Human Growth Factor Panel R2 - 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.

biolegend.com 1

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……………….........

PerformingtheAssayUsingaV-BottomPlate………………...

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

Chapter 5: DATA ACQUISITION AND ANALYSIS.........................18

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

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

Chapter 6: ASSAY CHARACTERIZATION......................................2.

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

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

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

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

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

3

3

3

4

6

6

7

8

9

9

9

10

11

12

12

15

18

18

18

19

20

20

20

21

21

22

Tel: 858-768-5800


2

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

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

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

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

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

23

24

25

29

33

biolegend.com 3


Chapter 1: KIT DESCRIPTION

Introduction

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

TheLEGENDplexTMHumanGrowthFactorPanelisabead-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 Panel is designed to allow flexible customizationwithin the panel. For mix and match within the panel, please visit https://www.biolegend.com/en-us/legendplex.

Thisassayisforresearchuseonly.

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.

Tel: 858-768-5800


4

Beads Usage

TheHumanGrowthFactorPanelincludestwosetsofbeads.Eachsethasauniquesizethatcanbeidentifiedonflowcytometerbasedontheirforwardscatter(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

biolegend.com 5


Figure3.BeadsBClassificationbyFL4

ForBeadsusageinthepanel,pleaserefertoTable1below:

Table1.BeadsIDandTargetInformation

Target Bead ID TopStandardConcentrations(ng/mL)

HumanAngiopoietin-2 A4

Note:Thetopstandardconcentrationsofanalytes

in this panel were set at variousconcentrations,but may be subject to change from lot to lot (pleasevisitbiolegend.com/en-us/legendplextodownloadalot-specificcertificateofanalysis).

HumanEGF A5

HumanEPO A6

HumanFGF-basic A7

HumanG-CSF A8

HumanGM-CSF A10

HumanHGF B2

HumanM-CSF B3

HumanPDGF-AA B4

HumanPDGF-BB B5

HumanSCF B6

HumanTGF-α B7

HumanVEGF B9

*BeadIDisusedtoassociateabeadpopulationtoaparticularanalytewhenusingtheLEGENDplex™dataanalysissoftwareprogram.Forfurtherinformationregardingtheuseoftheprogrampleasevisitbiolegend.com/en-us/legendplex.

B4 B5

B6 B7

B3

B9

B2

Tel: 858-768-5800


6

StorageInformation

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

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

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

Materials Supplied

TheLEGENDplexTMkitcontainsreagentsfor100testslistedinthetablebelow.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 77743LEGENDplexTMMatrixB1,Lyophilized 1 vial lyophilized 75024LEGENDplexTMAssayBuffer 1bottle 25 mL 77562LEGENDplexTMWashBuffer,20X 1bottle 25 mL 77564Filter plate 1 plate 76187Plate Sealers 4 sheets 78101

biolegend.com 7


Materials to be Provided by the End-User

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

Partiallistofcompatibleflowcytometers:

Flow Cytometer

Reporter Channel

ChannelEmission

ClassificationChannel

Channel Emission

Compensa-tionneeded?

BD FACSCaliburTM

(single laser)FL2 575 nm FL3 670 nm Yes

BD FACSCaliburTM

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

BD FACSArrayTM Yellow 575 nm Red 660 nm No*

BD FACSCantoTM

BD FACSCantoTM IIPE 575 nm APC 660 nm No*

BDTM LSR, LSR IIBD LSRFortessaTM PE 575-585

nm APC 660 nm No*

BD FACSAriaTM PE 575 nm APC 660 nm No*

*Compensationisnotrequiredforthespecifiedflowcytometerswhenset up properly, but is recommended for consistent results.

Forsettinguptheaboveflowcytometers,pleasefollowtheFlow Cytom-eter Setupguideinthismanualorvisit:www.biolegend.com/legendplex.

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

• Multichannelpipettescapableofdispensing5μLto200μL

• Reagentreservoirsformultichannelpipette

• Polypropylenemicrofugetubes(1.5mL)

• Laboratory vortex mixer

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

• Aluminum foil

• Absorbent pads or paper towels

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

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

Tel: 858-768-5800


8

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

Iftheassayisperformedinafilterplate:

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

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

• Ifneeded,additionalFilterplatescanbeorderedfromBioLegend(Cat#740377or740378).

IftheassayisruninaV-bottomplate:

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

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

• Precautions

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

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

• Matrix B1 for LEGENDplexTMkitscontainscomponentsofhumanoriginandshouldbehandledaspotentiallyhazardous.TherawmaterialhasbeenscreenedforinfectiousdiseasesandisnegativeforHIV,HBVandHCVusingFDA-approved test methods.

• Donotmixorsubstitutereagentsfromdifferentkitsorlots.Reagentsfromdifferentmanufacturersshouldnotbeusedwiththiskit.

• Donotusethiskitbeyonditsexpirationdate.

• SA-PEandPre-mixedBeadsarelight-sensitive.Minimizelightexposure.

biolegend.com 9

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.

Tel: 858-768-5800


10

PreparationofMatrixB1(forSerumorPlasmaSamplesOnly)

• Add 5.0 mL LEGENDplexTMAssayBuffertothebottlecontaininglyophilized MatrixB1.Allowatleast15minutesforcompletereconstitution.Vortexto

mixwell.LeftoverreconstitutedMatrixB1shouldbestoredat≤-70°Cforup to 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.ThiswillbeusedasthetopstandardC7.

Note:Thetopstandardconcentrationsofanalytesinthispanelweresetatvariousconcentrations,butmaybesubjecttochangefromlottolot(pleasevisitbiolegend.com/en-us/legendplextodownloadalot-specificcertificateofanalysis).

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

4. Add75µLofAssayBuffertoeachofthesixtubes.Prepare1:4dilutionofthetopstandardbytransferring25µLofthetopstandardC7totheC6tubeandmixwell.ThiswillbetheC6standard.

5. Inthesamemanner,performserial1:4dilutionstoobtainC5,C4,C3,C2andC1standards(seethetablebelowusingthetopstandardat1,000ng/mL as an example).AssayBufferwillbeusedasthe0pg/mLstandard(C0).

Tube/Standard

ID

Serial Dilution

Assay Buffertoadd (µL)

Standard to add

Final Conc.

(pg/mL)*

C7 -- -- -- 10,000

C6 1:4 75 25 µLofC7 2,500

C5 1:16 75 25 µLofC6 625

C4 1:64 75 25 µLofC5 156.3

C3 1:256 75 25 µLofC4 39.1

C2 1:1024 75 25 µLofC3 9.8

C1 1:4096 75 25 µLofC2 2.4

C0 -- 75 -- 0

biolegend.com 11


SampleDilution

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

Iffurthersampledilutionisdesired,dilutionshouldbedonewithMatrixB1 to 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.

Tel: 858-768-5800


12

Chapter 3: ASSAY PROCEDURE

TheLEGENDplexTMassaycanbeperformedeitherinafilterplate,orinaV-bottommicroplate.

• Thisprocedurerequiresavacuumfiltrationunitforwashing(seeMaterials to be Provided by the End-User, page 7).

• If the Filter plate assay procedure is not possible or if you prefer, the assay canbeperformedinaV-bottomplate.

Performing the Assay Using a Filter Plate

• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.

• Setthefilterplateonaninvertedplatecoveratalltimesduringassaysetupandincubationsteps,sothatthebottomoftheplatedoesnottouchanysurface.Touchingasurfacemaycauseleakage.

• Keeptheplateuprightduringtheentireassayprocedure,includingthewashing steps, to avoid losing beads.

• Theplateshouldbeplacedinthedarkorwrappedwithaluminumfoilforallincubationsteps.

• Standards and samples should be run in duplicate and arranged on the plateinaverticalconfigurationconvenientfordataacquisitionandanaly-sis(asshowninattachedPLATEMAP,page33).Besuretoloadstandardsinthefirsttwocolumns.Ifanautomationdeviceisusedforreading,theorientationandreadingsequenceshouldbecarefullyplanned.

1. Pre-wettheplatebyadding100μLofLEGENDplexTM1XWashBuffertoeachwellandletitsitfor1minuteatroomtemperature.Toremovetheexcess 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, load the plate as shown inthetablebelow(intheorderfromlefttoright):

AssayBuffer Matrix B1 Standard Sample*

StandardWells 25 µL --- 25 µL ---

Sample wells 25 µL --- --- 25 µL

biolegend.com 13


For measuring serum or plasma samples, load the plate as shown inthetablebelow(intheorderfromlefttoright):

AssayBuffer Matrix B1 Standard Sample*

StandardWells --- 25 µL 25 µL ---

Sample wells 25 µL --- --- 25 µL

*See SampleDilution

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

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 aplateshaker,secureitandshakeatapproximate500rpmfor2hoursatroom 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. Add150µLof1XWashBuffertoeachwell.Resuspendthebeadsonaplateshakerfor1minute.

11. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay(Note:Prolongedsamplestoragecanleadtoreducedsignal).

Iftheflowcytometerisequippedwithanautosampler,readtheplatedi-rectly using the autosampler. Please be sure to program the autosampler

Tel: 858-768-5800


14

to resuspend beads in the well immediately before taking samples. The probeheightmayneedtobeadjustedwhenusinganautosampler.

If an autosampler is not available, the samples can be transferred from the filterplatetomicroFACS(orFACS)tubes and read manually.

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

Vacuum to remove excess bu�er

Incubate 2 hours, RT, shaking

Capture beads

Biotinylated Detection Antibody

Analytes

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

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

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

BA

C

A B C

A B C

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

biolegend.com 15


PerformingtheAssayUsingaV-bottomPlate

• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.

• Keeptheplateuprightduringtheentireassayprocedure,includingthewashing steps, to avoid losing beads.

• Theplateshouldbeplacedinthedarkorwrappedwithaluminumfoilforallincubationsteps.

• Standards and samples should be run in duplicate and arranged on the plateinaverticalconfigurationconvenientfordataacquisitionandanalysis(asshowninattachedPLATEMAP,page33).Besuretoloadstandardsinthefirsttwocolumns.Ifanautomationdeviceisusedforreading,theori-entationandreadingsequenceshouldbecarefullyplanned.

1. Formeasuringtissueculturesupernatantsamples,load the plate as showninthetablebelow(intheorderfromlefttoright):

AssayBuffer AssayBuffer Standard Sample*


Sample wells --- 25 µL --- 25 µL For measuring serum or plasma samples, load the plate as shown in the

tablebelow(intheorderfromlefttoright):Matrix B1 AssayBuffer Standard Sample*


Sample wells --- 25 µL --- 25 µL *See SampleDilutiononpage12

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

3. Sealtheplatewithaplatesealer.Covertheentireplatewithaluminumfoil to protect the plate from light. Shakeat800rpmonaplateshakerfor2 hours at room temperature (Depending on the shaker, the speed may needtobeadjusted.Theoptimalspeedisonethatishighenoughtokeepbeadsinsuspensionduringincubation,butnottoohighthatitmaycausesample to spill from the wells).

4. Centrifugetheplateat1050rpm(~250g)for5minutes,usingaswingingbucketrotor(G.H3.8)withmicroplateadaptor(Please refer to Materials to be Provided by the End-User, page 7). Donotuseexcessivecentrifugation

Tel: 858-768-5800


16

speedasitmaymakeithardertoresuspendbeadsinlatersteps. Makesurethetimerofthecentrifugeworksproperlyandstandbytomake sure the centrifuge reaches preset speed.

5. Immediatelyaftercentrifugation,dumpthesupernatantintoabiohazardwastecontainerbyquicklyinvertingandflickingtheplateinonecontinu-ousandforcefulmotion.Thebeadspelletmayormaynotbevisibleafterdumping the supernatant. Loss of beads should not be a concern as the beadswillstayinthetipofthewellnicely.Blottheplateonastackofcleanpaper towel and drain the remaining liquid from the well as much as pos-sible. Be careful not to disturb the bead pellet.

Alternatively,removalofthesupernatantmaybecompletedusingamultichannelpipettesetat75µL.Trytoremoveasmuchliquidaspossiblewithout removing any beads. Be sure to changepipettetipsbetweeneachrow or column.

6. Washtheplatebydispensing200μLof1XWashBufferintoeachwellandincubate for one minute. Repeat step 4 and 5 above. A second wash is optional,butmayhelpreducebackground.

7. Add25µLofDetectionAntibodiestoeachwell.

8. Sealtheplatewithanewplatesealer.Covertheentireplatewithalumi-num foil to protect the plate from light. Shakeat800rpmonaplateshakerfor 1 hour at room temperature.

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

10. Sealtheplatewithanewplatesealer.Wraptheentireplatewithaluminumfoilandshaketheplateonaplateshakeratapproximate800rpmfor30minutes at room temperature.

11. Repeat step 4 and 5.

12. (Thiswashingstepisoptionalbuthelpstoreducethebackground.)Washthe plate by dispensing 200 μLof1XWashBufferintoeachwellandincu-bate for one minute. Repeat step 4 and 5 above.

13. Add150µLof1XWashBuffertoeachwell.Resuspendthebeadsbypipet-ting.

14. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay(Note:Prolongedsamplestoragecanleadtoreducedsignal).

Iftheflowcytometerisequippedwithanautosampler,thesamplescanberead directly. Please be sure to program the autosampler to resuspend beads in the well immediately before taking samples. The probe height mayneedtobeadjustedwhenusinganautosampler.

If an autosampler is not available, the samples can be transferred

biolegend.com 17

LEGENDplex™ Human Growth Factor PanelfromtheplatetomicroFACS(orFACS)tubesandreadmanually.

Assay Procedure Summary for V-bottom Plate

Incubate 2 hours, RT, shaking

Capture beads

Biotinylated Detection Antibody

Analytes

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

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

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

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

BA

CA B C

A B C

Tel: 858-768-5800


18

Chapter 4: FLOW CYTOMETER SETUP

Inordertogeneratereliabledata,theflowcytometermustbesetupproperlybeforedataacquisition.

Thesetupinstructionshavebeenremovedfromthismanualanduploadedontoour website to save paper.

Toaccessthesetupinstructions,pleasevisit:www.biolegend.com/legendplexandclickontheInstrument Setup tab.

Chapter 5: DATA ACQUISITION AND ANALYSIS

DataAcquisition

1. Beforereadingsamples,makesurethattheflowcytometerissetupprop-erly.

2. Createanewtemplateoropenanexistingtemplate(fordetailsonhowtocreateacytometer-specifictemplate,pleaserefertotheFlowCytometerSetupGuide).

3. Vortex each sample for 5 seconds before analysis.

4. Settheflowratetolow.Setthenumberofbeadstobeacquiredtoabout300peranalyte(e.g.,acquire900beadsfora3-plexassayor3,000beadsfora10-plexassay).Donotsettoacquiretotaleventsassamplesmaycontain large amounts of debris. Instead, create a large gate to include both BeadsAandBeadsB(gateA+B)andsettoacquirethenumberofeventsingateA+B.Thiswillexcludemajorityofthedebris.

Note:Donotacquiretoofewortoomanybeads.Toofewbeadsacquiredmay result in high CVs and too many beads acquired may result in slow data analysis later.

5. Read samples.

Whenreadingsamples,settheflowcytometertosetupmodefirstandwaituntilbeadpopulationisstabilizedbeforerecordingorswitchingtoacquisi-tionmode.

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

biolegend.com 19

LEGENDplex™ Human Growth Factor Panel Whennamingdatafiles,trytousesimplenameswithaconsecutivenum-

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

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

6. Proceed to data analysis using LEGENDplexTMDataAnalysisSoftwarewhendataacquisitioniscompleted.

Data Analysis

TheassayFCSfilesshouldbeanalyzedusingBioLegend’sLEGENDplex™dataanalysissoftware.TheprogramisofferedfreeofchargewiththepurchaseofanyLEGENDplex™assay.Forfurtherinformationregardingacccessto,anduseof the program please visit biolegend.com/en-us/legendplex.

Tel: 858-768-5800


20

Chapter 6: ASSAY CHARACTERIZATION

RepresentativeStandardCurve

ThisstandardcurvewasgeneratedusingtheLEGENDplexTMHumanGrowthFactorPanelfordemonstrationpurposeonly.Astandardcurvemustberun with each assay.

1.0

10.0

100.0

1000.0

10000.0

1.0 100.0 10000.0

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

AssaySensitivity

Theassaysensitivityorminimumdetectableconcentration(MDC)isthetheoreticallimitofdetectioncalculatedusingtheLEGENDplexTM Data AnalysisSoftwarebyapplyinga5-parametercurvefittingalgorithm.

Analyte MDC in Cell Culture Medium(pg/mL)

MDC in Serum(pg/mL)

HumanAng-2 4.9 5.4

HumanEGF 2.3 2.6

HumanEPO 4.6 4.7

HumanFGF-basic 7.3 7.6

HumanG-CSF 7.6 4.4

biolegend.com 21


HumanGM-CSF 3.3 4.4

HumanHGF 7.6 4.8

HumanM-CSF 0.9 0.7

HumanPDGF-AA 6.6 11.0

HumanPDGF-BB 6.9 3.6

HumanSCF 2.2 2.4

HumanTGF-α 6.6 5.4

HumanVEGF 9.9 11.5

Cross-Reactivity

Thefollowingrecombinantproteinsweretestedat50ng/mLusingtheLEGENDplexTMHumanGrowthFactorPanel.VEGFpaircandetectbothVEGF165 and VEGF121. SCF pair detects free SCF proteins. No or negligible cross-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.Thespikedsampleswere

Tel: 858-768-5800


22

thenassayed,andthemeasuredconcentrationswerecomparedwiththeexpected values.

Analyte% of Recovery in Cell Culture

Medium

% of Recov-ery in Serum

% of Recov-ery in Plasma

HumanAng-2 96% 45% 56%

HumanEGF 98% 82% 87%HumanEPO 93% 60% 67%HumanFGF-basic 94% 126% 104%HumanG-CSF 96% 84% 83%HumanGM-CSF 96% 118% 109%HumanHGF 99% 103% 84%HumanM-CSF 98% 51% 56%HumanPDGF-AA 98% 120% 117%HumanPDGF-BB 101% 133% NAHumanSCF 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-foldwithAssayBufferandspikedwithaknownconcentra-tionoftargetproteins.Thespikedsampleswereseriallydiluted1:2,1:4,1:8withMatrixB1andassayed.Themeasuredconcentrationsofseriallydilutedsampleswerecomparedwiththatofthespikedsamples.

biolegend.com 23


AnalyteLinearity in Cell

Culture Medium

Linearity in Serum

Linearity in Plasma

HumanAng-2 102% 116% 130%

HumanEGF 104% 102% 110%HumanEPO 105% 110% 131%HumanFGF-basic 100% 83% 87%HumanG-CSF 96% 96% 111%HumanGM-CSF 111% 86% 89%HumanHGF 102% 100% 113%HumanM-CSF 92% 126% 147%HumanPDGF-AA 96% 92% 114%HumanPDGF-BB 104% 98% 95%HumanSCF 106% 104% 102%HumanTGF-α 98% 97% 96%HumanVEGF 103% 108% 117%

Intra-Assay Precision

Twosampleswithdifferentconcentrationsoftargetproteinswereanalyzedinoneassaywith16replicatesforeachsample.Theintra-assayprecisionwas calculated as below.

Analyte Sample Mean(pg/mL) STDEV %CV

HumanAng-2Sample 1 38.8 1.3 3%

Sample 2 540.6 21.9 4%

HumanEGFSample 1 38.3 1.8 5%

Sample 2 579.2 39.8 7%

HumanEPOSample 1 38.6 3.2 8%

Sample 2 547.6 45.6 8%

HumanFGF-basicSample 1 38.4 1.8 5%

Sample 2 525.6 16.5 3%

Tel: 858-768-5800


24

HumanG-CSFSample 1 35.1 2.9 8%

Sample 2 565.9 46.4 8%

HumanGM-CSFSample 1 36.8 1.5 4%

Sample 2 529.0 26.3 5%

HumanHGFSample 1 38.2 2.1 6%

Sample 2 511.3 36.2 7%

HumanM-CSFSample 1 36.3 2.3 6%

Sample 2 518.4 22.0 4%

HumanPDGF-AASample 1 36.7 1.4 4%

Sample 2 485.7 24.1 5%

HumanPDGF-BBSample 1 39.4 2.8 7%

Sample 2 507.7 29.0 6%

HumanSCFSample 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

Twosampleswithdifferentconcentrationsoftargetproteinswereanalyzedinthreeindependentassayswith3replicatesforeachsample.Theinter-assay precision was calculated as below.

Analyte Sample Mean(pg/mL) STDEV %CV

HumanAng-2Sample 1 38.1 1.3 3%

Sample 2 601.1 60.6 10%

HumanEGFSample 1 41.5 2.0 5%

Sample 2 676.8 40.6 6%

HumanEPOSample 1 37.4 2.6 7%

Sample 2 659.1 62.0 9%

biolegend.com 25


HumanFGF-basicSample 1 40.0 1.3 3%

Sample 2 583.4 24.7 4%

HumanG-CSFSample 1 35.0 2.1 6%

Sample 2 668.8 59.2 9%

HumanGM-CSFSample 1 39.4 2.3 6%

Sample 2 638.6 36.2 6%

HumanHGFSample 1 38.9 2.2 6%

Sample 2 633.3 41.0 6%

HumanM-CSFSample 1 38.5 1.5 4%

Sample 2 618.9 24.3 4%

HumanPDGF-AASample 1 39.7 2.7 7%

Sample 2 569.7 41.1 7%

HumanPDGF-BBSample 1 39.6 2.1 5%

Sample 2 596.7 44.2 7%

HumanSCFSample 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)

HumanAng-2 39.2 - 2514 100% 904.2

HumanEGF ND - 44.0 55% 16.2

HumanEPO ND-104.7 70% 42.7

Tel: 858-768-5800


26

HumanFGF-basic ND-723.5 65% 160.7

HumanG-CSF ND - 181.1 90% 40.0

HumanGM-CSF ND-77.1 20% 33.7

HumanHGF 44.8 - 614.5 100% 207.4

HumanM-CSF ND - 150.0 20% 83.7

HumanPDGF-AA 218.2 - 3614 100% 986.4

HumanPDGF-BB 121.4 - 6091 100% 1405

HumanSCF 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)

HumanAng-2 154.8-2078 100% (pg/mL)

HumanEGF ND - 115.8 85% 35.9

HumanEPO ND-272.7 90% 39.3

HumanFGF-basic ND - 1019 90% 132.0

HumanG-CSF 15.7-170.9 100% 36.6

HumanGM-CSF ND - 40.6 25% 23.5

HumanHGF 36.3 - 1285 100% 311.1

HumanM-CSF ND -268.1 15% 140.4

HumanPDGF-AA 391.3 - 29802 100% 8772

HumanPDGF-BB 131.5-72120 100% 27243

HumanSCF 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

biolegend.com 27

LEGENDplex™ Human Growth Factor Panelcollectedafter1and4daysandassayedwiththeLEGENDplexTMHumanGrowthFactorkit.Theresults(allinpg/mL)aresummarizedbelow.

Analyte Control1 Day

PHA1 Day

PMA1 Day

LPS 1 Day

HumanAng-2 146 160 154 178

HumanEGF 52.5 54.1 55.7 54.5

HumanEPO ND ND ND ND

HumanFGF-basic ND ND 15.4 14.7

HumanG-CSF 34.8 2011 78.8 3297

HumanGM-CSF 5.4 8.0 228 7.9

HumanHGF 47.6 28.3 76.2 23.4

HumanM-CSF ND 7.7 106 2.0

HumanPDGF-AA 1101 1415 1454 1336

HumanPDGF-BB 5103 5100 5349 5139

HumanSCF 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

HumanAng-2 95.1 48.8 64.3 72.4

HumanEGF 61.3 61.6 78.1 67.2

HumanEPO ND ND 15.7 26.5

HumanFGF-basic ND 14.7 17.2 16.0

HumanG-CSF 39.1 2125 1565 4530

HumanGM-CSF 14.1 5.5 1157 7.8

HumanHGF 436 42.8 120 45.3

HumanM-CSF 23.7 3.9 1713 3.5

HumanPDGF-AA 1239 1830 3520 1576

HumanPDGF-BB 6778 7891 10247 8041

HumanSCF ND ND ND ND

Tel: 858-768-5800


28

HumanTGF-α 22.4 166 41.5 171

HumanVEGF 517 1580 177 2270ND=NotDetectable

HumanHUVC(1x106cells/mL)wereculturedundervariousconditions(50ng/mLIFN-γ+200ng/mLTNF-α;100ng/mLLPS).Supernatants were collectedafter1dayandassayedwiththeLEGENDplexTMHumanGrowthFactorkit.Theresults(allinpg/mL)aresummarizedbelow.

Analyte Control IFN-γ+TNF-α LPS

HumanAng-2 8588 6119 8694

HumanEGF 2128 2388 2798

HumanEPO 29.4 26.7 30.5

HumanFGF-basic 710 797 948

HumanG-CSF 68.9 752 1836

HumanGM-CSF 6.0 89.7 48.2

HumanHGF 11.9 94.3 13.5

HumanM-CSF 8.5 108.7 19.9

HumanPDGF-AA 398 518 396

HumanPDGF-BB 840 912 1288

HumanSCF ND ND ND

HumanTGF-α 8.4 9.3 10.6

HumanVEGF ND ND ND

ND=NotDetectable

biolegend.com 29


TROUBLESHOOTING

Problem Possible Cause Solution

Bead popula-tionshiftingupward or downward dur-ingacquisition

ThestrongPEsignalfrom 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

Vacuum pressure is insufficientorvacuummanifold 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,trythefollowing:

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.

Tel: 858-768-5800


30

Insufficientbead count or slow reading

Beads inappropriately prepared

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

Samples cause beads aggregationduetoparticulatematterorviscosity


Beads were lost during washing for in-tube assay

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

Vacuum pressure set too high

Adjust vacuum pressure such that 0.2 mL buffercanbesuctionedin3-5seconds.Donotexceed10”Hgofvacuum.

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.

biolegend.com 31


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.


Low or poor standard curve signal

Thestandardwasin-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.


Beadspopulationsarenot mixed properly

Makesureallbeadpopulationsaremixed.and in similar numbers.

Tel: 858-768-5800


32

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

Thestandardwasin-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.

biolegend.com 33


PLA

TE M

AP

(for

in-p

late

ass

ay)

1

2 3

4 5

6 7

8 9

10

11

12

A

C0

C4

Sa

mpl

e1

Sa

mpl

e5

Sa

mpl

e9

Sa

mpl

e 13

Sa

mpl

e17

Sa

mpl

e21

Sa

mpl

e25

Sa

mpl

e29

Sa

mpl

e33

Sa

mpl

e 37

B

C0

C4

Sa

mpl

e1

Sa

mpl

e5

Sa

mpl

e9

Sa

mpl

e 13

Sa

mpl

e17

Sa

mpl

e21

Sa

mpl

e25

Sa

mpl

e29

Sa

mpl

e33

Sa

mpl

e37

C

C1

C5

Sa

mpl

e2

Sa

mpl

e6

Sa

mpl

e10

Sa

mpl

e 14

Sa

mpl

e18

Sa

mpl

e22

Sa

mpl

e26

Sa

mpl

e30

Sa

mpl

e34

Sa

mpl

e38

D

C1

C5

Sa

mpl

e2

Sa

mpl

e6

Sa

mpl

e10

Sa

mpl

e 14

Sa

mpl

e18

Sa

mpl

e22

Sa

mpl

e26

Sa

mpl

e30

Sa

mpl

e34

Sa

mpl

e38

E

C2

C6

Sa

mpl

e3

Sa

mpl

e7

Sa

mpl

e11

Sa

mpl

e 15

Sa

mpl

e19

Sa

mpl

e23

Sa

mpl

e27

Sa

mpl

e31

Sa

mpl

e35

Sa

mpl

e39

F

C2

C6

Sa

mpl

e3

Sa

mpl

e7

Sa

mpl

e11

Sa

mpl

e 15

Sa

mpl

e19

Sa

mpl

e23

Sa

mpl

e27

Sa

mpl

e31

Sa

mpl

e35

Sa

mpl

e39

G

C3

C7

Sa

mpl

e4

Sa

mpl

e8

Sa

mpl

e12

Sa

mpl

e 16

Sa

mpl

e20

Sa

mpl

e24

Sa

mpl

e28

Sa

mpl

e32

Sa

mpl

e36

Sa

mpl

e40

H

C3

C7

Sa

mpl

e4

Sa

mpl

e8

Sa

mpl

e12

Sa

mpl

e 16

Sa

mpl

e20

Sa

mpl

e24

Sa

mpl

e28

Sa

mpl

e32

Sa

mpl

e36

Sa

mpl

e40

76319_R2

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

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

Enabling Legendary Discovery™

Documents

Human Growth Factor Panel R2 - LEGENDplex™