human growth factor panel - legendplex™

®

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. 740180

Human Growth Factor Panel(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 Growth Factor Panel

Table of Contents Page

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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..........................................

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

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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

Growthfactorsactivatecellproliferationanddifferentiationandregulatemanycellular processes including angiogenesis, hematopoiesis, tumorigenesis, me-tabolism,inflammationandtissuerepair.Theycouldalsobepotentialthera-peutictargetsanddiseasebiomarkers.Theaccuratemeasurementofgrowthfactorsiscriticalforabetterunderstanding of disease progression and related cellular processes.

The LEGENDplexTM Human Growth Factor Panel is a bead-based multiplexassay,usingfluorescence–encodedbeadssuitableforuseonvariousflowcytometers.Thispanelallowssimultaneousquantificationof13humangrowthfactors:An-giopoietin-2(Ang-2),EGF,EPO,FGF-basic,G-CSF,GM-CSF,HGF,M-CSF,PDGF-AA,PDGF-BB,SCF,TGF-αandVEGF.Thispanelprovideshighersensitivityandbroad-erdynamicrangethantraditionalELISAmethods.Thepanelhasbeenvalidatedfor use on serum, plasma and cell culture supernatant samples.

The Human Growth Factor Panelisdesignedtoallowflexiblecustomizationwith-in the panel. For mix and match within the panel, please visit www.biolegend.com/legendplex.

This assay is for research use only.

Principle of the Assay

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

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

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

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Beads Usage

The Human Growth Factor Panel includes two sets of beads. Each set has a uniquesizethatcanbeidentifiedonflowcytometerbasedontheirforwardscatter(FSC)andsidescatter(SSC)profiles(BeadsAandBeadsB,Figure1).Eachbeadsetcanbefurtherresolvedbasedontheirinternalfluorescenceintensities.TheinternaldyecanbedetectedusingFL3,FL4,orAPCchannel,dependingonthetypeofflowcytometerused.ThesmallerBeadsAconsistsof6beadpopulationsandthelargerBeadsBconsistsof7beadpopulations(Figure2-3).

Usingatotalof13beadpopulationsdistinguishedbysizeandinternalfluores-centdye,theHumanGrowthFactorPanelallowssimultaneousdetectionof13growthfactorsinonesampletest.Eachanalyteisassociatedwithaparticularbeadsetasindicated(Figures2-3andTable1).

Figure1.BeadsDifferentiatedbySize

Beads A = smaller beads

Beads B = larger beads

Figure2.BeadsAClassificationbyFL4

A5 A7 A8

A4

A6

A10

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Figure3.BeadsBClassificationbyFL4

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

Table1.BeadsIDandTargetInformation

Target Bead ID Top Standard Concentrations(ng/mL)

HumanAngiopoietin-2 A4 50

Human EGF A5 10

Human EPO A6 50

Human FGF-basic A7 50

Human G-CSF A8 50

Human GM-CSF A10 50

Human HGF B2 50

Human M-CSF B3 10

Human PDGF-AA B4 50

Human PDGF-BB B5 50

Human SCF B6 10

HumanTGF-α B7 50

HumanVEGF B9 50

*BeadIDisusedtoassociateabeadpopulationtoaparticularanalyteintheLEGENDplexTMDataAnalysisSoftware.TheassociationofanalyteandbeadIDwillbedefinedduringthegatingstepofthedataanalysis.

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

B4 B5

B6 B7

B3

B9

B2

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StorageInformation

Recommendedstorageforalloriginalkitcomponentsisbetween2°Cand8°C.DONOTFREEZEPre-mixedBeads,DetectionAntibodiesorSA-PE.

• Oncethestandardshavebeenreconstituted,immediatelytransfercon-tents into polypropylene vials. DO NOT STORE RECONSTITUTED STAN-DARDSINGLASSVIALS.

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

Materials Supplied

The LEGENDplexTMkitcontainsreagentsfor100testslistedinthetablebelow.Whenassayedinduplicate,thisisenoughforan8-pointstandardcurveand40samples.

Kit Components Quantity Volume Part #

Setup Beads 1: FITC Beads 1 vial 1 mL 77840

Setup Beads 2: PE Beads 1 vial 1 mL 77842Setup Beads 3: Raw Beads 1 vial 2 mL 77844Human Growth Factor Panel Pre-mixed Beads 1bottle 3.5 mL 76289

Human Growth Factor Panel Detec-tionAntibodies 1bottle 3.5 mL 76303

Human Growth Factor Panel Stan-dardCocktail,Lyophilized 1 vial lyophilized 76318

LEGENDplexTM SA-PE 1bottle 3.5 mL 77743LEGENDplexTMMatrixB,Lyophilized 1 vial lyophilized 77549LEGENDplexTMAssayBuffer 1bottle 25 mL 77562LEGENDplexTMWashBuffer,20X 1bottle 25 mL 77564Filter plate 1 plate 76187Plate Sealers 4 sheets 78101DataAnalysisSoftwareDongle 1 21217Human Growth Factor Panel Manual 1 76319

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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*

BDTMLSR,LSRIIBD LSRFortessaTM PE 575-585

nm APC 660 nm No*

BD FACSAriaTM PE 575 nm APC 660 nm No*

*Compensationisnotrequiredforthespecifiedflowcytometerswhensetupproperly,butisrecommendedforconsistentresults.

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

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

• Multichannelpipettescapableofdispensing5μLto200μL

• Reagentreservoirsformultichannelpipette

• Polypropylenemicrofugetubes(1.5mL)

• Laboratory vortex mixer

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

• Aluminum foil

• Absorbent pads or paper towels

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

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

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Iftheassayisrunusingthefilterplateprovided(recommended),

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

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

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

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

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

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

• Precautions

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

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

• Matrix B for LEGENDplexTMkitscontainscomponentsofhumanoriginandshouldbehandledaspotentiallyhazardous.TherawmaterialhasbeenscreenedforinfectiousdiseasesandisnegativeforHIV,HBVandHCVusingFDA-approved test methods.

• Donotmixorsubstitutereagentsfromdifferentkitsorlots.Reagentsfromdifferentmanufacturersshouldnotbeusedwiththiskit.

• Donotusethiskitbeyonditsexpirationdate.

• SA-PEandPre-mixedBeadsarelight-sensitive.Minimizelightexposure.

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LEGENDplex™ Human Growth Factor PanelChapter 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. If not pos-sible,aliquotandstoresamplesat≤-20°C.Avoidmultiple(>2)freeze/thawcycles.

ReagentPreparation

PreparationofAntibody-ImmobilizedBeads

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

PreparationofWashBuffer

• Bringthe20XWashBuffertoroomtemperatureandmixtobringallsaltsintosolution.

• Dilute25mLof20XWashBufferwith475mLdeionizedwater.Storeun-usedportionsbetween2°Cand8°Cforuptoonemonth.

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PreparationofMatrixB(forSerumorPlasmaSamplesOnly)

•Add 5.0 mL LEGENDplexTMAssayBuffertothebottlecontaininglyophilized MatrixB.Allowatleast15minutesforcompletereconstitution.Vortexto

mixwell.LeftoverreconstitutedMatrixBshouldbestoredat≤-70°Cforupto one month.

StandardPreparation

1. Priortouse,reconstitutethelyophilizedHumanGrowthFactorPanelStan-dardCocktailwith250µLAssayBuffer.

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:Analytesinthispanelhavedifferenttopstandardconcentrations(seetable1,page5fordetails).

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

4. Add75µLofAssayBuffertoeachofthesixtubes.Prepare1:4dilutionofthetopstandardbytransferring25µLofthetopstandardC7totheC6tube and mix well. This will be the C6 standard.

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

Tube/Standard

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 µLofC7 2,500 12,500

C5 1:16 75 25 µLofC6 625 3,125

C4 1:64 75 25 µLofC5 156.3 781.3

C3 1:256 75 25 µLofC4 39.1 195.3

C2 1:1024 75 25 µLofC3 9.8 48.8

C1 1:4096 75 25 µLofC2 2.4 12.2

C0 -- 75 -- 0 0

*TopstandardconcentrationofEGF,M-CSFandSCF is 10 ng/mL; **TopstandardconcentrationofAngiopoietin-2,EPO,FGF-basic,G-CSF,GM-CSF,

HGF,PDGF-AA,PDGF-BB,TGF-α,VEGFis50ng/mL.

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SampleDilution

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

Iffurthersampledilutionisdesired,dilutionshouldbedonewithMatrixBto ensure accurate measurement.

Addingserumorplasmasampleswithoutdilutionwillresultinlowas-sayaccuracyandpossibly,cloggingofthefilterplate.

• For cell culture supernatant samples, the levels of analyte can vary greatly fromsampletosample.Whilethesamplescanbetestedwithoutdilu-tions,apreliminaryexperimentmayberequiredtodeterminetheappro-priatedilutionfactor.

Ifsampledilutionisdesired,dilutionshouldbedonewithcorrespondingfresh cell culture medium to ensure accurate measurement.

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Chapter 3: ASSAY PROCEDURE

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

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

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

Performing the Assay Using a Filter Plate

• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.

• Setthefilterplateonaninvertedplatecoveratalltimesduringassaysetupandincubationstepssothatthebottomoftheplatedoesnottouchanysurface.Touchingasurfacemaycauseleakage.

• Keeptheplateuprightduringtheentireassayprocedure,includingthewashing steps, to avoid losing beads.

• Theplateshouldbeplacedinthedarkorwrappedwithaluminumfoilforallincubationsteps.

• Standards and samples should be run in duplicate and arranged on the plateinaverticalconfigurationconvenientfordataacquisitionandanaly-sis(asshowninattachedPLATEMAP,Page53-54).Besuretoloadstan-dardsinthefirsttwocolumns.Ifanautomationdeviceisusedforread-ing,theorientationandreadingsequenceshouldbecarefullyplanned.

1. Pre-wettheplatebyadding100μLofLEGENDplexTM1XWashBuffertoeach well and let it sit for 1 minute at room temperature. To remove the excess volume, place the plate on the vacuum manifold and apply vacuum. Donotexceed10”Hgofvacuum.Vacuumuntilwellsaredrained(5-10seconds).BlotexcessWashBufferfromthebottomoftheplatebypress-ingtheplateonastackofcleanpapertowels.Placetheplateontopoftheinverted plate cover.

For measuring cell culture supernatant samples:

• Add25µLoffreshcellculturemedium(nottheTCsamples)tostan-dard wells.

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• Add25µLofAssayBuffertosamplewells.• Add25µLofeachstandardtothestandardwells.• Add25µLofeachsampletothesamplewells(SeeDilutionof

Samples)

For measuring serum or plasma samples:

• Add25µLofMatrixBtothestandardwells.• Add 25 µLofAssayBuffertothesamplewells.• Add25µLofeachstandardtothestandardwells.• Add25µLofeachdilutedserumorplasmasampletothesample

wells(SeeDilutionofSamples).

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

3. Seal the plate with a plate sealer. Toavoidplateleaking,donotapplypositivepressuretothesealerwhensealingtheplate.Wraptheentireplate, including the inverted plate cover, with aluminum foil. Place the plateonaplateshaker,secureitandshakeatapproximate500rpmfor2hours 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,includingtheinvertedplatecover,withaluminumfoil.Placetheplateonaplateshakerandshakeatapproximately500rpmfor1houratroomtemperature.

7. Do not vacuum!Add25µLofSA-PEtoeachwelldirectly.

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

9. Repeat step 4 above.

10. Add200µLof1XWashBuffertoeachwell(or150µLifplateistobereadbyanautosampler.Highervolumemayresultinleakingofthefilterplateduring reading. Besuretosetthesamplevolumetobeanalyzedto70uLor less so that sample can be read again if needed).Resuspendthebeadsonaplateshakerfor1minute.

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11. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay (Note:Prolongedsamplestoragecanleadtoreducedsignal)

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

If an autosampler is not available, the samples need to be transferred from thefilterplatetoFACStubesandreadmanually.Inthiscase,thesamplevolumemayneedtobeincreasedfrom200µLto300µLbyaddingextra100µLof1XWashBuffertoeachtubetoavoidsamplerunningdrywhenreadonaflowcytometer.

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 Cell Culture Medium to standard wells25 μL Assay Bu�er to sample wells25 μL diluted standard to standard wells25 μL sample to sample wells25 μL pre-mixed beads to all wells

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

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 pre-mixed beads to all wells

BA

C

A B C

A B C

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Performing the Assay Using Microtubes or a 96-Well V- or U- bot-tom Microplate

• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.

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

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

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

1. For measuring cell culture supernatant samples:• Add25µLoffreshcellculturemedium(nottheTCsamples) to stan-

dardtubes/wells.• Add25µLofAssayBuffertosampletubes/wells.• Add25µLofeachstandardtothestandardtubes/wells.• Add25µLofeachsampletothesampletubes/wells(See Dilutionof

Samples).• Add25µLofthepre-mixedbeadstoalltubes/wells.• Add25µLDetectionAntibodiestoalltubes/wells.

For measuring serum or plasma samples:

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

wells(See DilutionofSamples).• Add25µLofthepre-mixedbeadstoalltubes/wells.• Add25µLDetectionAntibodiestoalltubes/wells.

Note: Thevolumeshouldbe100μLineachtube/well.Shakebeadsbottleintermittentlyduringtheadditiontoavoidbeadsettling.

2. Covertheentirerack/platewithaluminumfoiltoprotectthetubes/platefrom light. Shake(approximate1,000rpmforin-tubeassayand600rpmforin-plateassay)onaplateshakerfor2hoursatroomtemperature.

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

4. Covertheentirerack/platewithaluminumfoiltoprotectthetubes/platefrom light. Shake(approximate1,000rpmforin-tubeassayand600rpmforin-plateassay)onaplateshakerfor30minutesatroomtemperature.

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

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

7. Add200µLof1XWashBuffertoalltubes/well.Resuspendthebeadsbyvortexing(forin-tubeassay)orshakingonaplateshakerfor1minute(forin-plateassay).Centrifugethetubes/plateat1,000xg for 5 minutes, using aswingingbucketrotorwithmicroplateadaptor.Removethesupernatant.

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

9. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay(Note:Prolongedsamplestoragecanleadtoreducedsignal).

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

If an autosampler is not available, the samples can be read directly in FACS tubesorafterbeingtransferredfromthemicroplatetomicroFACStubes.Inthiscase,thesamplevolumemayneedtobeincreasedfrom200µLto300µLbyaddingextra100µLof1XWashBuffertoeachtube,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 Cell Culture Medium 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 pre-mixed beads to all tubes/wells25 μL Detection Antibodies to all tubes/wells

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

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 pre-mixed beads to all tubes/wells25 μL Detection Antibodies to all tubes/wells

A B

C

A

B

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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

(singlelaser) FL2 575 nm FL3 670 nm Yes

BD FACSCaliburTM

(duallaser) FL2 575 nm FL4 660 nm No*

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

BDTM LSR, LSRII,BD LSRFortessaTM PE 575-585

nm APC 660 nm No*

BD FACSAriaTM PE 575 nm APC 660 nm No*

*Compensationisnotrequiredforthespecifiedflowcytometerswhensetupproperly,butisrecommendedforconsistentresults.

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

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

1).Startuptheinstrumentfollowingthemanufacturer’srecommendations.

2).Createatemplatefordataacquisitionusingyourinstrument’sdata acquisitionsoftware.Atemplateisadocumentorworksheetwithdensityplotsthatallowstheusertoperformmachinesetupanddataacquisition.

3).SetupthePMTvoltagesofeachchanneltobeusedfordataacquisition usingtheSetupBeadsprovidedinthekit.

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

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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 VortexthevialoftheRawBeadsfor30secondstoresuspendthe beads.

3.2 Transfer400μLoftheRawBeadstoafreshFACStube.

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

Pauseandrestartacquisitionfrequentlyduringthesetupprocedure torefreshthebeadpopulationsafteradjustingsettings.

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LEGENDplex™ Human Growth Factor Panel Figure 6.

3.4 ContinueadjustingthesettingssothatBeadsAandBeadsBarewell separatedandtheFSCandSSCreadingsare>200.

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

(Figure6).

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

recommended).

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

Figure 7.

3.8 VortexthevialofSetupBeads2:PEBeadsfor30secondstoresus- pend the beads.

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3.9 Transfer400μLofthePEBeadstoafreshFACStube.

3.10ReplacetheRawBeadstubefromtheflowcytometerwiththe PE beads tube.

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

gate(Figure8).

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

Figure 8.

3.13 Save the document again for future use.

3.14Theflowcytometerisnowreadyforsampleacquisition.

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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 twogatesandlabelthemBeadsAandBeadsB(Figure9).

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 ontherightpanelbelow).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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LEGENDplex™ Human Growth Factor Panel3. SetupPMTVoltagesandCompensation

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

FollowtheinstructionsbelowforsettingupthePMTsettingsandcompen-sation:

3.1 VortexthevialoftheRawBeadsfor30secondstoresuspendthe beads.


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

Pauseandrestartacquisitionfrequentlyduringthesetupprocedure torefreshthebeadpopulations,afteradjustingsettings.

Figure 11.

3.4 ContinueadjustingthesettingssothatBeadsAandBeadsBarewell separatedandtheFSCandSSCreadingsare>200.

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

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

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Figure 12.

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

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

3.9 VortexthevialoftheSetupBeads1:FITCBeadsfor30secondsto resuspend the beads.

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

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

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

Note:FITCbeadsareonlyofsmallsize,fallingintheBeadsAgate.

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

Figure 14.

3.14VortexthevialofPEBeadsfor30secondstoresuspendthebeads.

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

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

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

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Figure 15.

3.18AdjusttheFL2settingsothatthemedianfluorescenceintensityofthe PEbeadsfallsbetweenthelot-specificrangelabeledonthePEBeads vial(Figure16).

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

Figure 16.

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

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

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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(Thesettingswillbesavedinthefinalstepofthissetupprocedureandanysettingssavedcanbeimportedtoanewexperiment.PleaserefertoStep2andStep3.9below).




A template for FACSDivaTMisaworksheetwithdensityplotsthatallowstheusertoperformmachinesetupanddataacquisition.

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

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

Tocreateanewtemplate,followtheinstructionsbelow:

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

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LEGENDplex™ Human Growth Factor Panel 2.2 Intheglobalworksheet,opentheworksheet.Createadotplotwith

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

Figure 19.

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

Figure 20.

2.4 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 VortexthevialofRawBeadsfor30secondstoresuspendthebeads.


3.3 Settheflowcytometerflowratetolow.RuntheRawBeads.Adjust thesettingsforFSCandSSCsothatbothbeadpopulationsarevisible (Figure21).

Pauseandrestartacquisitionfrequentlyduringthesetupprocedure torefreshthebeadspopulationsafteradjustingsettings.

Figure 21.

3.4 ContinueadjustingthesettingssothatBeadsAandBeadsBarewell separatedandtheFSCandSSCreadingsare>50(x1000).

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

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

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Beads Beads A+ Beads B

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

Figure 23.

3.8 AdjusttheAPCsettingssothatthetheAPCfluorescenceintensitiesof allbeadpopulationsarebetween1x102 and 5 x 104(Figure23).


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

3.10Theflowcytometerisnowreadyforsampleacquisition.

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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.

Theflowcytometersetupinstructionsarealsopostedonourwebsite: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,pleaserefertotheFlowCytometerSetupGuide).

3. Vortexeachsamplefor5secondsbeforeanalysis.

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

Note: Do not acquire too few or too many beads. Too few beads acquired mayresultinhighCVsandtoomanybeadsacquiredmayresultinslowdata analysis later.

Whenreadingsamples,settheflowcytometertosetupmodefirstandwaituntilbeadpopulationisstabilizedbeforeswitchingtoacquisitionmode.

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

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

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

5. Proceed to data analysis using LEGENDplexTMDataAnalysisSoftwarewhendataacquisitioniscompleted.

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Data Analysis

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

• Afterdownloading,installitonaPC(runningWindows7orWindows8)anduseitinconjunctionwiththeDataAnalysisSoftwareDongleincludedinthiskit.Thedonglehasalicensekeystoredinitandisneededtorunthesoftware.Tousethedongle,simplyplugitintheUSBportofthecomputeronwhichthedataanalysissoftwareisinstalled,priortolaunchingthesoftware.

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

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

biolegend.com 37


Chapter 6: FREQUENTLY ASKED QUESTIONS

Q. WhatisthedifferencebetweenLEGENDplexTM and Luminex®Assays?

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

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

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

Q. WhatarethecompatibleflowcytometersfortheLEGENDplex™assays?

In general, LEGENDplexTMassayscanbeusedonmostcommonflowcytom-eters, such as:

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

BD FACSAria™ BD FACSArrayTM

PleaserefertotheMATERIALSTOBEPROVIDEDBYEND-USERsectionfordetailsonthechannelconfigurationsofthecytometer.

Forflowcytometersnotlistedabove,theenduserneedstomakesure

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

TheFCSorListmodefilesfromthefollowinginstrumentsarecompatiblewith the LEGENDplex TMDataAnalysisSoftware.

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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™

BeckmanCoulter

CyAn™ ADP

FACSAria™ II FC 500

Accuri™ C6 Gallios™

ORFLO Moxi Flow™ MoFlo®Astrios™

CyteK DxP10™ MoFlo®XDP

Q. Whatistherightprocedureforrunningtheassay?

1. Performtheassayasinstructedinthiskitmanual.

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 Growth Factor Panel Ifaflowcytometerislistedinthecompatibleflowcytometerlistinthis

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

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

Q. Isthereaspecialsoftwarerequiredfordataanalysis?

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

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

Q. CanIselecttomeasureonlysomeanalyteswithinapanel?

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

Q. CanIselecttomeasureanalytesacrosspanels?

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

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

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

Othercomponents,suchasAssayBuffer,WashBuffer,Plate,andPlateSealerarenotlot-specificandcanthereforebeexchangedbetweendiffer-entkitsandlots.

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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.Ifsampleconcentra-tionsfallbelowtheminimumdetectionconcentration,itisconsiderednotdetectablebytheparticularassay.

Q. Duringdataacquisition,whydothebeadpopulations(definedbyFSCandSSC)sometimesappeartobedispersedorshifted?

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

Q. DoestheLEGENDplex™DataAnalysisSoftwarerunonAppleMacintosh?

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

Q. Whydoeseachkitincludeasoftwaredongle?

The dongle allows you to use our LEGENDplexTMsoftwarefordataanalysisand each dongle contains 1 million points. All dongles are the same.

Q. Iranoutofpointsonmydongle.CanIgetanewoneseparately?Or,canIuseadonglefromonekitforanotherkit?

Dongle is not sold separately. Contact BioLegend if you need a new dongle. Thedonglecanbeusedacrosskits.

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Chapter 7: ASSAY CHARACTERIZATION

RepresentativeStandardCurve

This standard curve was generated using the LEGENDplexTM Human Growth FactorPanelfordemonstrationpurposeonly.Astandardcurvemustberun with each assay.

AssaySensitivity

Theassaysensitivityorminimumdetectableconcentration(MDC)isthetheoreticallimitofdetectioncalculatedusingtheLEGENDplexTM Data AnalysisSoftwarebyapplyinga5-parametercurvefittingalgorithm.

Analyte MDC in Cell Culture Medium (pg/mL)

MDC in Serum (pg/mL)

Human Ang-2 4.9 5.4

Human EGF 2.3 2.6

Human EPO 4.6 4.7

Human FGF-basic 7.3 7.6

Human G-CSF 7.6 4.4

Human GM-CSF 3.3 4.4

Human HGF 7.6 4.8

Human M-CSF 0.9 0.7

Human PDGF-AA 6.6 11.0

1.0

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100.0

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10000.0

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MFI

Concentration (pg/mL)

Angiopoietin-2 EGF EPO FGF-basic G-CSF GM-CSF HGF M-CSF PDGF-AA PDGF-BB SCF TGF-α VEGF

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Human PDGF-BB 6.9 3.6

Human SCF 2.2 2.4

HumanTGF-α 6.6 5.4

HumanVEGF 9.9 11.5

Cross-Reactivity

Thefollowingrecombinantproteinsweretestedat50ng/mLusingtheLEGENDplexTM HumanGrowthFactorPanel.VEGFpaircandetectbothVEGF165andVEGF121.SCFpairdetectsfreeSCFproteins.Noornegligiblecross-reactivitywasfoundforallotheranalytes.

IL-27 IGF-II IL-13 IL-17F IL-23

IL-33 TGF-β1 IL-2 IL-4 IL-12p40

MIP-1β TGF-β2 IL-6 IL-21 IL-12p70

ENA-78 TGF-β3 IL-9 IL-22 IL-15

MCP-1 TNF-β IL-10 TSLP IL-18

RANTES PLGF-1 IFN-γ IL-1α IL-11

Ang-1 VEGF-c TNF-α IL-1β

IGF-I IL-5 IL-17A IFN-α

Groα IP-10 CCL11 MIP-1α

IL-8 TARC MIP-3α I-TAC

Accuracy (Spike Recovery)

Forspikerecoveryincellculturemedium,RPMIorDMEMwith10%FCSwasfirstdilutedtwo-foldwithAssayBufferandspikedwithtargetproteinsatthreedifferentlevelswithintheassayrange.Thespikedsampleswerethenassayed,andthemeasuredconcentrationswerecomparedwiththeexpected values.

Forspikerecoveryinserum(n=8)andplasma(n=8),sampleswerefirstdilutedtwo-foldwithAssayBufferandspikedwithtargetproteinsatthreedifferentlevelswithintheassayrange.Thespikedsampleswerethenas-sayed,andthemeasuredconcentrationswerecomparedwiththeexpectedvalues.

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Analyte% of Recovery in Cell Culture

Medium

% of Recov-ery in Serum

% of Recov-ery in Plasma

Human Ang-2 96% 45% 56%

Human EGF 98% 82% 87%Human EPO 93% 60% 67%Human FGF-basic 94% 126% 104%Human G-CSF 96% 84% 83%Human GM-CSF 96% 118% 109%Human HGF 99% 103% 84%Human M-CSF 98% 51% 56%Human PDGF-AA 98% 120% 117%Human PDGF-BB 101% 133% NAHuman SCF 100% 80% 80%HumanTGF-α 99% 78% 71%HumanVEGF 100% 75% 78%

NA=NotApplicableduetohighendogenouslevels

LinearityofDilution

Forspikelinearityincellculturemedium,RPMIorDMEMwith10%FCSwasfirstdilutedtwo-foldwithAssayBufferandspikedwithaknowncon-centrationoftargetproteins.Thespikedsampleswereseriallydiluted1:2,1:4,1:8withassaybufferandassayed.Themeasuredconcentrationsofseriallydilutedsampleswerecomparedwiththatofthespikedsamples.

Fortestinglinearityinserum(n=8)andplasma(n=8),sampleswerefirstdilutedtwo-foldwithAssayBufferandspikedwithaknownconcentrationoftargetproteins.Thespikedsampleswereseriallydiluted1:2,1:4,1:8withMatrixBandassayed.Themeasuredconcentrationsofseriallydilutedsampleswerecomparedwiththatofthespikedsamples.

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AnalyteLinearity in Cell

Culture Medium

Linearity in Serum

Linearity in Plasma

Human Ang-2 102% 116% 130%

Human EGF 104% 102% 110%Human EPO 105% 110% 131%Human FGF-basic 100% 83% 87%Human G-CSF 96% 96% 111%Human GM-CSF 111% 86% 89%Human HGF 102% 100% 113%Human M-CSF 92% 126% 147%Human PDGF-AA 96% 92% 114%Human PDGF-BB 104% 98% 95%Human SCF 106% 104% 102%HumanTGF-α 98% 97% 96%HumanVEGF 103% 108% 117%

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 Ang-2Sample 1 38.8 1.3 3%

Sample 2 540.6 21.9 4%

Human EGFSample 1 38.3 1.8 5%

Sample 2 579.2 39.8 7%

Human EPOSample 1 38.6 3.2 8%

Sample 2 547.6 45.6 8%

Human FGF-basicSample 1 38.4 1.8 5%

Sample 2 525.6 16.5 3%

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Human G-CSFSample 1 35.1 2.9 8%

Sample 2 565.9 46.4 8%

Human GM-CSFSample 1 36.8 1.5 4%

Sample 2 529.0 26.3 5%

Human HGFSample 1 38.2 2.1 6%

Sample 2 511.3 36.2 7%

Human M-CSFSample 1 36.3 2.3 6%

Sample 2 518.4 22.0 4%

Human PDGF-AASample 1 36.7 1.4 4%

Sample 2 485.7 24.1 5%

Human PDGF-BBSample 1 39.4 2.8 7%

Sample 2 507.7 29.0 6%

Human SCFSample 1 38.0 1.8 5%

Sample 2 482.3 25.3 5%

HumanTGF-αSample 1 38.1 2.0 5%

Sample 2 559.6 48.9 9%

HumanVEGFSample 1 37.2 1.8 5%

Sample 2 499.5 18.8 4%

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 Ang-2Sample 1 38.1 1.3 3%

Sample 2 601.1 60.6 10%

Human EGFSample 1 41.5 2.0 5%

Sample 2 676.8 40.6 6%

Human EPOSample 1 37.4 2.6 7%

Sample 2 659.1 62.0 9%

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Human FGF-basicSample 1 40.0 1.3 3%

Sample 2 583.4 24.7 4%

Human G-CSFSample 1 35.0 2.1 6%

Sample 2 668.8 59.2 9%

Human GM-CSFSample 1 39.4 2.3 6%

Sample 2 638.6 36.2 6%

Human HGFSample 1 38.9 2.2 6%

Sample 2 633.3 41.0 6%

Human M-CSFSample 1 38.5 1.5 4%

Sample 2 618.9 24.3 4%

Human PDGF-AASample 1 39.7 2.7 7%

Sample 2 569.7 41.1 7%

Human PDGF-BBSample 1 39.6 2.1 5%

Sample 2 596.7 44.2 7%

Human SCFSample 1 39.5 1.8 5%

Sample 2 586.4 52.4 9%

HumanTGF-αSample 1 38.4 1.8 5%

Sample 2 685.6 55.2 8%

HumanVEGFSample 1 37.8 1.4 4%

Sample 2 602.9 48.5 8%

Biological Samples

Serum

Normalhumanserumsamples(n=36)weretestedforendogenouslevelsofgrowthfactors.Theconcentrationsmeasuredareshownbelow:

Analyte Range (pg/mL)

% of Detectable

Mean of detectable

(pg/mL)Human Ang-2 39.2 - 2514 100% 904.2

Human EGF ND - 44.0 55% 16.2

Human EPO ND - 104.7 70% 42.7

Human FGF-basic ND - 723.5 65% 160.7

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Human G-CSF ND - 181.1 90% 40.0

Human GM-CSF ND - 77.1 20% 33.7

Human HGF 44.8 - 614.5 100% 207.4

Human M-CSF ND - 150.0 20% 83.7

Human PDGF-AA 218.2 - 3614 100% 986.4

Human PDGF-BB 121.4 - 6091 100% 1405

Human SCF ND - 203.3 45% 50.7

HumanTGF-α ND - 857.9 40% 244.9

HumanVEGF ND - 163.8 40% 84.7

ND=NotDetectable

Plasma

Normalhumanplasmasamples(n=20)weretestedforendogenouslevelsofgrowthfactors.Theconcentrationsmeasuredareshownbelow:

Analyte Range (pg/mL) % of Detectable

Mean of detectable

(pg/mL) Human Ang-2 154.8 - 2078 100% (pg/mL)

Human EGF ND - 115.8 85% 35.9

Human EPO ND - 272.7 90% 39.3

Human FGF-basic ND - 1019 90% 132.0

Human G-CSF 15.7 - 170.9 100% 36.6

Human GM-CSF ND - 40.6 25% 23.5

Human HGF 36.3 - 1285 100% 311.1

Human M-CSF ND -268.1 15% 140.4

Human PDGF-AA 391.3 - 29802 100% 8772

Human PDGF-BB 131.5 - 72120 100% 27243

Human SCF ND - 24.6 35% 13.6

HumanTGF-α ND - 727.6 45% 177.9

HumanVEGF ND - 373.2 65% 145.1

Cell Culture Supernatant

HumanPBMC(1x106cells/mL)wereculturedundervariousconditions(PHA,10µg/mL;PMA,50ng/mL;LPS, 50 ng/mL).Supernatants were collectedafter1and4daysandassayedwiththeLEGENDplexTM Human GrowthFactorkit.Theresults(allinpg/mL)aresummarizedbelow.

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Analyte Control1 Day

PHA1 Day

PMA1 Day

LPS 1 Day

Human Ang-2 146 160 154 178

Human EGF 52.5 54.1 55.7 54.5

Human EPO ND ND ND ND

Human FGF-basic ND ND 15.4 14.7

Human G-CSF 34.8 2011 78.8 3297

Human GM-CSF 5.4 8.0 228 7.9

Human HGF 47.6 28.3 76.2 23.4

Human M-CSF ND 7.7 106 2.0

Human PDGF-AA 1101 1415 1454 1336

Human PDGF-BB 5103 5100 5349 5139

Human SCF ND ND ND ND

HumanTGF-α 10.8 100 9.6 115

HumanVEGF 359 165 158 459ND=NotDetectable

Analyte Control4 Day

PHA4 Day

PMA4 Day

LPS 4 Day

Human Ang-2 95.1 48.8 64.3 72.4

Human EGF 61.3 61.6 78.1 67.2

Human EPO ND ND 15.7 26.5

Human FGF-basic ND 14.7 17.2 16.0

Human G-CSF 39.1 2125 1565 4530

Human GM-CSF 14.1 5.5 1157 7.8

Human HGF 436 42.8 120 45.3

Human M-CSF 23.7 3.9 1713 3.5

Human PDGF-AA 1239 1830 3520 1576

Human PDGF-BB 6778 7891 10247 8041

Human SCF ND ND ND ND

HumanTGF-α 22.4 166 41.5 171

HumanVEGF 517 1580 177 2270ND=NotDetectable

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LEGENDplex™ Human Growth Factor PanelHumanHUVC(1x106cells/mL)wereculturedundervariousconditions(50ng/mLIFN-γ+200ng/mLTNF-α;100ng/mLLPS).Supernatants were collectedafter1dayandassayedwiththeLEGENDplexTM Human Growth Factorkit.Theresults(allinpg/mL)aresummarizedbelow.

Analyte Control IFN-γ+TNF-α LPS

Human Ang-2 8588 6119 8694

Human EGF 2128 2388 2798

Human EPO 29.4 26.7 30.5

Human FGF-basic 710 797 948

Human G-CSF 68.9 752 1836

Human GM-CSF 6.0 89.7 48.2

Human HGF 11.9 94.3 13.5

Human M-CSF 8.5 108.7 19.9

Human PDGF-AA 398 518 396

Human PDGF-BB 840 912 1288

Human SCF ND ND ND

HumanTGF-α 8.4 9.3 10.6

HumanVEGF ND ND ND

ND=NotDetectable

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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.

OptimizeinstrumentsettingsusingKitSetupBeads,andmakeappropriatecom-pensationbetweenchannels.

Filter plate will not vacuum or some wells clogged

Vacuumpressureisinsufficientorvacuummanifold does not seal properly.

Increase vacuum pressure such that 0.2mLbuffercanbesuctionedin3-5seconds. Clean the vacuum manifold and makesurenodebrisonthemanifold.Press down the plate on the manifold to makeagoodseal.

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

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

Ifsomewellsarestillcloggedduringwashing, try the following:

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

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

3).Takeathinneedle(e.g.,insulinnee-dle),whileholdingtheplateupward,pokethelittleholeundereachofthecloggedwellsandvacuumagain.Donotpoketoohard or too deep as it may damage the filterandcauseleaking.

Filter plate was used without pre-wet.

Pre-wetplatewithwashbufferbeforerunning the assay.

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Insufficientbead count or slow reading

Beads inappropriately prepared

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

Samples cause beads aggregationduetoparticulatematterorviscosity

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

Beads were lost during washing for in-tube assay

Makesurebeadsarespundownbyvisu-allycheckthepellet(beadsareinlightblueorbluecolor).Beverycarefulwhenremoving supernatant during washing.

Probemaybepartiallyclogged

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

Plateleaked

Vacuumpressuresettoo high

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

Plate set directly on table or absorbent tow-elsduringincubationsorreagentadditions

Set plate on plate holder or raised edge sobottomoffilterisnottouchinganysurface.

Liquid present on the under side of the plate aftervacuum

Afterwashing,pressdownplatefirmlyonastackofcleanpapertowelstodrytheunderside of the plate.

Pipettetouchinganddamagedplatefilterduringadditions

Pipettetothesideofwells.

Highback-ground

Backgroundwellswerecontaminated

Avoidcross-wellcontaminationbychang-ingtipsbetweenpipettingwhenperform-ingtheassayusingamultichannelpipette.

InsufficientwashesThebackgroundmaybeduetonon-spe-cificbindingofSA-PE.Increasenumberofwashes.

Debris(FSC/SSC)duringsample acquisi-tion

Debris or platelet may existinsamplesolution

Centrifugesamplesbeforeanalyzingsamples. Remove platelet as much as possible.

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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

Makesureallreagentsarevacuumedoutcompletely in all wash steps.

Samples may contain particulatematters.

Centrifuge samples just prior to assay setup and use supernatant. If high lipid content is present, remove lipid layer aftercentrifugation.Samplemayneeddilutioniftooviscous.

Low or poor standard curve signal

The standard was in-correctlyreconstituted,stored or diluted

Followtheprotocoltoreconstitute,storeanddilutestandard.Doublecheckyourcalculation.

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

Makesuretheexperimenttogeneratethesamplesworked.Useproperpositivecontrols.

Samplesconcentrationshigher than highest standard point.

Dilutesamplesandanalyzeagain.

Standard curve was saturated at higher end of curve.

MakesurethePMTsettingforthere-porter channel is appropriate. Use shorter incubationtimeifincubationtimewastoo long

Missed beads populationsduring reading, ordistributionis unequal

Sample may cause some beads to ag-gregate.

Centrifuge samples just prior to assay setup and use supernatant. If high lipid content is present, remove lipid layer aftercentrifugation.Samplemayneeddilutioniftooviscous.

Beadspopulationsarenot mixed properly

Makesureallbeadpopulationsaremixed.and in similar numbers.

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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]

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