LEGENDplex™...LEGENDplex™ Mul˝-Analyte Flow Assay Kit Enabling Legendary Discovery™ For Accurate Quantification of Multiple uman Th (T helper Cell) Cytokines from Cell Culture

LEGENDplex™Mul�-Analyte Flow Assay Kit

Enabling Legendary Discovery™

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

Serum, Plasma and Other Biological Samples

Please read the entire manual before running the assay

BioLegend.com

LEGENDplex™Mul�-Analyte Flow Assay Kit


Human Proinflammatory Chemokine Panel 1

Mix and Match Subpanel

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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Human Proinflammatory Chemokine Panel 1 Mix and Match Subpanel

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

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

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

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

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

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

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

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

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

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

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

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

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

PLATE MAP....................................................................................

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Chapter 1: KIT DESCRIPTION

Introduction

Chemotacticcytokinesorchemokinesplaypivotalrolesinvariousprocessessuch as immune surveillance, organ development, angiogenesis, and immune responses.Expressionprofilingofchemokines,especiallythoseinvolvedininflammationandimmunedisorders,isimportantinachievingadeeperunder-standing of disease states.

TheHumanProinflammatoryChemokinePanel1isamultiplexbead-basedas-saypanel,usingfluorescence–encodedbeadssuitableforuseonvariousflowcytometers.Thispanelallowssimultaneousquantificationof13humanchemo-kines,including MCP-1 (CCL2), RANTES (CCL5), IP-10 (CXCL10), Eotaxin (CCL11), TARC(CCL17),MIP-1α(CCL3),MIP-1β(CCL4),MIG(CXCL9),MIP-3α(CCL20),ENA-78(CXCL5),GROα(CXCL1),I-TAC(CXCL11)andIL-8(CXCL8).ThisassaypanelprovideshigherdetectionsensitivitiesandbroaderdynamicrangesthantraditionalELISAmethods.Thepanelhasbeenvalidatedbydetectingexpectedchanges in biological samples.

TheLEGENDplex™HumanProinflammatoryChemokinePanel1(13-plex)isdesignedtoallowflexiblecustomizationwithinthepanel.Itcanalsobedividedintosubpanels.Thetablebelowshowsthepanelconfigurationandsamplediltuion requirement.

Cat # Plex Size Targets Sample Type Dilution Factor

740984,740985 13-plex

CCL2, CCL5, CXCL10, CCL11, CCL17, CCL3, CCL4, CXCL9, CCL20,

CXCL5, CXCL1, CXCL11, CXCL8

Tissue Culture Varies

741080, 741081 12-plex

CCL2, CXCL10, CCL11, CCL17, CCL3, CCL4,

CXCL9, CCL20, CXCL5, CXCL1, CXCL11, CXCL8


Serum/Plasma 2x

741082,741083 1-plex CCL5


Serum Plasma 50x

Please visit www.biolegend.com/legendplex formoreinformationonhowtomixandmatchwithinthepanel.

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Principle of the Assay

BioLegend’sLEGENDplexTM assays are bead-based immunoassays using the samebasicprincipleassandwichimmunoassays.

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

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

Beads Usage

TheHumanProinflammatoryChemokinePanelusestwosetsofbeads.Eachsethasauniquesizethatcanbeidentifiedbasedontheirforwardscatter(FSC)andsidescatter(SSC)profiles(BeadsAandBeadsB,Figure1).Eachbeadsetcanbefurtherresolvedbasedontheirinternalfluorescenceintensities.TheinternaldyecanbedetectedusingFL3,FL4,orAPCchannel,dependingonthetypeofflowcytometerused.ThesmallerBeadsAconsistsof6beadpopulationsandthelargerBeadsBconsistsof7beadpopulations(Figure2-3).

Usingatotalof13beadpopulationsdistinguishedbysizeandinternalfluores-centdye,theHumanProinflammatoryChemokinePanelallowssimultaneousdetectionof13chemokinesinasinglesample.Eachanalyteisassociatedwithaparticularbeadsetasindicated(Figures2-3andTable1).

Figure 1. Beads Differentiated by Size

Beads A = smaller beads

Beads B = larger bea

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Human Proinflammatory Chemokine Panel 1 Mix and Match Subpanel Figure 2. Beads A Classification by FL4

Figure 3. Beads B Classification by FL4

ForBeadsusageinthepanel,pleaserefertoTable1below:

Table 1. Beads ID*, Panel Target Selection and Top Standard Concentrations:

Target Bead ID

(13-Plex) (12-Plex) (1-Plex)Top Standard ConcentrationNo.740984,

740985No.741080,

741081No.741082,

741083

IL-8 A4 √ √ Note:Thetop standard concentrationsof analytes in thispanelwereset at various concentrations,but may be sub-ject to change from lot to lot

(please visit bio-legend.com/en-us/legendplex todownloadalot-specificcertificateof

analysis ).

IP-10 A5 √ √

Eotaxin A6 √ √

TARC A7 √ √

MCP-1 A8 √ √

RANTES A10 √ √

MIP-1α B2 √ √

MIG B3 √ √

ENA-78 B4 √ √

MIP-3α B5 √ √

GROα B6 √ √

I-TAC B7 √ √

MIP-1β B9 √ √

*BeadIDisusedtoassociateabeadpopulationtoaparticularanalytewhen

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using the LEGENDplex™dataanalysissoftwareprogram.Forfurtherinformationregarding the use of the program please visit biolegend.com/en-us/legendplex

Storage Information

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

• Oncethestandardshavebeenreconstituted,immediatelytransfercon-tentsintopolypropylenevials.DONOTSTORERECONSTITUTEDSTAN-DARDSINGLASSVIALS.

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

Materials Supplied

TheLEGENDplexTMkitcontainsreagentsfor100tests,listedinthetablebelow.Whenassayedinduplicate,thisisenoughforan8-pointstandardcurveand40samples.

For the Mix and Match Subpanels, individual beads are provided at 13X concen-tration.TheBufferSetcontainsSetupBeads,allBuffers,PlateSealers,Matrix,and SA-PE.

Kit Components Quantity Vol-ume Cat #

Capture Beads* (see tables below for more information) varies varies varies

LEGENDplex™HumanProinflammatoryChemokineDetectionAntibodies 1bottle 3.5 mL 740986

LEGENDplex™HumanProinflammatoryChemokineStandard 1 vial lyophi-

lized 740987

LEGENDplex™BufferSetA 1 740368Filter Plate* or V-bottomPlate** 1 Plate 740377*or

740379**

*Forkitwithfilterplate.**ForkitwithV-bottomplate.

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Human Proinflammatory Chemokine Panel 1 Mix and Match Subpanel Capture beads for Mix and Match Subpanels*:

Bead Name Quan-tity

Vol-ume Cat#

LEGENDplex™HumanCXCL8(IL-8)CaptureBeadA4,13X 1 vial 270 µL 740988

LEGENDplex™HumanCXCL10(IP-10)CaptureBeadA5,13X 1 vial 270 µL 740989

LEGENDplex™HumanCCL11(Eotaxin)CaptureBeadA6,13X 1 vial 270 µL 740990

LEGENDplex™HumanCCL17(TARC)CaptureBeadA7,13X 1 vial 270 µL 740991

LEGENDplex™HumanCCL2(MCP-1)CaptureBeadA8,13X 1 vial 270 µL 740813

LEGENDplex™HumanCCL5(RANTES)CaptureBeadA10,13X*

1 vial 270 µL 740992

LEGENDplex™HumanCCL3(MIP-1α)CaptureBeadB2,13X 1 vial 270 µL 740993

LEGENDplex™HumanCXCL9(MIG)CaptureBeadB3,13X 1 vial 270 µL 740994

LEGENDplex™HumanCXCL5(ENA-78)CaptureBeadB4,13X 1 vial 270 µL 740084

LEGENDplex™HumanCCL20(MIP-3α)CaptureBeadB5,13X 1 vial 270 µL 740995

LEGENDplex™HumanCXCL1(GROα)CaptureBeadB6,13X 1 vial 270 µL 740996

LEGENDplex™HumanCXCL11(I-TAC)CaptureBeadB7,13X 1 vial 270 µL 740997

LEGENDplex™HumanCCL4(MIP-1β)CaptureBeadB9,13X 1 vial 270 µL 740998

*For serum or plasma samples, mixing and matching of RANTES with other targets is not recommended due to high dilution requirement. A single plex is recommended for measuring RANTES in serum or plasma samples

LEGENDplex™ Buffer Set A (Cat#: 740368)

Components Quantity Volume Part #SetupBeads1:FITCBeads 1 vial 1 mL 77840SetupBeads2:PEBeads 1 vial 1 mL 77842SetupBeads3:RawBeads 1 vial 2 mL 77844LEGENDplexTM SA-PE 1bottle 3.5 mL 77743LEGENDplexTMMatrixB,Lyophilized 1 vial lyophilized 77549LEGENDplexTMAssayBuffer 1bottle 25 mL 77562LEGENDplexTMWashBuffer,20X 1bottle 25 mL 77564Plate Sealers 4sheets 78101

No plate is included in Buffer Set A. Plate need to be ordered separately. Please order the correct type of plate based on the preferred assay protocol (Cat# 740377 or 740378 for Filter Plate and Cat# 740379 for V-bottom Plate)

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Materials to be Provided by the End-User

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

Partial list of compatible flow cytometers:

Flow Cytometer

Reporter Channel

ChannelEmission

Classification Channel

Channel Emission

Compensa-tion needed?

BD FACSCaliburTM

(single laser)FL2 575 nm FL3 670 nm Yes

BD FACSCaliburTM

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

BD AccuriTM C6 FL2 585 nm FL4 675 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*

Beckman Coulter-CytoFLEX PE 585 nm APC 660 nm No*

Gallios PE 575 nm APC 660 nm No*

NovoCyte PE 572 nm APC 660 nm No*

*Compensation is not required for the specified flow cytometers when set up properly.

Forsettingupvariousflowcytometers,pleasevisit:www.biolegend.com/legendplex andclickontheInstrument Setup tab.

• Multichannelpipettescapableofdispensing5μLto200μL

• Reagentreservoirsformultichannelpipette

• Polypropylene microfuge tubes (1.5 mL)

• Laboratory vortex mixer

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

• Aluminum foil

• Absorbentpadsorpapertowels

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

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

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Human Proinflammatory Chemokine Panel 1 Mix and Match Subpanel • 1.1mLpolypropylenemicroFACStubes,in96-tuberack(e.g.,National

ScientificSupplyCo,catalog#TN0946-01R,orequivalent).

If the assay is performed in a filter plate;

• Avacuumfiltrationunit(MilliporeMultiScreen®HTSVacuumManifold,cat#MSVMHTS00orequivalent).Instructionsonhowtousethevacuummanifoldcanbefoundatthesupplier’swebsite.

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

• Ifneeded,additionalFilterplatecanbeorderedfromBioLegend(Cat#740377or740378).

If the assay is performed in a V-bottom plate;

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

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

Precautions

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

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

• MatrixBforLEGENDplexTMkitscontainscomponentsofhumanoriginandshouldbehandledaspotentiallyhazardous.TherawmaterialhasbeenscreenedforinfectiousdiseasesandisnegativeforHIV,HBVandHCVusingFDA-approved test methods.

• Donotmixorsubstitutereagentsfromdifferentkitsorlots.Reagentsfromdifferentmanufacturersshouldnotbeusedwiththiskit.

• Donotusethiskitbeyonditsexpirationdate.

• SA-PEandBeadsarelight-sensitive.Minimizelightexposure.

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Chapter 2: ASSAY PREPARATION

Sample Collection and Handling

Preparation of Serum Samples:

• Allowthebloodtoclotforatleast30minutesandcentrifugefor10min-utes at 1,000 x g.

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

• Whenusingfrozensamples,itisrecommendedthatsamplesarethawedcompletely,mixedandcentrifugedtoremoveparticulatespriortouse.

Preparation of Plasma Samples:

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

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

• Whenusingfrozensamples,itisrecommendedthatsamplesarethawedcompletely,mixedwellandcentrifugedtoremoveparticulates.

Preparation of Tissue Culture Supernatant:

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

Reagent Preparation

Preparation of Antibody-Immobilized Beads

Theindividualbeads(13X)shouldbemixedwitheachotheranddilutedto1XfinalconcentrationwithAssayBufferpriortouse.Tomixthebeads,followthestepsbelow(a5-plexsubpanelisusedasanexample):

1. Sonicate each bead vial for 1 minute in a sonicator bath and then vortex for 30 seconds to completely resuspend the beads

2. Calculate the amount of mixed and diluted beads needed for the assay. Prepareextratocompensateforpipettingloss.Eachreactionneeds25µLofmixedanddilutedbeads.For50reactions,prepare1.5mLofmixedbeads.For100reactions,prepare3mLofmixedbeads.

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Human Proinflammatory Chemokine Panel 1 Mix and Match Subpanel 3.Tomake1.5mlof5-plex1Xdilutedbeads,transfer115µLofeachof

the 5 individual beads (13X) to a fresh tube (total bead volume = 575 µL)andadd925µLofAssayBuffertomakethefinalvolumeof1.5 mL.

Preparation of Wash Buffer

• Bringthe20XWashBuffertoroomtemperatureandmixtobringallsaltsintosolution.

• Dilute25mLof20XWashBufferwith475mLdeionizedwater.Storeun-usedportionsbetween2°Cand8°Cforuptoonemonth.

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

• Add5.0mLLEGENDplexTMAssayBuffertothebottlecontaininglyophilized MatrixB.Allowatleast15minutesforcompletereconstitution.Vortexto

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

Standard Preparation

1. Priortouse,reconstitutethelyophilizedHumanProinflammatoryChemo-kinePanel1StandardCocktailwith250µLAssayBuffer.

2. Mixandallowthevialtositatroomtemperaturefor10minutes,andthen transfer the standard to an appropriately labeled polypropylene microfugetube.ThiswillbeusedasthetopstandardC7.

Note: The top standard concentrations of analytes in this panel were set at various concentrations, but may be subject to change from lot to lot (please visit biolegend.com/en-us/legendplex to download a lot-specific certificate of analysis ).

3. Label6polypropylenemicrofugetubesasC6,C5,C4,C3,C2andC1,respectively.

4.Add75µLofAssayBuffertoeachofthesixtubes.Prepare1:4dilutionofthetopstandardbytransferring25µLofthetopstandardC7totheC6tubeandmixwell.ThiswillbetheC6standard.

5. Inthesamemanner,performserial1:4dilutionstoobtainC5,C4,C3,C2and C1 standards (see the table below using 10ng/mL of top standard as an example).AssayBufferwillbeusedasthe0pg/mLstandard(C0).

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Tube/Standard ID

Serial Dilution

Assay Buffer to add (µL)

Standard to add

Final Conc. (pg/mL)

C7 -- -- -- 10,000

C6 1:4 75 25 µL of C7 2,500

C5 1:16 75 25 µLofC6 625

C4 1:64 75 25 µL of C5 156.3

C3 1:256 75 25 µLofC4 39.1

C2 1:1024 75 25 µL of C3 9.8

C1 1:4096 75 25 µL of C2 2.4

C0 -- 75 -- 0

Sample Dilution

• For measuring serum or plasma samples for all targets except RANTES, the samplesneedtobediluted2-foldwithAssayBufferbeforebeingtested(e.g.dilute50µLofsamplewith50µLofAssayBuffer).Iffurthesampledilutionisneeded,thesamplesshouldbediutedwithMatrixBprovidedinthekit.

• Formeasuringserumorplasmasamplesuisngthe1-plexRANTESkit,a50-folddilutionusingAssayBufferisrecommendedduetothehighconcentra-tionofRANTESinsamples.Iffurthesampledilutionisneeded,thesamplesshouldbediutedwithAssayBufferprovidedinthekit.

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

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

Ifsampledilutionisdesired,dilutionshouldbedonewithcorrespondingfreshcellculturemediumorAssayBuffertoensureaccuratemeasurement.

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Chapter 3: ASSAY PROCEDURE

TheLEGENDplexTM assaycanbeperformedinafilterplate,orinaV-bottomplate.

• Thein-filterplateassayprocedurerequiresavacuumfiltrationunitforwashing(seeMaterials to be Provided by the End-User, page 8).

• Ifthein-filterplateassayprocedureisnotpossibleorifyouprefer,theas-saycanbeperformedinaV-bottomplate.

Performing the Assay Using a Filter Plate

• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.

• Setthefilterplateonaninvertedplatecoveratalltimesduringassaysetupandincubationsteps,sothatthebottomoftheplatedoesnottouchanysurface.Touchingasurfacemaycauseleakage.

• Keeptheplateuprightduringtheentireassayprocedure,includingthewashingsteps,toavoidlosingbeads.

• Theplateshouldbeplacedinthedarkorwrappedwithaluminumfoilforallincubationsteps.

• Standards and samples should be run in duplicate and arranged on the plate in a vertical configuration convenient for data acquisition and analysis (as shown in attached PLATE MAP, page 33). Be sure to load stan-dards in the first two columns. If an automation device is used for read-ing, the orientation and reading sequence should be carefully planned.

1. Pre-wettheplatebyadding100μLofLEGENDplexTM1XWashBuffertoeachwellandletitsitfor1minuteatroomtemperature.Toremovetheexcess volume, place the plate on the vacuum manifold and apply vacuum. Donotexceed10”Hgofvacuum.Vacuumuntilwellsaredrained(5-10seconds).BlotexcessWashBufferfromthebottomoftheplatebypressingtheplateonastackofcleanpapertowels.Placetheplateontopoftheinverted plate cover.

For measuring cell culture supernatant samples, loadtheplateasshowninthetablebelow(intheorderfromlefttoright):

AssayBuffer Matrix B Standard Sample*

StandardWells 25 µL --- 25 µL ---

Samplewells 25 µL --- --- 25 µL

For measuring serum/plasma samples for all targets except RANTES, load theplateasshowninthetablebelow(intheorderfromlefttoright):

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StandardWells --- 25 µL 25 µL ---


For measuring serum/plasma samples for RANTES, load the plate as showninthetablebelow(intheorderfromlefttoright):




*See Sample Dilution

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

3. Sealtheplatewithaplatesealer.To avoid plate leaking, do not apply posi-tive pressure to the sealer when sealing the plate.Wraptheentireplate,includingtheinvertedplatecover,withaluminumfoil.Placetheplateonaplateshaker,secureitwitharubberbandandshakeatapproximate500rpm for 2 hours at room temperature.

4. Do not invert the plate! Place the plate on the vacuum manifold and apply vacuumasbeforeinStep1.Add200µLof1XWashBuffertoeachwell.RemoveWashBufferbyvacuumfiltration.BlotexcessWashBufferfromthebottomoftheplatewithanabsorbentpadorpapertowels. Repeat this washingsteponcemore.

5. Add25µLofDetectionAntibodiestoeachwell.

6. Sealtheplatewithafreshplatesealer.Wraptheentireplate,includingtheinvertedplatecover,withaluminumfoil.Placetheplateonaplateshakerandshakeatapproximately500rpmfor1houratroomtemperature.

7. Do not vacuum!Add25µLofSA-PEtoeachwelldirectly.

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

9. Repeatstep4above.

10. Add150µLof1XWashBuffertoeachwell.Resuspendthebeadsonaplateshakerfor1minute.

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11. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay(Note:Prolongedsamplestoragecanleadtoreducedsignal).

Iftheflowcytometerisequippedwithanautosampler,readtheplatedi-rectly using the autosampler. Please be sure to program the autosampler to resuspend beads in the well immediately before taking samples. The probe height may need to be adjusted when using an autosampler.

If an autosampler is not available, the samples can be transferred fromthefilterplateto micro FACS (or FACS) 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

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Performing the Assay Using a V-bottom Plate

• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.

• Keeptheplateuprightduringtheentireassayprocedure,includingthewashingsteps,toavoidlosingbeads.

• Theplateshouldbeplacedinthedarkorwrappedwithaluminumfoilforallincubationsteps.

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

1. For measuring cell culture supernatant samples, loadtheplateasshowninthetablebelow(intheorderfromlefttoright):



Samplewells 25 µL --- --- 25 µL For measuring serum/plasma samples for all targets except RANTES, load

theplateasshown inthetablebelow(intheorderfromlefttoright):AssayBuffer Matrix B Standard Sample*

StandardWells --- 25 µL 25 µL ---


For measuring serum/plasma samples for RANTES, load the plate as showninthetablebelow(intheorderfromlefttoright):




*See Sample Dilution

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

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need to be adjusted. The optimal speed is one that is high enough to keep beads in suspension during incubation, but not too high so it causes 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 8). Do not use excessive centrifuga-tionspeedasitmaymakeithardertoresuspendbeadsinlatersteps.Make sure the timer of the centrifuge works properly and standby to make sure the centrifuge reaches preset speed.

5. Immediatelyaftercentrifugation,dumpthesupernatantintoasinkbyquicklyinvertingandflickingtheplatein one continuous and forceful mo-tion.Donotworryaboutlosingbeadsevenifthepelletisnotvisible.Thebeadswillstayinthetipofthewellnicely.Blottheplateonastackofcleanpapertowelanddraintheremainingliquidfromthewellasmuchaspos-sible. Be careful not to disturb the bead pellet.

Alternatively,removalofthesupernatantmaybecompletedusingamultichannelpipettesetat75µL. Try to remove as much liquid as possible withoutremovinganybeads. Be sure to changepipettetipsbetweeneachroworcolumn.

6. Washtheplatebydispensing200μLof1XWashBufferintoeachwellandincubate for one minute. Repeatstep4and5above.Asecondwashisoptional,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. Repeatstep4,and5.

12. Washtheplatebydispensing200μLof1XWashBufferintoeachwellandincubate for one minute. Repeatstep4and5above.Thiswashingstepisoptionalbuthelpstoreducethebackground.

13. Add150µLof1XWashBuffertoeachwell.Resuspendthebeadsbypipet-ting.

14. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay(Note:Prolongedsamplestoragecanleadtoreducedsignal).

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Iftheflowcytometerisequippedwithanautosampler,thesamplescanberead directly. Please be sure to program the autosampler to resuspend beads in the well immediately before taking samples. The probe height may need to be adjusted when using an autosampler.

If an autosampler is not available, the samples can be transferred from the plate to micro FACS (or FACS) tubes and read manually.

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

C

A B C

A B C

biolegend.com 19


Chapter 4: FLOW CYTOMETER SETUP

Inordertogeneratereliabledata,theflowcytometermustbesetupproperlybeforedataacquisition.

Thesetupinstructionshavebeenremovedfromthismanualanduploadedontoourwebsitetosavepaper.

Toaccessthesetupinstructions,pleasevisit:www.biolegend.com/legendplex andclickontheInstrument Setup tab.

Chapter 5: DATA ACQUISITION AND ANALYSIS

Data Acquisition

1. Beforereadingsamples,makesurethattheflowcytometerissetupprop-erly.

2. Createanewtemplateoropenanexistingtemplate(fordetailsonhowtocreateacytometer-specifictemplate,pleaserefertotheFlowCytometerSetupGuide).

3. Vortex each sample for 5 seconds before analysis.

4.Settheflowratetolow.Setthenumberofbeadstobeacquiredtoabout300peranalyte(e.g.,acquire2,400beadsfora8-plexassayor4000beadsfor a 13-plex assay). Do not set to acquire total events as samples may contain large amounts of debris. Instead, create a large gate to include both Beads A and Beads B (gate A+B) and set to acquire the number of events in gateA+B.Thiswillexludemajorityofthedebris.

Note:Donotacquiretoofewortoomanybeads.ToofewbeadsacquiredmayresultinhighCVsandtoomanybeadsacquiredmayresultinslowdata analysis later.

5. Read samples.

Whenreadingsamples,settheflowcytometertosetupmodefirstandwaituntilbeadpopulationisstabilizedbeforerecordingorswitchingtoacquisi-tionmode.

TosimplifydataanalysisusingtheLEGENDplexTMDataAnalysisSoftware,readsamplesinthesameorderasshownonthePLATEMAPattachedatthe end of the manual. For an in-plate assay, read column by column (A1,

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B1, C1...A2, B2, C2...).

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

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

6.ProceedtodataanalysisusingLEGENDplexTMDataAnalysisSoftwarewhendataacquisitioniscompleted.

Data Analysis

TheassayFCSfilesshouldbeanalyzedusingBioLegend’sLEGENDplex™dataanalysissoftware.Theprogramisofferedfreeofchargewith the purchase of any LEGENDplex™assay.Forfurtherinformationregardingacccessto,anduseofthe program please visit biolegend.com/en-us/legendplex.

biolegend.com 21


Chapter 6: ASSAY CHARACTERIZATION

Representative Standard Curve

ThisstandardcurvewasgeneratedusingtheLEGENDplexTMHumanProinflammatoryChemokinePanel1fordemonstrationpurposeonly.Astandardcurvemustberunwitheachassay.

5

50

500

5000

1 10 100 1000 10000

MFI

Concentration (pg/mL)

IL-8 (A4)IP-10 (A5)Eotaxin (A6)TARC (A7)MCP-1 (A8)RANTES (A10)MIP-1α (B2)MIG (B3)ENA-78 (B4)MIP-3α (B5)GROα (B6)I-TAC (B7)MIP-1β (B9)

Assay Sensitivity

Theminimumdetectableconcentration(MDC)isthetheoreticallimitofdetectioncalculatedusingtheLEGENDplexTMDataAnalysisSoftwarebyapplyinga5-parametercurvefittingalgorithm.Assaysensitivitypresentedhere is MDC + 2 STDEV.

Analyte MDC in Cell Culture Medium (pg/mL)

MDC in Serum (pg/mL)

HumanIL-8 3.1+3.9 3.4+1.7

HumanIP-10 1.3+0.9 1.2+0.9

HumanEotaxin 1.7+0.4 2.5+4.8

HuamnTARC 0.9+1.0 1.0+0.9

HumanMCP-1 2.1+1.0 1.4+1.4

HumanRANTES 0.4+0.2 0.3+0.3

HumanMIP-1α 4.7+3.1 3.8+2.6

HumanMIG 0.3+0.2 0.5+0.2

HumanENA-78 0.7+0.3 0.6+0.6

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HumanMIP-3α 0.4+0.6 0.4+0.4

HumanGROα 0.6+0.3 0.5+0.4

HumanI-TAC 0.4+0.2 0.4+0.3

HumanMIP-1β 0.3+0.1 0.3+0.3

Cross-Reactivity

Thefollowinghumanrecombinantproteinsweretestedat50ng/mLusingtheLEGENDplexTM HumanProinflammatoryChemokinePanel1.CXCL2andCXCL3showedacrossof4%and5%withCXCL1,respectively. No or negligiblecross-reactivitywasfoundbetweenallotheranalytes.

CCLL22 IFN-y CCL28 CX3CL1 CCL14 IL-17A

CCL23 CXCL7 IL-10 IL-2 CCL13 IL-15

CCL24 CXCL14 CCL18 IL-1R CCL8 IL-13

CCL25 CXCL13 CXCL6 IL-1B CCL15 IL-12

CCL26 CXCL16 CCL1 IL-10 CCL19

CCL27 CXCL17 CCL7 IL-17F CXCL4

Accuracy (Spike Recovery)

Forspikerecoveryincellculturemedium,targetproteinswithknownconcentrationswerespikedintocellculturemedium(RPMIandDMEMwith10%FCS)atthreedifferentlevelswithintheassayrange.Thespikedsampleswerethenassayed,andthemeasuredconcentrationswerecom-paredwithexpectedvalues.

Forspikerecoveryinserum,asamplewithknownhighconcentrationsoftargetproteinswasspikedintounknownserumsamples.Thespikedsampleswerethenassayed,andthemeasuredconcentrationswerecom-paredwithexpectedvalues.

Analyte % of Recovery in Cell Culture Medium

% of Recovery in Serum

HumanIL-8 102.74% 92.29%HumanIP-10 111.97% 92.34%HumanEotaxin 111.45% 93.98%HuamnTARC 116.74% 88.70%

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HumanMCP-1 113.75% 118.98%HumanRANTES 111.75% 112.80%HumanMIP-1α 111.22% 97.52%HumanMIG 126.32% 167.83%HumanENA-78 109.71% 127.79%HumanMIP-3α 118.13% 93.29%HumanGROα 111.34% 150.36%HumanI-TAC 114.68% 109.47%HumanMIP-1β 115.77% 109.23%

Linearity of Dilution

Fortestinglinearityofdilution,serumsampleswerefirstdilutedtwo-foldwithAssayBuffer,thenseriallydiluted1:2,1:4,1:8withMatrixBandas-sayed.Themeasuredconcentrationsofseriallydilutedsampleswerethencomparedwiththatofthetwo-folddilutedsamples.

Analyte Linearity of Dilution Analyte Linearity of

DilutionHumanIL-8 133.7% HumanMIG 160.0%

HumanIP-10 121.3% HumanENA-78 114.4%

HumanEotaxin 157.2% HumanMIP-3α 113.9%

HuamnTARC 123.5% HumanGROα 98.6%

HumanMCP-1 107.8% HumanI-TAC 91.6%

HumanRANTES 110.1% HumanMIP-1β 117.1%

HumanMIP-1α 108.9%

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Intra-Assay Precision

Twosampleswithdifferentconcentrationsoftargetproteinswereanalyzedinoneassaywith16replicatesforeachsample.Theintra-assayprecisionwascalculatedasbelow.

Analyte Sample Mean (pg/mL) STDEV %CV

HumanIL-8Sample 1 379.3 15.4 4.1%

Sample 2 1204.5 62.9 5.2%

HumanIP-10Sample 1 83.3 1.7 2.1%

Sample 2 264.4 7.6 2.9%

HumanEotaxinSample 1 55.9 1.7 3.0%

Sample 2 217.6 6.6 3.0%

HumanTARCSample 1 104.2 4.5 4.3%

Sample 2 323.7 13.1 4.1%

HumanMCP-1Sample 1 89.5 2.3 2.6%

Sample 2 326.2 8.1 2.5%

HumanRANTESSample 1 19.8 0.6 3.1%

Sample 2 65.8 2.0 3.0%

HumanMIP-1αSample 1 286.2 7.7 2.7%

Sample 2 1027.8 20.6 2.0%

HumanMIGSample 1 51.6 2.7 5.2%

Sample 2 183.9 14.3 7.8%

HumanENA-78Sample 1 70.0 3.2 4.6%

Sample 2 264.4 9.3 3.5%


Sample 2 94.5 3.3 3.5%

HumanGROαSample 1 46.9 1.3 2.8%

Sample 2 135.9 4.7 3.4%

HumanI-TACSample 1 27.6 0.8 2.7%

Sample 2 96.1 3.9 4.1%

HumanMIP-1βSample 1 40.5 1.8 4.5%

Sample 2 110.5 4.7 4.3%

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Inter-Assay Precision

Twosampleswithdifferentconcentrationsoftargetproteinswereana-lyzedinfiveindependentassaysbyfivedifferentpeople.Theinter-assayprecisionwascalculatedasbelow.

Analyte Sample Mean (pg/mL) STDEV %CV

HumanIL-8Sample 1 373.5 64.6 17.3%

Sample 2 1401.2 195.5 14.0%

HumanIP-10Sample 1 117.3 16.2 13.8%

Sample 2 438.4 29.2 6.7%

HumanEotaxinSample 1 62.3 13.2 21.2%

Sample 2 247.4 40.3 16.3%

HumanTARCSample 1 108.3 12.6 11.6%

Sample 2 396.2 51.9 13.1%

HumanMCP-1Sample 1 92.3 16.2 17.6%

Sample 2 365.5 50.6 13.9%

HumanRANTESSample 1 20.6 3.0 14.7%

Sample 2 73.1 6.9 9.4%


Sample 2 1131.7 138.4 12.2%

HumanMIGSample 1 106.5 20.7 19.5%

Sample 2 489.1 90.7 18.5%

HumanENA-78Sample 1 76.0 14.7 19.3%

Sample 2 334.2 44.5 13.3%


Sample 2 120.4 14.7 12.2%

HumanGROαSample 1 48.8 7.9 16.3%

Sample 2 170.0 10.6 6.3%

HumanI-TACSample 1 38.9 6.8 17.6%

Sample 2 161.9 17.8 11.0%

HumanMIP-1βSample 1 38.3 6.1 15.9%

Sample 2 128.3 12.0 9.3%

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Biological Samples

Serum and Plasma (Samples are not paired.)

Normalhumanserumsamples(n=20)weretestedforendogenouslevelsoftheChemokines.Theconcentrationsmeasuredareshownbelow:

Analyte Range (pg/ml)

No. of Detectable

% of Detectable

Mean (pg/mL)

HumanIL-8 ND-458 17 85% 88

HumanIP-10 48-996 20 100% 167

HumanEotaxin ND-524 18 90% 110

HuamnTARC 74-1786 20 100% 423

HumanMCP-1 76-538 20 100% 303

HumanRANTES 6907-17093 20 100% 8753

HumanMIP-1α ND-167 7 35% 24

HumanMIG 26-223 20 100% 62

HumanENA-78 112-1083 20 100% 362

HumanMIP-3α ND-33 17 85% 11

HumanGROα 24-628 20 100% 229

HumanI-TAC ND-367 19 95% 47

HumanMIP-1β 7--66 20 100% 23

ND = Non-detectable

Normalhumanplasmasamples(n=60))weretestedforendogenouslevelsofchemokines.Theconcentrationsmeasuredareshownbelow:

Analyte Range (pg/mL)

No. of Detectable

% of Detectable

Mean (pg/mL)

HumanIL-8 ND-2704 54 90% 273

HumanIP-10 13-1700 60 100% 168

HumanEotaxin ND-18866 53 88% 513

HuamnTARC 3-253 60 100% 75

HumanMCP-1 37-351 60 100% 141

HumanRANTES 671-11294 60 100% 3704

HumanMIP-1α ND-924 32 53% 160

HumanMIG 8-491 60 100% 75

HumanENA-78 6-326 60 100% 68

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HumanMIP-3α ND-193 48 80% 27

HumanGROα ND-359 52 87% 59

HumanI-TAC ND-1356 58 97% 95

HumanMIP-1β ND-57 56 93% 6

ND = Non-detectable

Cell Culture Supernatant

HumanPBMC(1x106cells/mL)wereculturedundervariousconditions(LPS,1ug/mL;LPS,1ug/mL;IFN-y,100ng/mL;CD3,1µg/mLplate-coated;CD28,1µg/mLsoluble;).Supernatantswerecollectedafter48hoursandassayedwiththeLEGENDplexTMHumanProinflammatoryChemokinePanel1kit.Theresults(allinpg/mL)aresummarizedbelow.

Analyte Control CD3+CD28 LPS LPS+IFN-y

HumanIL-8 886 138502 50046 29338

HumanIP-10 1747 4218 ND 154

HumanEotaxin 3 119 40 39

HuamnTARC 13 3818 30 46

HumanMCP-1 2859 6253 9045 2297

HumanRANTES 225 2626 433 557

HumanMIP-1α 35 35736 4192 4323

HumanMIG 692 13299 ND 186

HumanENA-78 118 1295 3089 87

HumanMIP-3α ND 826 42 21

HumanGROα 70 3105 3159 1152

HumanI-TAC 2 ND ND ND

HumanMIP-1β 31 9157 971 987

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TROUBLESHOOTING

Problem Possible Cause Solution

Bead popula-tionshiftingupwardordownwarddur-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.

Filterplatewillnot vacuum orsomewellsclogged

Vacuum pressure is insufficientorvacuummanifold does not seal properly.

Increase vacuum pressure such that 0.2mLbuffercanbesuctionedin3-5seconds. Clean the vacuum manifold and makesurenodebrisonthemanifold.Pressdowntheplateonthemanifoldtomakeagoodseal.

Samples have insoluble particlesorsampleistoo viscous (e.g., serum and plasma samples)

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

Ifsomewellsarestillcloggedduringwashing,trythefollowing:

1).Addbuffertoallthewells,pipetteupanddownthecloggedwellsandvacuumagain.

2).Useapieceofcleanwipe,wipetheun-dersideofthecloggedwellsandvacuumagain.

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

Filterplatewasusedwithoutpre-wet.

Pre-wetplatewithwashbufferbeforerunning the assay.

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Insufficientbead count or slowreading

Beads inappropriately prepared

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

Samples cause beads aggregationduetoparticulatematterorviscosity.


Beadswerelostduringwashingforin-tubeassay

Makesurebeadsarespundownbyvisu-allycheckthepellet(beadsareinlightblueorbluecolor).Beverycarefulwhenremovingsupernatantduringwashing.

Probe might be par-tiallyclogged.

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 tableorabsorbenttow-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) during sample acquisi-tion

Debris or platelet may exist in sample solu-tion.

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.

Platewashingwasnotuniform

Makesureallreagentsarevacuumedoutcompletelyinallwashsteps.

Samples may contain particulatematters.


Loworpoorstandard curve signal

Thestandardwasin-correctlyreconstituted,stored or diluted

Followtheprotocoltoreconstitute,storeanddilutestandard.Doublecheckyourcalculation.

Wrongorshortincuba-tiontime

Ensurethetimeofallincubationswasappropriate.

Signals too high, standard curves satu-rated

PMT value for FL2/PE set too high

MakesurethePMTsettingforthere-porter channel is appropriate

Plateincubationtimewastoolong Useshorterincubationtime.

Sample read-ings are out of range

Samples contain no or belowdetectablelevelsof analyte

Makesuretheexperimenttogeneratethesamplesworked.Useproperpositivecontrols.

Samplesconcentrationshigher than highest standard point.

Dilutesamplesandanalyzeagain.

Standardcurvewassaturated 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.

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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.

Platewashingwasnotuniform

Makesureallreagentsarevacuumedoutcompletelyinallwashsteps.

Samples may contain particulatematters.


Loworpoorstandard curve signal

Thestandardwasin-correctlyreconstituted,stored or diluted

Followtheprotocoltoreconstitute,storeanddilutestandard.Doublecheckyourcalculation.

Wrongorshortincuba-tiontime

Ensurethetimeofallincubationswasappropriate.

Signals too high, standard curves satu-rated

PMT value for FL2/PE set too high

MakesurethePMTsettingforthere-porter channel is appropriate

Plateincubationtimewastoolong Useshorterincubationtime.

Sample read-ings are out of range

Samples contain no or belowdetectablelevelsof analyte

Makesuretheexperimenttogeneratethesamplesworked.Useproperpositivecontrols.

Samplesconcentrationshigher than highest standard point.

Dilutesamplesandanalyzeagain.

Standardcurvewassaturated at higher end of curve.

MakesurethePMTsettingforthere-porter channel is appropriate. Use shorter incubationtimeifincubationtimewastoolong

Missed beads populationsduring reading, ordistributionis unequal

Sample may cause some beads to ag-gregate.


Beadspopulationsarenot mixed properly

Makesureallbeadpopulationsaremixed.and in similar numbers.

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biolegend.com 33


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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


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Documents

LEGENDplex™...LEGENDplex™ Mul˝-Analyte Flow Assay Kit Enabling Legendary Discovery™ For Accurate Quantification of Multiple uman Th (T helper Cell) Cytokines from Cell Culture