LEGENDplex™ · LEEDple Mouse Cytokine Release Syndrome Mi and Match Subpanel Chapter 1: KIT DESCRIPTION Introduction Cytokine release syndrome is a systemic inflammatory response

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

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LEGENDplex™Mul�-Analyte Flow Assay Kit


Mouse Cytokine Release Syndrome 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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LEGENDplex™ Mouse Cytokine Release Syndrome 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……………………………………...................

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

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

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

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Chapter 1: KIT DESCRIPTIONIntroduction

CytokinereleasesyndromeisasystemicinflammatoryresponsesyndromecausedbyadverseeffectsofmonoclonalorCARTcellimmunotherapy.CRScanalsoariseasacomplicationduringinfectioninresponsetosepsis.Regardlessofitsinitialetiology,CRSischaracterizedbyanauto-amplifyingcascadeofleukocyteactivationwhichdrivestheproductionandreleaseofseveralinflammatorybiomarkers.

TheLEGENDplexTMMouseCytokineReleaseSyndromePanel(13-plex)isabead-basedmultiplexassaypanel,usingfluorescence-encodedbeadssuitableforuseonvariousflowcytometers.Itallowsforsimultaneousquantificationof13keybiomarkers involved in cytokine release syndrome such as IFN-γ,IL-10,CCL4(MIP-1β),IFN-α,CXCL9(MIG),CXCL10(IP-10),TNF-α,IL-6,VEGF,IL-4,CCL3(MIP-1α),CCL2(MCP-1),andGM-CSF.ThisassaypanelprovideshigherdetectionsensitivityandbroaderdynamicrangethantraditionalELISAmethods.Thepanelhasbeenvalidatedforuseoncellculturesupernatant,serum,andplasmasamples.

TheMouseCytokineReleaseSyndromePanelisdesignedtoallowflexiblecustomizationwithinthepanel.Pleasevisitwww.biolegend.com/legendplex formoreinformationonpaneldesignandhowtomixandmatchwithinthepanel.

Thisassayisforresearchuseonly

Principle of the Assay

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

Beadsaredifferentiatedbysizeandinternalfluorescenceintensities.Thesur-faceofeachbeadsetisfirstconjugatedwithspecificantibodies,andthenusedascapturebeadsforthatparticularanalyte.Whenaselectedpanelofcapturebeadsaremixedandincubatedwithasamplecontainingtargetanalytes,eachanalytewillbindtoitsspecificcapturebeads.Afterwashing,abiotinylatedde-tectionantibodycocktailisadded,andeachdetectionantibodyinthecocktailwillbindtoitsspecificanalyteboundonthecapturebeads,thusformingcap-turebead-analyte-detectionantibodysandwiches.Streptavidin-phycoerythrin(SA-PE)issubsequentlyadded,whichwillbindtothebiotinylateddetectionantibodies,providingfluorescentsignalintensitiesinproportiontotheamountof bound analytes.

Sincethebeadsaredifferentiatedbysizeandinternalfluorescenceintensityonaflowcytometer,analyte-specificpopulationscanbesegregatedandPE

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fluorescentsignalquantified.Theconcentrationofaparticularanalyteisdeter-mined using a standard curve generated in the same assay.

Beads Usage

TheMouse Cytokine Release Syndrome Panelusestwosetsofbeads.Eachsethasauniquesizethatcanbeidentifiedbasedontheirforwardscatter(FSC)andsidescatter(SSC)profiles(BeadsAandBeadsB,Figure1).Eachbeadsetcanbefurtherresolvedbasedontheirinternalfluorescenceintensities.TheinternaldyecanbedetectedusingeithertheFL3,FL4,orAPCchannels,dependingonthetypeofflowcytometerused.ThesmallerABeadsconsistsof6beadpopulations(A4,A5,A6,A7,A8,A10)andthelargerBBeadsconsistsof7beadpopulations(B2,B3,B4,B5,B6,B7,B9)(Figure2-3).Usingatotalof13beadpopulationsdistinguishedbysizeandinternalfluores-centdye,theMouse Cytokine Release Syndrome Panel allowssimultaneousdetectionof13proteinsinasinglesample.Eachanalyteisassociatedwithaparticularbeadsetasindicated(Figures2-3andTable1).

Figure 1. Beads Differentiated by Size

Beads A = smaller beads

Beads B = larger beads

Figure 2. Beads A Classification by FL4

A5 A7 A8

A4

A6

A10

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Figure 3. Beads B Classification by FL4

ForBeadsusageinthefullpanel,pleaserefertoTable1below.

Table 1. Panel Targets and Bead ID*

Target Bead ID Top Standard Concentrations

IFN-γ A4

Thetopstandardconcentration

ofeachtargetmayvaryandmaysubject

to change from lot to lot. Please refer to

thelot-specificCertificateofAnalysisfor

thisinformation.

IL-10 A5

CCL4 (MIP-1β) A6

IFN-α A7

CXCL9 (MIG) A8

CXCL10 (IP-10) A10

TNF-α B2

IL-6 B3

VEGF B4

IL-4 B5

CCL3 (MIP-1α) B6

CCL2 (MCP-1) B7

GM-CSF B9

*BeadIDisusedtoassociateabeadpopulationtoaparticularanalyteintheLEGENDplexTMDataAnalysisSoftware.TheassociationofanalyteandbeadIDwillbedefinedduringthegatingstepofthedataanalysis.

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

B4 B5

B6 B7

B3

B9

B2

Tel: 858-768-58006


Storage Information

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

• Oncethestandardshavebeensufficientlyreconstituted,immediatelytransfercontentsintopolypropylenevials.DONOTSTORERECONSTITUT-EDSTANDARDSINGLASSVIALS.

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

Materials Supplied

TheLEGENDplexTMkitcontainsreagentsfor100tests,listedinthetablebelow.Whenassayedinduplicate,thisisenoughforan8-pointstandardcurveand40samples.

FortheMixandMatchSubpanels,individualbeadsareprovidedat13Xconcen-tration.TheBufferSetcontainsSetupBeads,allBuffers,PlateSealers,Matrix,SA-PEandDataAnalysisSoftwareDongle.AmanualisalsoprovidedforeachMixandMatchsubpanel.

Kit Components Quantity Volume Cat #Capture Beads* (see tables below for more information) Varies Varies Varies

Mouse Cytokine Release Syndrome PanelDetectionAntibodies 1bottle 3.5mL 741025

Mouse Cytokine Release Syndrome Panel Standard 1 vial Lyophilized 741026

LEGENDplexTMBufferSetI 1 -- 740623

Filter Plate* or V-bottomPlate** 1 plate -- 740377*or

740379**

Mouse Cytokine Release Syndrome PanelMixandMatchSubpanelManual 1 -- 750001064

*Forkitwithfilterplate.**ForkitwithV-bottomplate.Onlyoneplateispro-vided for each kit.

Capture beads for Mix and Match SubpanelsBead Name Quantity Volume Cat #

LEGENDplex™MouseIFN-γCaptureBead,A4,13X 1 vial 270μL 740065

LEGENDplex™MouseIL-10CaptureBeadA5,13X 1 vial 270μL 741013

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LEGENDplex™MouseCCL4(MIP-1β)CaptureBeadA6,13X 1 vial 270μL 741014

LEGENDplex™MouseIFN-αCaptureBeadA7,13X 1 vial 270μL 741015

LEGENDplex™MouseCXCL9(MIG)Cap-tureBeadA8,13X 1 vial 270μL 741016

LEGENDplex™MouseCXCL10(IP-10)CaptureBeadA10,13X 1 vial 270μL 741017

LEGENDplex™MouseTNF-αCaptureBeadB2,13X 1 vial 270μL 741018

LEGENDplex™MouseIL-6CaptureBeadB3,13X 1 vial 270μL 740860

LEGENDplex™MouseVEGFCaptureBeadB4,13X 1 vial 270μL 741019

LEGENDplex™MouseIL-4CaptureBeadB5,13X 1 vial 270μL 741020

LEGENDplex™MouseCCL3(MIP-1α)CaptureBeadB6,13X 1 vial 270μL 741021

LEGENDplex™MouseCCL2(MCP-1)CaptureBeadB7,13X 1 vial 270μL 741012

LEGENDplex™MouseGM-CSFCaptureBead,B9,13X 1 vial 270μL 740163

LEGENDplexTM Buffer Set I (Cat#: 740623)Components Quantity Volume Part #

SetupBeads1:FITCBeads 1 vial 1 mL 77840

Setup Beads 2: PE Beads 1 vial 1 mL 77842

SetupBeads3:RawBeads 1 vial 2 mL 77844

LEGENDplexTM SA-PE 1bottle 3.5mL 77743

LEGENDplexTMMatrixA,Lyophilized 1 vial Lyophilized 75306

LEGENDplexTMAssayBuffer 1bottle 25mL 77562

LEGENDplexTMWashBuffer,20X 1bottle 25mL 77564

DataAnalysisSoftwareDongle 1 -- 21217

Plate Sealers 4sheets -- 78101

No plate is included in Buffer Set I. 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)

Tel: 858-768-58008


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

ReporterEmission

Classification Channel

Channel Emission

Compen-sation

needed?

BD FACSCaliburTM FL2 575nm FL4 660nm No*

BD AccuriTMC6 FL2 585nm FL4 675nm No*

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

BDTMLSR,LSRIIBD LSRFortessaTM PE 575nm APC 660nm No*

GalliosTM PE 575nm APC 660nm No*

CytoFLEX PE 585nm APC 660nm No*

NovoCyte PE 572nm APC 660nm No*

AttuneTMNxT PE 574nm APC 670nm No*

Guava®easyCyte YLW 583nm RED2 661nm No*

SonySH800 PE 585nm APC 665nm No**Compensation is not required for the specified flow cytometers when set up properly.

Forsettingupvariousflowcytometers,pleasevisit:www.biolegend.com/legendplex and click on the Instrument Setup tab.

• Multichannelpipettescapableofdispensing5μLto200μL

• Reagentreservoirsformultichannelpipette

• Polypropylenemicrofugetubes(1.5mL)

• MicroFACStubes,1.1mL(iftheflowcytometerdoesnotcontainanautos-ampler)

• Laboratoryvortexmixer

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

• Aluminum foil

• Absorbentpadsorpapertowels

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

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

If the assay is performed in a filter plate:

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

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

• Ifneeded,additionalFilterplatescanbeorderedfromBioLegend(Cat#740377or740378).

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

• 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.

• Donotmixorsubstitutereagentsfromdifferentkitsorlots.Reagentsfromdifferentmanufacturersshouldnotbeusedwiththiskit.

• Donotusethiskitbeyonditsexpirationdate.

• SA-PEandbeadsarelight-sensitive.Minimizelightexposure.

Tel: 858-768-580010


Chapter 2: ASSAY PREPARATION

Sample Collection and Handling

Preparation of Serum Samples:

• Allowthebloodtoclotforatleast30minutesandcentrifugefor20min-utesat1,000xg.

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

• Whenusingfrozensamples,itisrecommendedthatsamplesbethawedcompletely,mixedandcentrifugedtoremoveparticulatespriortouse.

Preparation of Plasma Samples:

• Plasmacollectionshouldbecollectedusingananti-coagulant(e.g.,EDTA,Citrate).Centrifugefor20minutesat1,000xgwithin30minutesofbloodcollection.*Heparinplasmaisnotrecommendedforthispanel.

• Removeplasmaandassayimmediately,oraliquotandstoresamplesat≤-20°C.Avoidmultiple(>2)freeze/thawcycles.

• Whenusingfrozensamples,itisrecommendedthatsamplesbethawedcompletely,mixedwellandcentrifugedtoremoveparticulates.

Preparation of Cell Culture Supernatant:

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

Reagent Preparation

Preparation of Antibody-Immobilized Beads

1. Theindividualbeads(13X)needtobecombinedwithoneanotheranddilutedwithAssayBuffertocreatea1Xworkingsolutionofbeadspriortouse.

2. Sonicateeachbeadvialfor1minuteinasonicatorbathandthenvortexfor30 seconds to completely resuspend the beads.

3. Calculateandpreparea1Xbeadsworkingsolutionbasedonthedesirednumberofreactionsandplex-sizeofyourassay(i.e.thenumberofindi-vidualbeadvials)followingthestepsdescribedbelow.

A.Totalvolume(µL)=30x(numberofreactions)

B.Volumeneededfromeach13Xbeadsvial(µL)=2.3 x(numberof

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reactions)

C.AssayBufferneeded(µL)=A–Bx(numberofindividualbeadsvialstobemixed)

Note:calculationsfortotalvolumeincludea20%excesstoaccountforanylossduringpipetting.

Example: to prepare 50 reactions for a 5-plex assay

A.Totalvolume(µL)=30x50=1500µL

B.Volumeperbeadsvialneeded(µL)=2.3x50=115µL

C.AssayBufferneeded(µL)=A–Bx(numberofindividualbeadsvials)=1500–(115x5)=925µL

Combine115µLofeachbeadsvial(5vials)with925µLofassaybuffertogetthedesiredfinalvolumeof1500µLof1Xworkingsolutionofbeads.

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

• Add5.0mLLEGENDplexTMAssayBuffertothebottlecontaininglyophilized MatrixA.Allowatleast15minutesforcompletereconstitution.Vortexto

mixwell.LeftoverreconstitutedMatrixAshouldbestoredat≤-70°Cforupto one month.

Preparation of Wash Buffer• Bringthe20XWashBuffertoroomtemperatureandmixtobringallsalts

intosolution.

• Dilute25mLof20XWashBufferwith475mLdeionizedwater.Storeun-usedportionsbetween2°Cand8°Cforuptoonemonth.

Standard Preparation

1. Priortouse,reconstitutethelyophilizedMouseCytokineReleaseSyn-drome Panel Standardwith250μLLEGENDplexTMAssayBuffer.

2. Mixandallowthevialtositatroomtemperaturefor15minutes,andthentransfer the standard to an appropriately labeled polypropylene microcen-trifugetube.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 (see lot-specific Certificate of Analysis provided in the kit box for details).

3. Label6polypropylenemicrocentrifugetubesasC6,C5,C4,C3,C2andC1,respectively.

4.Add75µLofAssayBuffertoeachofthesixtubes.Prepare1:4dilutionofthetopstandardbytransferring25µLofthetopstandardC7totheC6

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tubeandmixwell.ThiswillbetheC6standard.

5.Inthesamemanner,performserial1:4dilutionstoobtainC5,C4,C3,C2and C1 standards (see the table below using the top standard at 10,000 pg/mL as an example).AssayBufferwillbeusedasthe0pg/mLstandard(C0).

Tube/Standard ID

Serial Dilution

Assay Buffer to add (µL)

Standard to add

Final Conc. (pg/mL)

C7 -- -- -- 10,000

C6 1:4 75 25µLofC7 2,500

C5 1:16 75 25µLofC6 625

C4 1:64 75 25µLofC5 156.25

C3 1:256 75 25µLofC4 39.01

C2 1:1024 75 25µLofC3 9.77

C1 1:4096 75 25µLofC2 2.44

C0 -- 75 -- 0

Sample Dilution

• Serumorplasmasamplesmustbediluted2-foldwithLEGENDplexTMAssay Bufferbeforebeingtested(e.g.dilute50uLofsamplewith50uLofLEG-ENDplexTMAssayBuffer).Iffurtherdilutionisdesired,dilutionshouldbedonewithMatrixAtoensureaccuratemeasurement.

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

• Forcellculturesupernatantsamples,thelevelsofanalytecanvarygreatlyfrom sample to sample. Totestcellculturesupernatantsamples,a prelimi-naryexperimentmayberequiredtodeterminetheappropriatedilutionfactor.Iffurtherdilutionisdesired,dilutionshouldbedonewithcorre-spondingfreshcellculturemediumorAssayBufferasadiluenttoensureaccurate measurement.

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Chapter 3: ASSAY PROCEDURE

TheLEGENDplexTMassaycanbeperformedinafilterplate,orinaV-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 34). Be sure to load standards in the first two columns. If an automation device is used for reading, the orientation and reading sequence should be carefully planned.

1. Pre-wettheplatebyadding100μLofLEGENDplexTM1XWashBuffertoeachwellandletitsitfor1minuteatroomtemperature.Toremovetheexcessvolume,placetheplateonthevacuummanifoldandapplyvacuum.Donotexceed10”Hgofvacuum.Vacuumuntilwellsaredrained(5-10seconds).BlotexcessWashBufferfromthebottomoftheplatebypressingtheplateonastackofcleanpapertowels.Placetheplateontopof the inverted plate cover.

2. Loadtheplateasshowninthetablebelow(intheorderfromlefttoright)For measuring cell culture supernatant samples:

Cell Culture Medium orAssayBuffer Standard Sample*

StandardWells 25µL 25µL --Samplewells 25µL -- 25µL

For measuring serum or plasma samples:MatrixA AssayBuffer Standard Sample*

StandardWells 25µL --- 25µL ---

Samplewells --- 25µL --- 25µL *See Sample Dilution on page 12

Tel: 858-768-580014


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

4. 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.

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

6. Add25µLofDetectionAntibodiestoeachwell.

7. Sealtheplatewithafreshplatesealer.Wraptheentireplate,includingtheinvertedplatecover,withaluminumfoil.Placetheplateonaplateshakerandshakeatapproximately500rpmfor1houratroomtemperature.

8. Do not vacuum!Add25µLofSA-PEtoeachwelldirectly.

9. Sealtheplatewithafreshplatesealer.Wraptheentireplate,includingtheinvertedplatecover,withaluminumfoil.Placetheplateonaplateshakerandshakeatapproximate500rpmfor30minutesatroomtemperature.

10. Repeatstep5above.

11. Add150µLof1XWashBuffertoeachwell.Resuspendthebeadsonaplateshaker for 1 minute.

12. 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.

Ifanautosamplerisnotavailable,thesamplescanbetransferredfromthefilterplatetomicroFACS(orFACS)tubes and read manually.

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

Vacuum to remove excess bu�er

Incubate 2 hours, RT, shaking

Capture beads

Biotinylated Detection Antibody

Analytes

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

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

For cell culture supernatant samples, Add to the plate:25 μL Assay Bu�er to all wells25 μL diluted standard to standard wells25 μL sample to sample wells25 μL mixed beads to all wells

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

For serum and plasma samples,Add to the plate:25 μL Matrix A to standard wells25 μL Assay Bu�er to sample wells25 μL diluted standard to standard wells25 μL sample to sample wells25 μL mixed beads to all wells

BA

C

A B C

A B C

Tel: 858-768-580016


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,page34).Besuretoloadstandardsinthefirsttwocolumns.Ifanautomationdeviceisusedforreading,theori-entationandreadingsequenceshouldbecarefullyplanned.

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

Cell Culture Medium orAssayBuffer Standard Sample*

StandardWells 25µL 25µL --Samplewells 25µL -- 25µL

For measuring serum or plasma samples:

MatrixA AssayBuffer Standard Sample*

StandardWells 25µL --- 25µL ---

Samplewells --- 25µL --- 25µL *See Sample Dilution on page 12

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

3. Sealtheplatewithaplatesealer.Covertheentireplatewithaluminumfoil to protect the plate from light. Shakeat800rpmon a plate shaker for 2 hours at room temperature (Depending on the shaker, the speed may need to be adjusted. The optimal speed is one that is high enough to keep beads in suspension during incubation, but not too high that it may cause sample 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 8). Donotuseexcessivecentrifuga-tionspeedasitmaymakeithardertoresuspendbeadsinlatersteps.Make sure the timer of the centrifuge works properly and standby to make sure

the centrifuge reaches preset speed.

5. Immediatelyaftercentrifugation,dumpthesupernatantintoabiohazardwastecontainerbyquicklyinvertingandflickingtheplatein one continu-ous and forceful motion.Thebeadspelletmayormaynotbevisibleafterdumpingthesupernatant.Blottheplateonastackofcleanpapertowelanddraintheremainingliquidfromthewellasmuchaspossible.Becarefulnot to disturb the bead pellet.

Alternatively,removalofthesupernatantmaybecompletedusingamultichannelpipettesetat75µL.Trytoremoveasmuchliquidaspossiblewithoutremovinganybeads. 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. Shakeat800rpmon a plate shaker for 1 hour at room temperature.

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

10. Sealtheplatewithanewplatesealer.Wraptheentireplatewithaluminumfoilandshaketheplateonaplateshakeratapproximate800rpmfor30minutes at room temperature.

11. Repeatstep4,and5.

12. (Thiswashingstepisoptionalbuthelpstoreducethebackground.)Washthe plate by dispensing 200 μLof1XWashBufferintoeachwellandincu-bate for one minute. Repeatstep4and5above.

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 may need to be adjusted when using an autosampler.

Ifanautosamplerisnotavailable,thesamplescanbetransferredfromtheplatetomicroFACS(orFACS)tubesandreadmanually.

17

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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 Wash 1 time Add 25 μL Detection AntibodiesIncubate 1 hr, RT, shaking

For cell culture supernatant samples, Add to the plate:25 μL Assay Bu�er to all wells25 μL diluted standard to standard wells25 μL sample to sample wells25 μL mixed beads to all wells

For serum and plasma samples,Add to the plate:25 μL Matrix A to standard wells25 μL Assay Bu�er to sample wells25 μL diluted standard to standard wells25 μL sample to sample wells25 μL mixed beads to all wells

BA

C

A B C

A B C

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

biolegend.com19


Chapter 4: FLOW CYTOMETER SETUP

Inordertogeneratereliabledata,theflowcytometermustbesetupproperlybeforedataacquisition.

Thesetupinstructionshavebeenremovedfromthismanualanduploadedontoourwebsitetosavepaper.

Toaccessthesetupinstructions,pleasevisit:www.biolegend.com/legendplex and click on the Instrument Setup tab.

Chapter 5: DATA ACQUISITION AND ANALYSIS

Data Acquisition

1. Beforereadingsamples,makesurethattheflowcytometerissetupprop-erly.

2. Createanewtemplateoropenanexistingtemplate(fordetailsonhowtocreateacytometer-specifictemplate,pleaserefertotheFlowCytometerSetupGuide).

3. Vortexeachsamplefor5secondsbeforeanalysis.

4.Settheflowratetolow.Setthenumberofbeadstobeacquiredtoabout300peranalyte(e.g.,acquire2,400beadsfora8-plexassayor3,000beadsfora13-plexassay).Donotsettoacquiretotaleventsassamplesmaycontainlargeamountsofdebris.Instead,createalargegatetoincludebothBeadsAandBeadsB(gateA+B)andsettoacquirethenumberofeventsingateA+B.Thiswillexludemajorityofthedebris.

Note:Donotacquiretoofewortoomanybeads.ToofewbeadsacquiredmayresultinhighCVsandtoomanybeadsacquiredmayresultinslowdata analysis later.

5.Readsamples.

Whenreadingsamples,settheflowcytometertosetupmodefirstandwaituntilbeadpopulationisstabilizedbeforerecordingorswitchingtoacquisi-tionmode.

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

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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,createaseparatefolderforeachassay.

6.ProceedtodataanalysisusingLEGENDplexTMDataAnalysisSoftwarewhendataacquisitioniscompleted.

Data Analysis

• TheFCSfilegeneratedonaflowcytometershouldbeanalyzedusingBio-Legend’sLEGENDplexTMDataAnalysisSoftware.TheLEGENDplexTM Data AnalysisSoftwarecanbedownloadedforfreehere:www.biolegend.com/legendplex.

• ForPCusers,installthesoftwareonaPCrunningWindows7orWin-dows8anduseitinconjunctionwiththeDataAnalysisSoftwareDongleincludedinthiskit.Thedonglehasalicensekeystoredinitandisneededtorunthesoftware.Tousethedongle,simplyplugitintheUSBportofthecomputeronwhichthedataanalysissoftwareisinstalled,priortolaunch-ingthesoftware.

• ForMacusers,installonaMacOSXversion10.7(Lion)orlaterandyouwillbepromotedtorequestasoftwarelicensekeyafterthesoftwareinstallation.

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

biolegend.com21


Chapter 6: ASSAY CHARACTERIZATIONRepresentative Standard Curve

ThisstandardcurvewasgeneratedusingtheLEGENDplexTM Mouse CytokineReleaseSyndromePanelfordemonstrationpurposesonly.Astandardcurvemustberunwitheachassay.

Assay Sensitivity

Theassaysensitivityorminimumdetectableconcentration(MDC)isthetheoreticallimitofdetectioncalculatedusingtheLEGENDplexTM Data AnalysisSoftwarebyapplyinga5-parametercurvefittingalgorithm.Theformulaforassaysensitivitypresentedbelowis(MDC+2STDEV).

Analyte

Sensitivity in Cell Culture Media

(pg/mL) (N = 19)

Sensitivity in Serum (pg/mL) (N = 14)

MouseIFN-γ 1.8+2.2 1.3 + 1.0

Mouse IL-10 4.1+4.9 4.8+3.3

MouseCCL4(MIP-1β) 6.9+5.1 5.9+4.1

MouseIFN-α 0.6+0.5 0.8+0.7

MouseCXCL9(MIG) 10.7+10.2 8.1+6.3

MouseCXCL10(IP-10) 16.4+13.0 14.6+8.2

MouseTNF-α 0.6+0.7 1.0+0.7

1.0

10.0

100.0

1000.0

10000.0

1 10 100 1000 10000 100000 1000000

MFI

Concentration (pg/mL)

mIFN-y

mIL-10

mCCL4 (MIP-1b)

mIFNα

mCXCL9 (MIG)

mCXCL10 (IP-10)

mTNF-a

mIL-6

mVEGF

mIL-4

mCCL3 (MIP-1α)

mCCL2 (MCP-1)

mGM-CSF

Tel: 858-768-580022


MouseIL-6 0.8+1.0 0.9+1.0

MouseVEGF 4.5+4.3 4.6+4.3

MouseIL-4 0.9+1.2 0.7+0.7

MouseCCL3(MIP-1α) 2.1+2.9 2.0 + 2.1

MouseCCL2(MCP-1) 6.8+8.0 12.5+20.6

MouseGM-CSF 1.3 + 1.2 1.8+4.1

Cross-ReactivityTargetmouseproteinsweretestedindividuallyattheindicatedconcentra-tionsbelowusingtheLEGENDplexTMMouse Cytokine Release Syndrome Panel,withnegligiblecross-reactivityobservedfornon-intendedtargets.

Analyte Conc. (pg/mL)

MouseIFN-γ 200,000Mouse IL-10 300,000

MouseCCL4(MIP-1β) 500,000MouseIFN-α 100,000

MouseCXCL9(MIG) 500,000MouseCXCL10(IP-10) 1,000,000

MouseTNF-α 100,000MouseIL-6 100,000

MouseVEGF 200,000MouseIL-4 100,000

MouseCCL3(MIP-1α) 200,000MouseCCL2(MCP-1) 500,000

MouseGM-CSF 100,000

Thefollowingrecombinantproteinsweretestedindividuallyatatleast50ng/mL.Noornegligiblecross-reactivitywasfound.

Mouse

MMP-2 IL-9 CXCL11 VEGF120

CCL10 VEGF120 IL-17A/F IFN-β1

IL-1α IL-21 IL-22 IL-23

IL-5 IL-7

biolegend.com23


Human

IFN-γ IL-10 CCL4 IFN-α2

CXCL9(MIG) CXCL10(IP-10) TNF-α IL-6

VEGF-165 IL-4 CCL3 (MIP-1α) CCL2

GM-CSF

Accuracy (Spike Recovery)

Forspikerecoveryincellculturemedium,serum,andplasma. Sampleswerespikedwithtargetrecombinantproteinsatthreedifferentlevelswithintheassayrange.Thespikedsampleswerethenassayed,andthemeasuredconcentrationswerecomparedwiththeexpectedvalues.

Analyte% of Recovery in Cell Culture

(N=2)

% of Recovery in Serum (N=8)

% of Recovery in Plasma

(N=16)

MouseIFN-γ 97% 74% 76%

Mouse IL-10 107% 75% 71%

MouseCCL4(MIP-1β) 105% 100% 101%

MouseIFN-α 95% 90% 95%

MouseCXCL9(MIG) 97% 106% 94%

MouseCXCL10(IP-10) 93% 61% 80%

MouseTNF-α 97% 79% 81%

MouseIL-6 99% 85% 90%

MouseVEGF 134% 67% 65%

MouseIL-4 104% 79% 81%

Mouse CCL3 (MIP-1α) 101% 92% 93%

Mouse CCL2 (MCP-1) 112% 93% 111%

MouseGM-CSF 98% 91% 89%

Linearity of Dilution

Serumandplasmasampleswereinitiallydiluted2-foldwithAssayBuffer,thenseriallydiluted2,4,8foldstillwithMatrixAandassayed.

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Cellculturesampleswerespikedwithtargetproteinswithknowncon-centrationsintheassayrange,thenseriallydiluted2,4,8foldwithAssayBufferandassayed.

Themeasuredconcentrationsofseriallydilutedsampleswerethencom-paredwiththeconcentrationofthelowestdilutionbasedonserialdilutionfactor used.

Analyte% Linearity

Cell Culture (N=2) Serum (N=8) Plasma (N=16)

MouseIFN-γ 101% 120% 107%

Mouse IL-10 111% 136% 125%

MouseCCL4(MIP-1β) 114% 104% 88%

MouseIFN-α 89% 96% 80%

MouseCXCL9(MIG) 112% 126% 112%

MouseCXCL10(IP-10) 91% 177% 114%

MouseTNF-α 97% 104% 93%

MouseIL-6 101% 117% 100%

MouseVEGF 169% 132% 116%

MouseIL-4 114% 124% 109%

MouseCCL3(MIP-1α) 103% 95% 85%

MouseCCL2(MCP-1) 127% 96% 86%

MouseGM-CSF 100% 102% 96%

Intra-Assay Precision

Twosampleswithdifferentconcentrationsofeachtargetproteinwereanalyzedinoneassaywith16replicatespersample.Theintra-assayprecisionisshownbelow.

Analyte Sample Mean (pg/mL) STDEV %CV

MouseIFN-γSample 1 73.4 6.98 10%

Sample 2 346.4 19.3 6%

Mouse IL-10Sample 1 115.9 16.46 14%

Sample 2 450.2 32.7 7%

MouseCCL4(MIP-1β)Sample 1 152.1 13.80 9%

Sample 2 1040.6 117.7 11%

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MouseIFN-αSample 1 39.5 4.43 11%

Sample 2 192.5 17.8 9%

MouseCXCL9(MIG)Sample 1 250.3 30.36 12%

Sample 2 677.9 64.8 10%

MouseCXCL10(IP-10)Sample 1 154.1 12.20 8%

Sample 2 571.6 28.6 5%

MouseTNF-αSample 1 36.2 3.39 9%

Sample 2 168.0 14.5 9%

MouseIL-6Sample 1 39.1 6.01 15%

Sample 2 195.4 20.4 10%

MouseVEGFSample 1 31.5 6.25 20%

Sample 2 144.3 7.6 5%


Sample 2 171.9 13.8 8%

MouseCCL3(MIP-1α)Sample 1 65.3 6.77 10%

Sample 2 314.9 26.6 8%

MouseCCL2(MCP-1)Sample 1 124.7 10.59 8%

Sample 2 544.1 25.3 5%

MouseGM-CSFSample 1 39.0 4.48 11%

Sample 2 182.2 17.9 10%

Inter-Assay Precision

Twosampleswithdifferentconcentrationsofeachtargetproteinwereanalyzedintenindependentassayswithfourreplicatespersample.Theinter-assayprecisionisshownbelow.

Analyte Sample Mean (pg/mL) STDEV %CV

MouseIFN-γSample 1 77.5 3.4 12%

Sample 2 314.1 35.1 11%

Mouse IL-10Sample 1 122.3 19.3 16%

Sample 2 406 46.3 11%

MouseCCL4(MIP-1β)Sample 1 180.4 23.7 13%

Sample 2 311.5 213.7 23%

MouseIFN-αSample 1 45.3 5.6 12%

Sample 2 186.2 23.2 12%

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MouseCXCL9(MIG)Sample 1 317.9 88.3 28%

Sample 2 656.2 156.1 24%

MouseCXCL10(IP-10)Sample 1 132.2 21 16%

Sample 2 478.7 70.1 15%

MouseTNF-αSample 1 41 7.6 19%

Sample 2 159.8 37.5 13%


Sample 2 185.6 37.5 20%

MouseVEGFSample 1 32.3 7.8 24%

Sample 2 130.7 8.8 7%


Sample 2 161.7 20.7 13%

MouseCCL3(MIP-1α)Sample 1 71.5 13.0 18%

Sample 2 304.1 38.6 13%

MouseCCL2(MCP-1)Sample 1 121.6 19.1 16%

Sample 2 517.6 47.0 9%

MouseGM-CSFSample 1 45.5 7.2 16%

Sample 2 177.7 19.7 11%

Biological Samples

Thevaluesinthissectionareprovidedforreferenceonly.Theassayspro-vided in this kit are intended for research use only. Serum Serumsamplesofthefollowingstrainswerepooledfromaminimumoftenmiceeach,andtestedfortheendogenouslevelsoftheproteins.Theconcentrationsareshownbelow(inpg/mL).

Analyte Range (pg/mL) % DetectableMean

(pg/mL)

MouseIFN-γ 10.1-77.6 100% 35.6

Mouse IL-10 14.4-24.4 75% 13.6

MouseCCL4(MIP-1β) 13.8-93.9 100% 45.5

MouseIFN-α 3.6-204.4 100% 61.9

MouseCXCL9(MIG) 38.5-126.5 50% 41.3

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MouseCXCL10(IP-10) 133.2-1019.3 100% 572.8

MouseTNF-α 27.5-130.8 100% 62.6

MouseIL-6 7.8-85.5 100% 39.9

MouseVEGF 205.1-205.1 25% 205.1

IL-4 5.8-5.8 25% 5.8

MouseCCL3(MIP-1α) 5.9-8.5 100% 7.3

MouseCCL2(MCP-1) 132.9-683.8 100% 356.8

MouseGM-CSF 0.8-10.8 100% 6.03 Plasma Plasmasamplesofthefollowingstrainswerepooledfromaminimumoftenmiceeach,andtestedfortheendogenouslevelsoftheproteins.Theconcentrationsareshownbelow(inpg/mL).

Analyte Range (pg/mL) % DetectableMean (pg/

mL)

MouseIFN-γ 2.14-43.9 100% 16.5

Mouse IL-10 15.1-32.3 37.5% 9.8

MouseCCL4(MIP-1β) 8.5-64.0 100% 26.2

MouseIFN-α 2.4-315.0 100% 50.4

MouseCXCL9(MIG) 20.6-72.7 75% 35.2

MouseCXCL10(IP-10) 198.9-1238.7 100% 533.7

MouseTNF-α 25.2-84.3 100% 43.9

MouseIL-6 7.5-85.5 100% 54.2

MouseVEGF 18.7-167.2 50% 148.1

MouseIL-4 1.36-3.82 37.5% 2.3

MouseCCL3(MIP-1α) 4.26-6.4 100% 5.3

MouseCCL2(MCP-1) 125.2-441.9 100% 243.7

MouseGM-CSF 0.8-6.8 87.5% 2.6 Cell Culture Supernatant Mousesplenocytecells(1x10⁶cells/mL)wereculturedundervariousconditions(LPS1µg/mL,Anti-CD3/CD28bothat1µg/mL,CellActivationCocktail(Cat#423301),LPS+IFN-γprimed1µg/mL+100ng/mL,R8482µg/

mL,PHA1µg/mL,PolyI:C1µg/mL,ConA10µg/mL,ConA+IL-210µg/mL+10ng/mL).Supernatantswerecollectedafter72hoursandassayedwiththeLEGENDplexTM Mouse Cytokine Release Syndrome Panel.Theresults(allinpg/mL)aresummarizedbelow.

AnalyteControl

(pg/mL)LPS

(pg/mL)

CD3 + CD 28

(pg/mL)

PMA + LONO

(pg/mL)MouseIFN-γ ND 612.2 33749.2 25028.5

Mouse IL-10 ND 909.8 1205.9 170.4

MouseCCL4(MIP-1β) 64.7 373.5 127041.7 4156.3

MouseIFN-α ND 1.8 1.9 1.1

MouseCXCL9(MIG) ND 11.1 458.2 150.2

MouseCXCL10(IP-10) ND 259.0 423.1 131.0

MouseTNF-α 4.9 304.5 1719.4 1083.4

MouseIL-6 ND 328.9 219.5 90.2

MouseVEGF ND 175.1 531.5 258.7

MouseIL-4 ND ND 123.1 5.3

MouseCCL3(MIP-1α) 14.7 371.6 32313.3 5301.1

MouseCCL2(MCP-1) ND 19.0 ND 92.4

MouseGM-CSF ND 29.8 10209.3 2502.2

ND=Non-detectable

AnalyteLPS +

IFNy stm(pg/mL)

R848 stm(pg/mL)

PHA stm(pg/mL)

Poly I:C stm(pg/mL)

MouseIFN-γ 28823.8 195.2 ND 6.9

Mouse IL-10 945.1 1417.8 ND 38.9

MouseCCL4(MIP-1β) 146.2 835.8 147.1 785.6

MouseIFN-α 4.6 1.1 ND 5.9

MouseCXCL9(MIG) 78.5 38.6 ND 16.4

MouseCXCL10(IP-10) 3537.3 148.0 19.2 418.9

MouseTNF-α 595.8 380.8 29.7 50.5

MouseIL-6 351.2 598.0 2.1 14.0

MouseVEGF 88.1 138.6 0.0 ND

28

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MouseIL-4 ND ND ND ND

MouseCCL3(MIP-1α) 71.4 549.3 86.8 435.3

MouseCCL2(MCP-1) 19.0 85.5 9.7 0.0

MouseGM-CSF 202.3 7.8 ND 1.9

ND=Non-detectable

AnalyteControl

(pg/mL)ConA stm (pg/mL)

ConA + IL-2 stm

(pg/mL)MouseIFN-γ ND 516.0 8149.5

Mouse IL-10 ND 32.6 74.1

MouseCCL4(MIP-1β) 161.4 1386.5 1245.7

MouseIFN-α ND ND 0.7

MouseCXCL9(MIG) ND 48.2 94.6

MouseCXCL10(IP-10) ND 154.2 267.7

MouseTNF-α 8.0 165.3 330.2

MouseIL-6 0.7 63.6 61.6

MouseVEGF 6.3 16.2 27.6

MouseIL-4 ND 93.3 115.7

MouseCCL3(MIP-1α) 68.9 1105.0 1285.4

MouseCCL2(MCP-1) 10.3 698.6 163.2

MouseGM-CSF 2.6 201.3 856.4

ND=Non-detectable

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TROUBLESHOOTING

Problem Possible Cause Solution

Bead popula-tionshiftingupwardordownwarddur-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.

Filterplatewillnot vacuum orsomewellsclogged

Vacuum pressure is insufficientorvacuummanifold does not seal properly.

Increase vacuum pressure such that 0.2 mLbuffercanbesuctionedin3-5seconds.Clean the vacuum manifold and make sure nodebrisonthemanifold.Pressdowntheplate on the manifold to make a good seal.

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

Centrifugesamplesjustpriortoassaysetup and use supernatant. If high lipid contentispresent,removelipidlayeraftercentrifugation.Samplemayneeddilutionif too viscous.

Ifsomewellsarestillcloggedduringwash-ing,trythefollowing:

1).Addbuffertoallthewells,pipetteupanddownthecloggedwellsandvacuumagain.

2).Useapieceofcleanwipe,wipetheun-dersideofthecloggedwellsandvacuumagain.

3).Takeathinneedle(e.g.,insulinneedle),whileholdingtheplateupward,pokethelittleholeundereachofthecloggedwellsand vacuum again. Do not poke too hard ortoodeepasitmaydamagethefilterand cause leaking.

Filterplatewasusedwithoutpre-wet.

Pre-wetplatewithwashbufferbeforerun-ning the assay.

biolegend.com31


Insufficientbead count or slowreading

Beads inappropriately prepared

Sonicatebeadvialsandvortexjustpriortoaddition.Agitatemixedbeadsintermit-tentlyinreservoirwhilepipettingthisintothe plate.

Samples cause beads aggregationduetoparticulatematterorviscosity.


Beadswerelostduringwashingforin-tubeassay

Makesurebeadsarespundownbyvisu-ally check the pellet (beads are in light blueorbluecolor).Beverycarefulwhenremovingsupernatantduringwashing.

Probe might be par-tiallyclogged.

Sampleprobemayneedtobecleaned,orifneeded,probeshouldberemovedandsonicated.

Plate leaked

Vacuum pressure set too high

Adjustvacuumpressuresuchthat0.2mLbuffercanbesuctionedin3-5seconds.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-specificbindingofSA-PE.Increasenumberofwashes.

Debris(FSC/SSC)duringsample acquisi-tion

Debris or platelet may existinsamplesolu-tion.

Centrifugesamplesbeforeanalyzingsamples. Remove platelet as much as possible.

Tel: 858-768-580032


Variationbe-tweenduplicate samples

Beadsaggregation SonicateandvortextheBeadspriortouse.

Multichannelpipettemay not be calibrated or inconsistent pipet-ting

CalibratePipette.Ensuregoodpipettingpractice.Primepipettebeforeusemayhelp.

Platewashingwasnotuniform

Make sure all reagents are vacuumed out completelyinallwashsteps.

Samples may contain particulatematters.


Loworpoorstandard curve signal

Thestandardwasin-correctlyreconstituted,stored or diluted

Followtheprotocoltoreconstitute,storeand dilute standard. Double check your calculation.

Wrongorshortincuba-tiontime

Ensurethetimeofallincubationswasappropriate.

Signals too high,standardcurves satu-rated

PMTvalueforFL2/PEset 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 populationsduringreading,ordistributionis unequal

Sample may cause some beads to ag-gregate.


Beadspopulationsarenotmixedproperly

Makesureallbeadpopulationsaremixed.and in similar numbers.

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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™ · LEEDple Mouse Cytokine Release Syndrome Mi and Match Subpanel Chapter 1: KIT DESCRIPTION Introduction Cytokine release syndrome is a systemic inflammatory response