdilution,serumsamples(n=6)werefirstdilutedtwo-foldwithAssayBuffer,thenseriallydiluted1:2,1:4,1:8withMatrixBandassayed.Themeasuredconcentrationsofseriallydilutedsampleswerethen compared with that of the two-fold diluted samples.

Analyte Linearity of Dilution Analyte Linearity of

DilutionHuman TSLP 100% Human IL-12p70 111%HumanIL-1α 86% Human IL-15 110%HumanIL-1β 105% Human IL-18 110%Human GM-CSF 92% Human IL-11 104%HumanIFN-α2 64% Human IL-27 125%Human IL-23 99% Human IL-33 116%HumanIL-12p40 58%

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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

Human TSLPSample 1 627.0 42.3 7%Sample 2 39.7 2.6 6%

HumanIL-1αSample 1 667.4 56.9 9%Sample 2 37.1 2.1 6%

HumanIL-1βSample 1 630.8 28.9 5%Sample 2 37.5 3.2 8%

Human GM-CSFSample 1 679.7 57.0 8%Sample 2 38.6 2.3 6%

HumanIFN-α2Sample 1 646.4 46.2 7%Sample 2 37.4 2.3 6%

Human IL-23Sample 1 634.3 53.7 8%Sample 2 42.3 2.1 5%

Human IL-12p40

Sample 1 626.4 48.4 8%Sample 2 40.0 3.1 8%

Human IL-12p70

Sample 1 703.5 58.0 8%Sample 2 42.9 3.9 9%






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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

Human TSLPSample 1 633.6 42.4 7%Sample 2 39.6 3.0 8%

HumanIL-1αSample 1 640.2 55.3 9%Sample 2 37.1 3.3 9%

HumanIL-1βSample 1 619.7 44.6 7%Sample 2 37.4 3.7 10%

Human GM-CSFSample 1 697.9 53.1 8%Sample 2 38.1 2.8 7%

HumanIFN-α2Sample 1 655.2 45.3 7%Sample 2 36.7 2.6 7%


Human IL-12p40

Sample 1 595.1 31.7 5%Sample 2 39.2 2.4 6%

Human IL-12p70

Sample 1 722.7 54.9 8%Sample 2 42.9 4.1 10%






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Biological Samples

Serum and Plasma (Samples are paired.)

Normalhumanserumsamples(n=20)weretestedforendogenouslevelsofthecytokines.Theconcentrationsmeasuredareshownbelow:

Analyte Range (pg/ml)

No. of Detectable

% of Detectable

Mean* (pg/mL)

Human TSLP ND-152.1 10 50% 25.7

HumanIL-1α ND-251.8 9 45% 40.0

HumanIL-1β ND-91.2 3 15% 39.3

Human GM-CSF ND-173.6 2 10% 93.4

HumanIFN-α2 ND-352.6 7 35% 103.9

Human IL-23 ND-204.4 4 20% 112.3

HumanIL-12p40 ND-801.8 15 75% 121.8

Human IL-12p70 ND-41.6 3 15% 20.5

Human IL-15 ND-196.8 11 55% 25.7

Human IL-18 33-406.3 20 100% 133.3

Human IL-11 ND-127.1 5 25% 44.4

Human IL-27 ND-541.1 4 20% 51.0

Human IL-33 ND-536.4 4 20% 161.8

ND=Non-detectable*The mean is calculated based on detectable samples only.

Normalhumanplasmasamples(n=20)weretestedforendogenouslevelsofcytokines.Theconcentrationsmeasuredareshownbelow:

Analyte Range (pg/mL)

No. of Detectable

% of Detectable

Mean* (pg/mL)

Human TSLP ND-104.9 10 50% 14.1

HumanIL-1α ND-171.9 6 30% 39.3

HumanIL-1β ND-64.2 4 20% 19.0

Human GM-CSF ND-113.7 2 10% 58.6

HumanIFN-α2 ND-256.8 9 45% 49.4

Human IL-23 ND-201.5 3 15% 79.1

HumanIL-12p40 ND-475.5 17 85% 63.2

Human IL-12p70 ND-26.0 5 25% 7.6

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Human IL-15 ND-140.4 14 70% 20.7

Human IL-18 29.9-409.9 20 100% 142.7

Human IL-11 ND-58.3 4 20% 21.4

Human IL-27 ND-323.8 8 40% 47.5

Human IL-33 ND-383.7 1 5% 383.7

ND=Non-detectable*The mean is calculated based on detectable samples only.

Cell Culture Supernatant

Human PBMC (1 x 106cells/mL)wereculturedundervariousconditions(LPS,1µg/mL;PolyIC,50µg/mL;CPG,5µg/mL;R488,2µg/mL;IFN-γ,100ng/mL).Supernatantswerecollectedafter42hoursandassayedwiththe LEGENDplexTMHumanCytokinePanel2kit.Theresults(allinpg/mL)are summarized below.

Analyte Control LPS Poly IC CPG R488 LPS+IFN-γ*

Human TSLP ND ND ND ND ND 2.7

HumanIL-1α 1.5 480.6 1.6 1.5 159.1 2825.9

HumanIL-1β 0.8 1515.4 9.5 0.8 859.2 4571.7

Human GM-CSF

1.3 20.9 4.0 1.3 5.5 23.1

HumanIFN-α2 ND ND 51.7 ND 334.2 12.8

Human IL-23 ND 5.3 4.2 ND 5.7 2410.4

Human IL-12p40

ND 44.0 16.5 1.5 196.6 7889.8

Human IL-12p70

ND ND 43.0 ND 3.2 >10,000

Human IL-15 ND 2.4 2.3 2.0 2.6 6.5

Human IL-18 ND 15.2 3.4 1.3 14.1 38.9

Human IL-11 0.7 4.7 1.6 1.4 5.5 10.5

Human IL-27 ND ND 1.2 ND 1.4 54.3

Human IL-33 ND ND ND ND 1.1 2.9 ND=Non-detectable*CellswereprimedwithIFN-γfor2hoursbeforeLPSstimulation.

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48

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.

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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.

Centrifuge samples before analyzing samples. Remove platelet as much as possible.

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50

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.

Dilute samples and analyze again.

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.

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LEGENDplex™ Kits are manufactured by BioLegend 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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