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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
Enabling Legendary Discovery™
Non-Human Primate (NHP) Th Cytokine Panel 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.
biolegend.com 1
NHP Th Cytokine 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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NHP Th Cytokine Mix and Match Subpanel
Chapter 1: KIT DESCRIPTION
Introduction
Thelper(Th)cellsplayimportantrolesinregulatingimmuneresponses.Theysecretecytokinestostimulatevariouseffectorcells,suchascytotoxicTcells,Bcellsandmacrophages.AccuratemeasurementofThcytokineexpressioniscriticaltoidentifythecorrespondingThcellsandforin-depthunderstandingofthe immune responses.
The LEGENDplexTM Non-HumanPrimate(NHP) Th Cytokine Panel is a bead-basedmultiplexassay,usingfluorescence–encodedbeadssuitableforuseonvariousflowcytometers.Thispanelallowssimultaneousquantificationof10NHPcytokines,includingIL-2,4,5,6,10,13,17A,21,IFN-γandTNF-α,whicharecollectivelysecretedbyTh1,Th2,Th17,andTfollicularcells.Thisassaypro-videshighsensitivitiesandbroaddynamicrange.The panel has been validated foruseonserum,plasmaandcellculturesupernatantsamplesinbothRhesusand Cynomolgus species.
TheNHPThCytokinePanelisdesignedtoallowflexiblecustomizationwithinthepanel.Itcanalsobedividedintocelltype-specificsubpanels.Pleaserefertothetargetselectiontableforpanel-specifictargetinformation(Table1).Formixandmatchwithinthepanel,please visit www.biolegend.com/legendplex.
This assay is for research use only.
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.
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Beads Usage
TheNHPThCytokinePanelusestwosetsofbeads.Eachsethasauniquesizethatcanbeidentifiedbasedontheirforwardscatter(FSC)andsidescatter(SSC)profiles(BeadsAandBeadsB,Figure1).Eachbeadsetcanbefurtherresolvedbasedontheirinternalfluorescenceintensities.Theinternaldyecanbede-tectedusingFL3,FL4,orAPCchannel,dependingonthetypeofflowcytometerused.ThesmallerBeadsAconsistsof5beadpopulationsandthelargerBeadsBconsistsof5beadpopulations(Figure2-3).
Usingatotalof10beadpopulationsdistinguishedbysizeandinternalfluo-rescentdye,theNHPThCytokinePanelallowssimultaneousdetectionof10cytokinesinasinglesample.Eachanalyteisassociatedwithaparticularbeadset as indicated (Figures 2-3 and Table 1).
Figure1.BeadsDifferentiatedbySize
Beads A = smaller beads
Beads B = larger beads
Figure2.BeadsAClassificationbyFL4
A5 A7 A8
A4
A6
A10
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Figure3.BeadsBClassificationbyFL4
ForBeadsusageinvariouspanels,pleaserefertoTable1below:
Table1.BeadsIDandPanel-SpecificTargetSelection
Target Bead ID
Th1Cat. No.740381*
&740391**
Th2Cat. No.740382*
&740392**
Th1/Th2Cat. No.740383*
&740393**
Th17Cat. No.740384*
&740394**
TfhCat. No.740385*
&740395**
Top Standard concentration
(ng/mL)
IL-2 A4 √ v The top standard
concentrationof each target may vary and may be sub-
ject to change from lot to lot. Please refer to thelot-specificCertificateofAnalysis for
this informa-tion
IL-5 A5 √ √
IL-6 A6 √ √ √ √ √
IL-10 A7 √ √ √ √ √
IL-13 A10 √ √
TNF-α B2 √ √ √ √ √
IFN-γ B3 √ √ √ √
IL-4 B4 √ √ √
IL-17A B5 √
IL-21 B6 √ √
*Cat#forkitswithfilterplates.**Cat#forkitswithV-bottomplates
BeadIDisusedtoassociateabeadpopulationtoaparticularanalyteintheLEGENDplexTMDataAnalysisSoftware.TheassociationofanalyteandbeadIDwillbedefinedduringthegatingstepofthedataanalysis.
WhenenteringanalyteandbeadIDinfomationduringthegatingstep,alwaysenterinthesequentialorderofthebeadID(e.g,A4,A5,A6...B2,B3,B4...). PleaserefertotheLEGENDplexTMDataAnalysisSoftwareUserGuideandOnlineHelpfordetails(www.biolegend.com/legendplex).
B4 B5
B6 B7
B3
B9
B2
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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
TheLEGENDplexTMkitcontainsreagentsfor100tests,listedinthetablebelow.Whenassayedinduplicate,thisisenoughforan8-pointstandardcurveand40samples.
FortheMixandMatchSubpanels,individualbeadsareprovidedat13Xconcen-tration.TheBufferSetcontainsSetupBeads,allBuffers,PlateSealers,Matrix,SA-PEandDataAnalysisSoftwareDongle.AmanualisprovidedforeachMixand Match subpanel.
Kit Components Quantity Volume Cat #
Capture Beads* (see tables below for moreinformation) varies 270µL varies
LEGENDplex™NHPThCytokineDe-tectionAntibodies 1bottle 3.5mL 740315
LEGENDplex™NHPThCytokineStandard 1 vial lyophilized 740314
LEGENDplex™BufferSetA 1 740368Filter Plate* or V-bottomPlate** 1 plate 740377*or
740379**NHPThCytokineMixandMatchCustom Subpanel Manual 1 76761
*Forkitwithfilterplate.**ForkitwithV-bottomplate.
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Capture beads for Mix and Match Subpanels:
Bead Name Quan-tity
Vol-ume Cat#
LEGENDplex™NHPIL-2CaptureBeadA4,13X 1 vial 270µL 740304LEGENDplex™NHPIL-5CaptureBeadA5,13X 1 vial 270µL 740305LEGENDplex™NHPIL-6CaptureBeadA6,13X 1 vial 270µL 740306LEGENDplex™NHPIL-10CaptureBeadA7,13X 1 vial 270µL 740307LEGENDplex™NHPIL-13CaptureBeadA10,13X 1 vial 270µL 740308LEGENDplex™NHPTNF-αCaptureBeadB2,13X 1 vial 270µL 740309LEGENDplex™NHPIFN-γCaptureBeadB3,13X 1 vial 270µL 740310LEGENDplex™NHPIL-4CaptureBeadB4,13X 1 vial 270µL 740311LEGENDplex™NHPIL-17ACaptureBeadB5,13X 1 vial 270µL 740312LEGENDplex™NHPIL-21CaptureBeadB6,13X 1 vial 270µL 740313
Please refer to BeadsIDandPanel-SpecificTargetSelectiontable(Table1),to see which capture beads are included in each panel.
LEGENDplex™BufferSetA(Cat#:740368)
Component 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 3.5mL 77743LEGENDplexTMMatrixB,Lyophilized 1 vial lyophilized 77549LEGENDplexTMAssayBuffer 1bottle 25mL 77562LEGENDplexTMWashBuffer,20X 1bottle 25mL 77564DataAnalysisSoftwareDongle 1 21217Plate Sealers 4sheets 78101
NoplateisincludedinBufferSetA.Plateneedtobeorderedseparately.Please order the correct type of plate based on the preferred assay protocol (Cat#740377forFilterPlateandCat#740379forV-bottomPlate)
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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*
BDTMLSR,LSRIIBD LSRFortessaTM PE 575-585
nm APC 660 nm No*
BD FACSAriaTM PE 575 nm APC 660 nm No*
*Compensationisnotrequiredforthespecifiedflowcytometerswhensetupproperly,butisrecommendedforconsistentresults.
Forsettingupvariousflowcytometers,pleasevisit:www.biolegend.com/legendplex and click on the Instrument Setup tab.
• Multichannelpipettescapableofdispensing5μLto200μL
• Reagentreservoirsformultichannelpipette
• Polypropylenemicrofugetubes(1.5mL)
• Laboratoryvortexmixer
• Sonicatorbath(e.g.,BransonUltrasonicCleanermodel#B200,orequiva-lent)
• Aluminum foil
• Absorbentpadsorpapertowels
• Plateshaker(e.g.,Lab-LineInstrumentsmodel#4625,orequivalent)
• Tabletopcentrifuges(e.g.,Eppendorfcentrifuge5415C,orequivalent)
• 1.1mLpolypropylenemicroFACStubes,in96-tuberack(e.g.,NationalScientificSupplyCo,catalog#TN0946-01R,orequivalent).
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NHP Th Cytokine Mix and Match SubpanelIftheassayisperformedinafilterplate;
• Avacuumfiltrationunit(MilliporeMultiScreen®HTSVacuumManifold,cat#MSVMHTS00orequivalent).Instructionsonhowtousethevacuummanifoldcanbefoundatthesupplier’swebsite.
• Avacuumsource(minivacuumpumporlinevacuum,e.g.,MilliporeVacuumPump,catalog#WP6111560,orequivalent).
• Ifneeded,additionalFilterplatecanbeorderedfromBioLegend(Cat#740377or740378).
IftheassayisperformedinaV-bottomplate;
• 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.
• MatrixBforLEGENDplexTM kits contains components of human origin and shouldbehandledaspotentiallyhazardous.TherawmaterialhasbeenscreenedforinfectiousdiseasesandisnegativeforHIV,HBVandHCVusingFDA-approved test methods.
• Donotmixorsubstitutereagentsfromdifferentkitsorlots.Reagentsfromdifferentmanufacturersshouldnotbeusedwiththiskit.
• Donotusethiskitbeyonditsexpirationdate.
• SA-PEandBeadsarelight-sensitive.Minimizelightexposure.
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Chapter 2: ASSAY PREPARATION
SampleCollectionandHandling
PreparationofSerumSamples:
• Allowthebloodtoclotforatleast30minutesandcentrifugefor10min-utesat1,000xg.
• Remove serum and assay immediately or aliquot and store samples at ≤-20°C.Avoidmultiple(>2)freeze/thawcycles.
• Whenusingfrozensamples,itisrecommendedthatsamplesarethawedcompletely,mixedandcentrifugedtoremoveparticulatespriortouse.
PreparationofPlasmaSamples:
• PlasmacollectionusingEDTAasananti-coagulantisrecommended.Centri-fugefor10minutesat1,000xgwithin30minutesofbloodcollection.
• Removeplasmaandassayimmediately,oraliquotandstoresamplesat≤-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
Theindividualbeads(13X)shouldbemixedanddilutedto1XwithAssayBufferpriortouse.Tomixthebeads,followthestepsbelow(a5-plexsub-panelisusedasanexample):
1. Sonicate the beads vials for 1 minute in a sonicator bath and then vortexfor30secondspriortouse.
2.Calculatetheamountofmixedanddilutedbeadsneededfortheassay. Prepareextratocompensateforpipettingloss.Eachreactionneeds 25µLofmixedanddilutedbeads.For50reactions,prepare1.5mLof mixedbeads.For96reactions,prepare3mLofmixedbeads.
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NHP Th Cytokine Mix and Match Subpanel3.Tomake1.5mlof5-plex1Xdilutedbeads,transfer115µLofeachof
the5individualbeads(13X)toafreshtube(totalbeadvolume=575µL)andadd925µLofAssayBuffertomakethefinalvolumeof1.5mL.
PreparationofWashBuffer
• Bringthe20XWashBuffertoroomtemperatureandmixtobringallsaltsintosolution.
• Dilute25mLof20XWashBufferwith475mLdeionizedwater.Storeun-usedportionsbetween2°Cand8°Cforuptoonemonth.
PreparationofMatrixB(forSerumorPlasmaSamplesOnly)
• Add10.0mLLEGENDplexTMAssayBuffertothebottlecontaininglyophi-lizedMatrixB.Allowatleast15minutesforcompletereconstitution.Vortextomixwell.LeftoverreconstitutedMatrixBshouldbestoredat≤-70°Cforuptoonemonth.
StandardPreparation
1. Priortouse,reconstitutethelyophilizedNHPThCytokineStandardwith250µLAssayBuffer.
2. Mixandallowthevialtositatroomtemperaturefor10minutes,andthen transfer the standard to an appropriately labeled polypropylene microcentrifugetube.ThiswillbeusedasthetopstandardC7.
3. Label6polypropylenemicrocentrifugetubesasC6,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 concentrationasanexample).AssayBufferwillbeusedasthe0pg/mLstandard(C0).
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Tube/Standard
ID
Serial Dilution
Assay Buffertoadd (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 156.3
C3 1:256 75 25µLofC4 39.1
C2 1:1024 75 25µL of C3 9.8
C1 1:4096 75 25µL of C2 2.4
C0 -- 75 -- 0
SampleDilution
• Serumorplasmasamplesmustbediluted4-foldwithAssayBufferbeforebeingtested(e.g.dilute50µLofsamplewith150µLofAssayBuffer).
Iffurthersampledilutionisdesired,dilutionshouldbedonewithMatrixBto ensure accurate measurement.
Addingserumorplasmasampleswithoutdilutionwillresultinlowassayaccuracyandpossibly,cloggingofthefilterplate.
• Forcellculturesupernatantsamples,thelevelsofanalytecanvarygreatlyfromsampletosample.Whilethesamplescanbetestedwithoutdilutions,apreliminaryexperimentmayberequiredtodeterminetheappropriatedilutionfactor.
Ifsampledilutionisdesired,dilutionshouldbedonewithcorrespondingfreshcellculturemediumorAssayBuffertoensureaccuratemeasurement.
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Chapter 3: ASSAY PROCEDURE
TheLEGENDplexTM assaycanbeperformedinafilterplate,orinaV-bottomplate.
• Thein-filterplateassayprocedurerequiresavacuumfiltrationunitforwashing(seeMaterialstobeProvidedbytheEnd-User,page8). If you haveperformedbead-basedmultiplexassaysbefore,yourlabmayalreadyhavethevacuumfiltrationunitsetup.
• Ifthein-filterplateassayprocedureisnotpossibleorifyouprefer,theas-saycanbeperformedinaV-bottomplate.
Performing the Assay Using a Filter Plate
• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.
• Setthefilterplateonaninvertedplatecoveratalltimesduringassaysetupandincubationsteps,sothatthebottomoftheplatedoesnottouchany surface. Touching a surface may cause leakage.
• Keeptheplateuprightduringtheentireassayprocedure,includingthewashingsteps,toavoidlosingbeads.
• Theplateshouldbeplacedinthedarkorwrappedwithaluminumfoilforallincubationsteps.
• Standards and samples should be run in duplicate and arranged on the plateinaverticalconfigurationconvenientfordataacquisitionandanalysis(asshowninattachedPLATEMAP,page32).Besuretoloadstan-dardsinthefirsttwocolumns.Ifanautomationdeviceisusedforread-ing,theorientationandreadingsequenceshouldbecarefullyplanned.
1. Pre-wettheplatebyadding100μLofLEGENDplexTM1XWashBuffertoeachwellandletitsitfor1minuteatroomtemperature.Toremovetheexcessvolume,placetheplateonthevacuummanifoldandapplyvacuum.Donotexceed10”Hgofvacuum.Vacuumuntilwellsaredrained(5-10seconds).BlotexcessWashBufferfromthebottomoftheplatebypressingtheplateonastackofcleanpapertowels.Placetheplateontopoftheinverted plate cover.
Formeasuringcellculturesupernatantsamples,loadtheplateasshowninthetablebelow(intheorderfromlefttoright):
AssayBuffer MatrixB Standard Sample*
StandardWells 25µL --- 25µL ---
Samplewells 25µL --- --- 25µL
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Formeasuringserumsamples,loadtheplateasshowninthetablebelow(intheorderfromlefttoright):
AssayBuffer MatrixB Standard Sample*
StandardWells --- 25µL 25µL ---
Samplewells 25µL --- --- 25µL
*See SampleDilution
2. Vortexmixedbeadsbottlefor30seconds.Add25μLofmixedbeadstoeachwell.Thevolumeshouldbe75μLineachwellafterbeadsaddition.(Note:Duringadditionofthebeads,shakemixedbeadsbottleintermit-tentlytoavoidbeadsettling).
3. Sealtheplatewithaplatesealer.Toavoidplateleaking,donotapplyposi-tivepressuretothesealerwhensealingtheplate.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.Resuspendthebeadsonaplateshaker for 1 minute.
11. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay(Note:Prolongedsamplestoragecanleadtoreducedsignal).
Iftheflowcytometerisequippedwithanautosampler,readtheplatedirectly using the autosampler. Please be sure to program the autosampler
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NHP Th Cytokine Mix and Match Subpanel
to resuspend beads in the well immediately before taking samples. The probe height may need to be adjusted when using an autosampler.
Ifanautosamplerisnotavailable,thesamplescanbetransferredfromthefilterplateto 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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PerformingtheAssayUsingaV-bottomPlate
• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.
• Keeptheplateuprightduringtheentireassayprocedure,includingthewashingsteps,toavoidlosingbeads.
• Theplateshouldbeplacedinthedarkorwrappedwithaluminumfoilforallincubationsteps.
• Standards and samples should be run in duplicate and arranged on the plateinaverticalconfigurationconvenientfordataacquisitionandanalysis(asshowninattachedPLATEMAP,page32).Besuretoloadstandardsinthefirsttwocolumns.Ifanautomationdeviceisusedforreading,theori-entationandreadingsequenceshouldbecarefullyplanned.
1. For measuring cell culture supernatant samples,loadtheplateasshowninthetablebelow(intheorderfromlefttoright):
AssayBuffer MatrixB Standard Sample*
StandardWells 25µL --- 25µL ---
Samplewells 25µL --- --- 25µL Formeasuringserumsamples,loadtheplateasshown inthetablebe-
low(intheorderfromlefttoright):AssayBuffer MatrixB Standard Sample*
StandardWells --- 25µL 25µL ---
Samplewells 25µL --- --- 25µL
*See SampleDilution
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 (Dependingontheshaker,thespeedmayneedtobeadjusted.Theoptimalspeedisonethatishighenoughtokeepbeadsinsuspensionduringincubation,butnottoohighsoitcausesspillfrom the wells).
4. Centrifugetheplateat1050rpm(~250g)for5minutes,usingaswingingbucketrotor(G.H3.8)withmicroplateadaptor(Please refer to Materials to beProvidedbytheEnd-User,page8).Donotuseexcessivecentrifugationspeed as it may make it harder to resuspend beads in later steps. Make
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NHP Th Cytokine Mix and Match Subpanel
surethetimerofthecentrifugeworksproperlyandstandbytomakesurethe centrifuge reaches preset speed.
5. Immediatelyaftercentrifugation,dumpthesupernatantintoasinkbyquicklyinvertingandflickingtheplateinonecontinuousandforcefulmo-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. 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. Washtheplatebydispensing200μLof1XWashBufferintoeachwellandincubate for one minute. Repeatstep4and5above.Thiswashingstepisoptionalbuthelpstoreducethebackground.
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,thesamplescanbetransferredfromtheplate to micro FACS (or FACS) tubes and read manually.
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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, 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
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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
DataAcquisition
1. Beforereadingsamples,makesurethattheflowcytometerissetupprop-erly.
2. Createanewtemplateoropenanexistingtemplate(fordetailsonhowtocreateacytometer-specifictemplate,pleaserefertotheFlowCytometerSetup Guide).
3. Vortexeachsamplefor5secondsbeforeanalysis.
4.Settheflowratetolow.Setthenumberofbeadstobeacquiredtoabout300peranalyte(e.g.,acquire2,400beadsfora8-plexassayor4000beadsfora13-plexassay).Donotsettoacquiretotaleventsassamplesmaycontainlargeamountsofdebris.Instead,createalargegatetoincludebothBeads 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.Readsamples.
Whenreadingsamples,settheflowcytometertosetupmodefirstandwaituntilbeadpopulationisstabilizedbeforerecordingorswitchingtoacquisi-tionmode.
TosimplifydataanalysisusingtheLEGENDplexTMDataAnalysisSoftware,readsamplesinthesameorderasshownonthePLATEMAPattachedattheendofthemanual.Foranin-plateassay,readcolumnbycolumn(A1,B1,C1...A2,B2,C2...).
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20
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
• TheFCSfilegeneratedonaflowcytometershouldbeanalyzedusingBioLegend’sLEGENDplexTMDataAnalysisSoftware.TheLEGENDplexTM Data AnalysisSoftwarecanbedownloadedforfreehere:www.biolegend.com/legendplex.
• ForPCusers,installthesoftwareonaPCrunningWindows7orWindows8anduseitinconjunctionwiththeDataAnalysisSoftwareDongleincludedin this kit. The dongle has a license key stored in it and is needed to run the software.Tousethedongle,simplyplugitintheUSBportofthecomputeronwhichthedataanalysissoftwareisinstalled,priortolaunchingthesoftware.
• ForMacusers,installonaMacrunningMacOSXversion10.7(Lion)orlaterandyouwillbepromotedtorequestasoftwarelicensekeyafterthesoftwareinstallation.
• FollowtheLEGENDplexTMDataAnalysisSoftwareUserGuideandOnlineHelptousethesoftware(www.bioLegend.com/legendplex;orpress F1 for
online help at any step of the data analysis).
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Chapter 6: ASSAY CHARACTERIZATION
RepresentativeStandardCurve
ThisstandardcurvewasgeneratedusingtheLEGENDplexTMNHPThCyto-kinePanelfordemonstrationpurposeonly.Astandardcurvemustberunwitheachassay.
1
10
100
1000
10000
1 10 100 1000 10000 100000
MFI
Concentration (pg/mL)
IL-2 IL-5 IL-6 IL-10 IL-13 TNF-α IFN-γ IL-4 IL-17A IL-21
AssaySensitivity
Theassaysensitivityorminimumdetectableconcentration(MDC)isthetheoreticallimitofdetectioncalculatedusingtheLEGENDplexTM Data AnalysisSoftwarebyapplyinga5-parametercurvefittingalgorithm.
Analyte MDC in Cell Culture Medium (pg/mL)
MDC in Serum (pg/mL)
NHPIL-2 1.7 0.8
NHPIL-5 1.0 0.7
NHPIL-6 0.8 1.0
NHPIL-10 8.1 4.9
NHPIL-13 0.9 0.8
NHPTNF-α 0.8 0.9
NHPIFN-γ 0.9 0.8
NHPIL-4 0.8 0.7
NHPIL-17A 2.0 1.5
NHPIL-21 1.9 1.8
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Cross-Reactivity
Therewasnoornegligiblecross-reactivityfoundfortheanalytesinthispanel.
Accuracy (Spike Recovery)
Forspikerecoveryincellculturemedium,RPMIorDMEMwith10%FCSwasspikedwithtargetproteinsatthreedifferentlevelswithintheassayrange.Thespikedsampleswerethenassayed,andthemeasuredconcen-trationswerecomparedwiththeexpectedvalues.
Forspikerecoveryinserumandplasma,bothRhesusandCynomolgussamplesweretested.Serum(n=12)orplasma(n=8)werefirstdilutedfour-foldwithAssayBufferandspikedwithtargetproteinsatthreedifferentlevelswithintheassayrange.Thespikedsampleswerethenassayed,andthemeasuredconcentrationswerecomparedwiththeexpectedvalues.
Analyte% of Recovery in Cell Culture
Medium
% of Recovery in Serum
% of Recovery in Plasma
NHPIL-2 106% 76% 79%NHPIL-5 98% 97% 90%NHPIL-6 102% 78% 87%NHPIL-10 85% 71% 68%NHPIL-13 87% 66% 70%NHPTNF-α 92% 62% 70%NHPIFN-γ 85% 82% 68%NHPIL-4 88% 80% 64%NHPIL-17A 88% 78% 82%NHPIL-21 97% 134% 127%
LinearityofDilution
Forspikelinearityincellculturemedium,RPMIorDMEMwith10%FCSwasfirstspikedwithaknownconcentrationoftargetproteins.Thespikedsampleswereseriallydiluted1:2,1:4,1:8withassaybufferandassayed.Themeasuredconcentrationsofseriallydilutedsampleswerecomparedwiththatofthespikedsamples.
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Forspikelinearityinserumandplasma,bothRhesusandCynomolgussamplesweretested.Serum(n=12)orplasma(n=8)werefirstdilutedfour-foldwithAssayBufferandspikedwithaknownconcentrationoftargetproteins.Thespikedsampleswereseriallydiluted1:2,1:4,1:8withMatrixBandassayed.Themeasuredconcentrationsofseriallydilutedsampleswerecomparedwiththatofthespikedsamples.
AnalyteLinearity of
DilutioninCellCulture Medium
Linearity of Dilutionin
Serum
Linearity of Dilutionin
PlasmaNHPIL-2 109% 110% 131%NHPIL-5 95% 109% 94%NHPIL-6 96% 105% 100%NHPIL-10 100% 124% 117%NHPIL-13 118% 127% 129%NHPTNF-α 107% 138% 126%NHPIFN-γ 105% 139% 121%NHPIL-4 105% 132% 119%NHPIL-17A 102% 135% 126%NHPIL-21 97% 127% 109%
Intra-Assay Precision
Twosampleswithdifferentconcentrationsoftargetproteinswereanalyzedinoneassaywith16replicatesforeachsample.Theintra-assayprecisionwascalculatedasbelow.
Analyte Sample Mean (pg/mL) STDEV %CV
NHPIL-2Sample 1 33.3 2.6 8%Sample 2 443.8 32.9 7%
NHPIL-5Sample 1 42.5 3.1 7%Sample 2 546.6 35.9 7%
NHPIL-6Sample 1 38.9 3.9 10%Sample 2 590.1 52.2 9%
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NHPIL-10Sample 1 108.3 8.8 8%Sample 2 1567.1 163.9 10%
NHPIL-13Sample 1 38.0 3.7 10%Sample 2 525.4 47.9 9%
NHPTNF-αSample 1 41.2 3.2 8%Sample 2 572.4 64.5 11%
NHPIFN-γSample 1 40.3 2.9 7%Sample 2 521.0 44.6 9%
NHPIL-4Sample 1 37.8 2.9 8%Sample 2 557.9 51.1 9%
NHPIL-17ASample 1 39.2 2.8 7%Sample 2 526.1 37.4 7%
NHPIL-21Sample 1 32.7 2.0 6%Sample 2 462.9 30.8 7%
Inter-Assay Precision
Twosampleswithdifferentconcentrationsoftargetproteinswereanalyzedinthreeindependentassayswith3replicatesforeachsample.Theinter-assayprecisionwascalculatedasbelow.
Analyte Sample Mean (pg/mL) STDEV %CV
NHPIL-2Sample 1 31.8 3.4 11%Sample 2 470.5 46.0 10%
NHPIL-5Sample 1 40.7 3.4 8%Sample 2 571.7 49.0 9%
NHPIL-6Sample 1 38.0 3.7 10%Sample 2 594.8 56.4 9%
NHPIL-10Sample 1 113.1 17.1 15%Sample 2 1772.3 280.2 16%
NHPIL-13Sample 1 37.4 3.3 9%Sample 2 557.5 59.2 11%
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NHPTNF-αSample 1 41.5 3.1 8%Sample 2 595.6 64.3 11%
NHPIFN-γSample 1 40.2 3.0 7%Sample 2 570.0 67.9 12%
NHPIL-4Sample 1 36.7 2.9 8%Sample 2 569.9 45.9 8%
NHPIL-17ASample 1 39.1 2.7 7%Sample 2 553.2 56.4 10%
NHPIL-21Sample 1 29.1 4.3 15%Sample 2 483.7 37.4 8%
Biological Samples
Serum and Plasma (Samples are not paired)
NormalRhesus(n=16)andCynomolgus(n=16)serumsamplesweretestedforendogenouslevelsoftheThcytokines.Theconcentrationsmeasuredareshownbelow:
Analyte Range (pg/ml)
No. of Detectable
% of Detectable
Mean (pg/mL)
NHPIL-2 5.2-63.3 32 100% 13.5
NHPIL-5 ND-32.4 21 66% 7.4
NHPIL-6 ND-439.0 23 72% 25.7
NHPIL-10 ND-48.4 6 19% 28.3
NHPIL-13 ND-9.5 4 13% 7.1
NHPTNF-α ND-8.7 8 25% 3.7
NHPIFN-γ ND-20.7 29 91% 4.7
NHPIL-4 ND-16.5 3 9% 8.6
NHPIL-17A ND-17.0 5 16% 10.9
NHPIL-21 ND-125.8 24 75% 26.1
ND=Non-detectable
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NormalRhesus(n=4)andCynomolgus(n=4)plasmasamplesweretestedforendogenouslevelsofThcytokines.Theconcentrationsmeasuredareshownbelow:
Analyte Range (pg/mL)
No. of Detectable
% of Detectable
Mean (pg/mL)
NHPIL-2 6.1-26.4 8 100% 13.3
NHPIL-5 ND-18.0 6 75% 6.5
NHPIL-6 ND-14.5 3 38% 7.9
NHPIL-10 ND 0 0% ND
NHPIL-13 ND 0 0% ND
NHPTNF-α ND 0 0% ND
NHPIFN-γ ND-7.0 7 88% 4.1
NHPIL-4 ND 0 0% ND
NHPIL-17A ND 0 0% ND
NHPIL-21 ND-129.9 6 75% 35.0
ND=Non-detectable
Cell Culture Supernatant
RhesusorCynomolgusPBMCs(1x106cells/mL)wereculturedundervariousconditions(PHA,5µg/mL;PMA,20ng/mL;Ionomycin(I),500ng/mL;LPS,1µg/mL;IFN-γ,100ng/mL).Supernatantswerecollectedaf-ter72hoursandassayedwiththeLEGENDplexTMNHPThCytokinePanel.Theresults(allinpg/mL)aresummarizedbelow.
Analyte Control PHA PMA + I LPS IFN-γ+LPS
Rhesus IL-2 18.2 1.8 7173.4 4.3 8.1
RhesusIL-5 1.7 23.9 57.2 ND 0.9
RhesusIL-6 5.2 4282.0 118.0 3774.4 14444.2
RhesusIL-10 3.3 139.4 568.3 197.1 29.1
Rhesus IL-13 3.1 112.3 347.9 6.9 8.3
RhesusTNF-α 7.6 84.6 846.4 94.8 2816.9
RhesusIFN-γ ND 20.7 2351.0 ND 9841.0
RhesusIL-4 ND 1.9 4.2 ND 0.6
RhesusIL-17A 1.7 33.6 68.4 3.6 5.8
Rhesus IL-21 4.3 36.3 22.5 33.4 61.7
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Analyte Control PHA PMA + I LPS IFN-γ+LPS
Cynomolgus IL-2 16.2 15.0 5261.0 8.9 19.3
CynomolgusIL-5 3.0 106.7 132.1 1.3 2.4
CynomolgusIL-6 2.4 3833.3 100.3 3497.4 9289.3
CynomolgusIL-10 ND 86.6 233.2 75.3 37.6
Cynomolgus IL-13 1.2 212.8 270.8 2.1 3.0
CynomolgusTNF-α 8.6 53.3 358.2 53.3 621.7
CynomolgusIFN-γ ND 165.2 1607.1 ND 5315.0
CynomolgusIL-4 ND 16.6 11.6 ND 1.0
CynomolgusIL-17A ND 91.6 91.0 2.5 4.8
Cynomolgus IL-21 ND 47.6 27.5 39.3 79.6ND=Non-detectable
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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.
Increasevacuumpressuresuchthat0.2mLbuffercanbesuctionedin3-5seconds.Clean the vacuum manifold and make sure nodebrisonthemanifold.Pressdowntheplate on the manifold to make a good seal.
Samples have insoluble particlesorsampleistooviscous(e.g.,serumand plasma samples)
Centrifuge samples just prior to assay setup 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.
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Insufficientbead count or slowreading
Beads inappropriately prepared
Sonicatebeadvialsandvortexjustpriortoaddition.Agitatemixedbeadsintermit-tentlyinreservoirwhilepipettingthisintothe plate.
Samples cause beads aggregationduetoparticulatematterorviscosity.
Centrifuge samples just prior to assay setup and use supernatant. If high lipid contentispresent,removelipidlayeraftercentrifugation.Samplemayneeddilutionif too viscous.
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.
InsufficientwashesThe background may be due to non-specificbindingofSA-PE.Increasenumberofwashes.
Debris(FSC/SSC) during sample acquisi-tion
Debris or platelet may existinsamplesolu-tion.
Centrifugesamplesbeforeanalyzingsamples. Remove platelet as much as possible.
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NHP Th Cytokine Mix and Match Subpanel
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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.
Centrifuge samples just prior to assay setup and use supernatant. If high lipid contentispresent,removelipidlayeraftercentrifugation.Samplemayneeddilutionif too viscous.
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.
Centrifuge samples just prior to assay setup and use supernatant. If high lipid contentispresent,removelipidlayeraftercentrifugation.Samplemayneeddilutionif too viscous.
Beadspopulationsarenotmixedproperly
Makesureallbeadpopulationsaremixed.and in similar numbers.
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76761_V03
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]
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