legendplex™ · measurement of the adipokines is critical for abetter understanding of disease...
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®
LEGENDplex™Mul�-Analyte Flow Assay Kit
For Accurate Quantification of Multiple Human Th(T helper Cell) Cytokines from Cell Culture Supernatant,
Serum, Plasma and Other Biological Samples
Please read the entire manual before running the assay
BioLegend.com
®
LEGENDplex™Mul�-Analyte Flow Assay Kit
Cat. No. 740233
Non-human Primate (NHP) Adipokine Panel
(13-plex)
For Cell Culture and Urine Samples
Please read the entire manual before running the assay.
BioLegend.com
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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™ NHP Adipokine Panel
Table of Contents Page
Chapter 1: KIT DESCRIPTION..................................................
Introduction……………………………………………..........................
PrincipleoftheAssay……………………....……………....….…......
BeadsUsage...........................................………..……………...
StorageInformation…………………………………….......…..........
MaterialsSupplied………………….....……………….................…
MaterialstobeProvidedbytheEnd-User……...........……...
Precautions.................................……………………................
Chapter 2: ASSAY PREPARATION.............................................
SampleCollectionandHandling…………………………............
ReagentPreparation…………………………………………...............
StandardPreparation..........................................................
SampleDilution…………...........…….......................................
Chapter 3: ASSAY PROCEDURE..................................................
PerformingtheAssayUsingaFilterPlate……………….........
Performing the Assay Using Microtubes, or a 96-Well V- orU-bottomMicroplate…..................................................
Chapter 4: FLOW CYTOMETER SETUP.......................................
Setup Procedure for FACSCaliburTM with Dual Laser..........
Setup Procedure for FACSCaliburTM with a Single Laser.....
Setup Procedure for BD FACSAriaTM, FACSCantoTM and LSR Series..........................................................................
Setup Procedure for Other Flow Cytometers ...................
Chapter 5: DATA ACQUISITION AND ANALYSIS.........................
DataAcquisition..................................................................
Data Analysis.....................................................................
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Chapter 6: FREQUENTLY ASKED QUESTIONS...........................
Chapter 7: ASSAY CHARACTERIZATION..........................................
StandardCurve.………………………………………………………........
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AssaySensitivity...……………………………………………………..…..
Cross-Reactivity……………………………………………………..........
Accuracy.............................................................................
LinearityofDilution………………………………………………..........
Intra-AssayPrecision……………………………………...................
Inter-AssayPrecision……………………………………...................
BiologicalSamples…………………………………………….………....
TROUBLESHOOTING...........................……………………………………...
PLATE MAP...............……………………………………………………...............
RACK MAP....................................................................................
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LEGENDplex™ NHP Adipokine Panel
Chapter 1: KIT DESCRIPTION
Introduction
Adiposetissue(AT)playsanactiveroleinregulatingmetabolismandenergyhomeostasis. AT expresses and secretes numerous soluble factors, including cytokines,chemokinesandhormones,collectivelyreferredtoasadipokinesherein. Altered levels of these proteins have been associated with a variety of metabolicdiseasesandinflammatory-autoimmuneconditions.Theaccuratemeasurementoftheadipokinesiscriticalforabetterunderstanding of disease progression and immune responses.
The LEGENDplexTM NHP Adipokine Panel is a bead-based multiplexassay, uing fluorescence–encodedbeadssuitableforuseonvariousflowcytometers.Thispanelallowssimultaneousquantificationof13adipokinesfromRhesus or Cyno-molgus monkey,includingadiponectin,adipsin,RBP4,MCP-1,IL-1β,IP-10,IL-10,IL-8,leptin,IL-6,IFN-γ,resistinandTNF-α.Thispanelprovideshighsensitiv-ity and broad dynamic range. The panel has been validated for use on urine and cell culture supernatant samples. The panel is not suitable for serum or plasma samplesduetodifferentsampledilutionrequirementsfordifferentanalytes.
The NHP Adipokine Panel isdesignedtoallowflexiblecustomization.Formixandmatch within the panel, please visit www.biolegend.com/legendplex.
This assay is for research use only.
Principle of the Assay
BioLegend’s LEGENDplexTM assays are bead-based immunoassays using the same basic principle as sandwich immunoassays.
Beadsaredifferentiatedbysizeandinternalfluorescenceintensities.Eachbeadisconjugatedwithaspecificantibodyonitssurfaceandservesasthecapturebeadforthatparticularanalyte.Whenaselectedpanelofcapturebeadsismixedandincubatedwithasamplecontainingtargetanalytesspecifictothecaptureantibodies,eachanalytewillbindtoitsspecificcapturebeads.Afterwashing,abiotinylateddetectionantibodycocktailisadded,andeachdetec-tionantibodyinthecocktailwillbindtoitsspecificanalyteboundonthecap-turebeads,thusformingcapturebead-analyte-detectionantibodysandwiches.Streptavidin-phycoerythrin(SA-PE)issubsequentlyadded,whichwillbindtothebiotinylateddetectionantibodies,providingfluorescentsignalintensitiesinproportiontotheamountofboundanalytes.
Sincethebeadsaredifferentiatedbysizeandinternalfluorescenceintensityonaflowcytometer,analyte-specificpopulationscanbesegregatedandquanti-fiedbythePEfluorescentsignal.Theconcentrationofaparticularanalyteisdetemined by a standard curve generated in the same assay.
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Beads Usage
The NHP Adipokine Panelincludestwosetsofbeads.Eachsethasauniquesizethatcanbeidentifiedonflowcytometerbasedontheirforwardscatter(FSC)andsidescatter(SSC)profiles(BeadsAandBeadsB,Figure1).Eachbeadsetcanbefurtherresolvedbasedontheirinternalfluorescenceintensities.TheinternaldyecanbedetectedusingFL3,FL4,orAPCchannel,dependingonthetypeofflowcytometerused.ThesmallerBeadsAconsistsof6beadpopula-tionsandthelargerBeadsBconsistsof7beadpopulations(Figure2-3).
Usingatotalof13beadpopulationsdistinguishedbysizeandinternalfluo-rescent dye, the NHP Adipokine Panelallowssimultaneousdetectionof13adipokinesinonesampletest.Eachanalyteisassociatedwithaparticularbeadset as indicated in Table 1.
Figure1.BeadsDifferentiatedbySize
Beads A = smaller beads
Beads B = larger beads
Figure2.BeadsAClassificationbyFL4
A5 A7 A8
A4
A6
A10
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LEGENDplex™ NHP Adipokine Panel
Figure3.BeadsBClassificationbyFL4
For Beads usage in the panel, please refer to Table 1 below:
Table1.BeadsIDandTargetInformation
Target Bead ID Top Standard Concentrations(ng/mL)
NHPAdiponectin A4 200
NHP Adipsin A5 50
NHPRBP4 A6 50
NHP MCP-1 A7 10
NHPIL-1β A8 10
NHP IP-10 A10 10
NHP IL-10 B2 10
NHP IL-8 B3 10
NHPLeptin B4 10
NHP IL-6 B5 10
NHPIFN-γ B6 10
NHPResistin B7 10
NHPTNF-α B9 10
*BeadIDisusedtoassociateabeadpopulationtoaparticularanalyteintheLEGENDplexTMDataAnalysisSoftware.TheassociationofanalyteandbeadIDwillbedefinedduringthegatingstepofthedataanalysis.
WhenenteringanalyteandbeadIDinfomationduringthegatingstep,alwaysenterinthesequentialorderofthebeadID(e.g,A4,A5,A6...B2,B3,B4...).Please refer to the LEGENDplexTMDataAnalysisSoftwareUserGuideandOnlineHelpfordetails(www.biolegend.com/legendplex).
B4 B5
B6 B7 B3
B9
B2
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StorageInformation
Recommended storage for all original kit components is between 2°C and 8°C. DONOTFREEZEPre-mixedBeads,DetectionAntibodiesorSA-PE.
• Oncethestandardshavebeenreconstituted,immediatelytransfercon-tents into polypropylene vials. DO NOT STORE RECONSTITUTED STAN-DARDS IN GLASS VIALS.
• Uponreconstitution,theleftoverstandardshouldbestoredat≤-70°Cforusewithinonemonth.Avoidmultiple(>2)freeze-thawcycles.Discardanyleftoverdilutedstandards.
Materials Supplied
The LEGENDplexTM kit contains reagents for 100 tests listed in the table below. Whenassayedinduplicate,thisisenoughforan8-pointstandardcurveand40samples.
Kit Components Quantity Volume Part #
Setup Beads 1: FITC Beads 1 vial 1 mL 77840
Setup Beads 2: PE Beads 1 vial 1 mL 77842Setup Beads 3: Raw Beads 1 vial 2 mL 77844
NHP Adipokine Panel Premixed Beads 1bottle 3.5 mL 76601
NHPAdipokinePanelDetectionAntibodies 1bottle 3.5 mL 76604
NHP Adipokine Panel Standard Cock-tail,Lyophilized 1 vial lyophilized 76607
LEGENDplexTM SA-PE 1bottle 3.5 mL 77743LEGENDplexTMAssayBuffer 1bottle 25 mL 77562LEGENDplexTMWashBuffer,20X 1bottle 25 mL 77564Filter plate 1 plate 76187Plate Sealers 4sheets 78101DataAnalysisSoftwareDongle 1 21217NHP Adipokine Panel Manual 1 76599
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Materials to be Provided by the End-User
• Aflowcytometerequippedwithtwolasers(e.g.,a488nmbluelaseror532nmgreenlaseranda633-635nmredlaser)capableofdistinguishing575 nm and 660 nm oraflowcytometerequippedwithonelaser(e.g.,488nmbluelaser)capableofdistinguishing575nmand670nm.
Partiallistofcompatibleflowcytometers:
Flow Cytometer
Reporter Channel
ChannelEmission
ClassificationChannel
Channel Emission
Compensa-tionneeded?
BD FACSCaliburTM(single laser)
FL2 575 nm FL3 670 nm Yes
BD FACSCaliburTM(dual laser)
FL2 575 nm FL4 660 nm No*
BD FACSArrayTM Yellow 575 nm Red 660 nm No*
BD FACSCantoTM
BD FACSCantoTM IIPE 575 nm APC 660 nm No*
BDTMLSR,LSRIIBD LSRFortessaTM
PE 575-585 nm APC 660 nm No*
BD FACSAriaTM PE 575 nm APC 660 nm No*
*Compensationisnotrequiredforthespecifiedflowcytometerswhensetupproperly,butisrecommendedforconsistentresults.
Forsettinguptheaboveflowcytometers,pleasefollowtheFlow Cytom-eter Setup guide in this manual or visit: www.biolegend.com/legendplex.
Forflowcytometersnotlistedhere,theend-userneedstosetupthemachine following similar guidelines. Please refer to Setup Procedure for Other Flow CytometerssectioninChapter4.
• Multichannelpipettescapableofdispensing5μLto200μL
• Reagentreservoirsformultichannelpipette
• Polypropylenemicrofugetubes(1.5mL)
• Laboratory vortex mixer
• Sonicatorbath(e.g.,BransonUltrasonicCleanermodel#B200,orequiva-lent)
• Aluminum foil
• Absorbent pads or paper towels
• Plateshaker(e.g.,Lab-LineInstrumentsmodel#4625,orequivalent)
• Tabletopcentrifuges(e.g.,Eppendorfcentrifuge5415C,orequivalent)
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Iftheassayisrunusingthefilterplateprovided(recommended),
• Avacuumfiltrationunit(MilliporeMultiScreen®HTSVacuumManifold,catalog#MSVMHTS00orequivalent).Instructionsonhowtousethevacuum manifold can be found at the supplier’s website.
• Avacuumsource(minivacuumpumporlinevacuum,e.g.,MilliporeVacuumPump,catalog#WP6111560,orequivalent)
Iftheassayisruninmicrotubes,V-orU-bottom96-wellplate(optional),
• 1.1mLpolypropylenemicroFACStubes,in96-tuberack(e.g.,NationalScientificSupplyCo,catalog#TN0946-01R,orequivalent).
• Centrifugewithaswingingbucketadaptorformicrotiterplatesormicro-tuberacks(e.g.,BeckmanCoulterAllegraTM 6R Centrifuge with MICROPLUS CARRIERadaptorforGH3.8andJS4.3Rotors).
• 96-well,V-orU-bottom,clearpolypropylenemicroplate(lowproteinbind-ing,e.g.,grenierbio-one,Catalog#651201orequivalent).
• Precautions
• Sodiumazidehasbeenaddedtosomereagentsasapreservative.Al-thoughtheconcentrationsarelow,sodiumazidemayreactwithleadandcopperplumbingtoformhighlyexplosivemetalazides.Ondisposal,flushwithalargevolumeofwatertopreventazidebuild-up.
• Donotmixorsubstitutereagentsfromdifferentkitsorlots.Reagentsfromdifferentmanufacturersshouldnotbeusedwiththiskit.
• Donotusethiskitbeyonditsexpirationdate.
• SA-PEandBeadsarelight-sensitive.Minimizelightexposure.
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LEGENDplex™ NHP Adipokine PanelChapter 2: ASSAY PREPARATION
SampleCollectionandHandling
PreparationofTissueCultureSupernatantorUrineSamples:
• Centrifuge the sample to remove debris and assay immediately. If not pos-sible,aliquotandstoresamplesat≤-20°C.Avoidmultiple(>2)freeze/thawcycles.
ReagentPreparation
PreparationofAntibody-ImmobilizedBeads
SonicatethePre-mixedBeadsbottlefor1minuteinasonicatorbathandthen vortex for 30 seconds prior to use. If no sonicator bath is available, in-creasethevortexingtimeto1minutetocompletelyresuspendthebeads.
PreparationofWashBuffer
• Bringthe20XWashBuffertoroomtemperatureandmixtobringallsaltsintosolution.
• Dilute25mLof20XWashBufferwith475mLdeionizedwater.Storeun-usedportionsbetween2°Cand8°Cforuptoonemonth.
StandardPreparation
1. Priortouse,reconstitutethelyophilizedNHPAdipokinePanelStandardCocktailwith250µLAssayBuffer.
2. Mix and allow the vial to sit at room temperature for 10 minutes, and then transfer the standard to an appropriately labeled polypropylene microfuge tube. This will be used as the top standard C7.
3. Label6polypropylenemicrofugetubesasC6,C5,C4,C3,C2andC1,re-spectively.
4.Add75µLofAssayBuffertoeachofthesixtubes.Prepare1:4dilutionofthe top standard by transferring 25 µL of the top standard C7 to the C6 tube and mix well. This will be the C6 standard.
5. Inthesamemanner,performserial1:4dilutionstoobtainC5,C4,C3,C2andC1standards(see the tables below).AssayBufferwillbeusedasthe0pg/mLstandard(C0).
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Tube Serial Dilution
Assay Buffertoadd(µL)
Standard to add
Final Conc.
(pg/mL)*
Final Conc.
(pg/mL)**
Final Conc.
(pg/mL)***
C7 -- -- -- 10,000 50,000 200,000
C6 1:4 75 25 µL of C7 2,500 12,500 50,000
C5 1:16 75 25 µL of C6 625 3,125 12,500
C4 1:64 75 25 µL of C5 156.3 781.3 3,125
C3 1:256 75 25 µLofC4 39.1 195.3 781.3
C2 1:1024 75 25 µL of C3 9.8 48.8 195.3
C1 1:4096 75 25 µL of C2 2.4 12.2 48.8
C0 -- 75 -- 0 0 0
*TopstandardconcentrationofMCP-1,IL-1β,IP-10,IL-10,IL-8,Leptin,IL-6,IFN-γ,ResistinandTNF-αis10ng/mL.
**TopstandardconcentrationofAdipsinandRBP4is50ng/mL.
***TopstandardconcentrationofAdiponectinis200ng/mL.
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LEGENDplex™ NHP Adipokine Panel
SampleDilution
• For cell culture supernatant samples, the levels of analyte can vary greatly from sample to sample. While the samples can be tested without dilu-tions,apreliminaryexperimentmayberequiredtodeterminetheappro-priatedilutionfactor.
Ifsampledilutionisdesired,dilutionshouldbedonewithcorrespondingfreshcellculturemediumorAssayBuffertoensureaccuratemeasure-ment.
• Urinesamplesshouldbedilutedtwo-foldwithAssayBufferbeforetesting(e.g.dilute50µLofsamplewith50µLofAssayBuffer).Furtherdilutionisneededifthesampleconcentrationisabovetheupperlimitofthestandard range.
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Chapter 3: ASSAY PROCEDURE
The LEGENDplexTM assaycanbeperformedeitherinafilterplate,inmicrotubes,orinaV-orU-bottommicroplate.
• Thein-filterplateassayprocedureishighlyrecommendedduetoitsgoodsample to sample consistency, assay robustness and ease of handling. This procedurerequiresavacuumfiltrationunitforwashing(seeMaterials to beProvidedbytheend-user,Page7).IfyouhaveperformedaLuminex®-basedmultiplexassaybefore,yourlabshouldalreadyhavethevacuumfiltrationunitsetup.
• Ifthein-filterplateassayprocedureisnotpossible,orifyouprefer,theas-saycanbeperformedinmicrotubesorinaV-orU-bottommicroplate.Forin-tubeassay,werecommendusingmicroFACStubes(seeMaterials to be Providedbytheend-user,Page7).
Performing the Assay Using a Filter Plate
• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.
• Setthefilterplateonaninvertedplatecoveratalltimesduringassaysetupandincubationstepssothatthebottomoftheplatedoesnottouchanysurface. Touching a surface may cause leakage.
• Keeptheplateuprightduringtheentireassayprocedure,includingthewashing steps, to avoid losing beads.
• The plate should be placed in the dark or wrapped with aluminum foil for allincubationsteps.
• Standards and samples should be run in duplicate and arranged on the plateinaverticalconfigurationconvenientfordataacquisitionandanaly-sis(asshowninattachedPLATEMAP,Page52-53).Besuretoloadstan-dardsinthefirsttwocolumns.Ifanautomationdeviceisusedforread-ing,theorientationandreadingsequenceshouldbecarefullyplanned.
1. Pre-wettheplatebyadding100μLofLEGENDplexTM1XWashBuffertoeach well and let it sit for 1 minute at room temperature. To remove the excess volume, place the plate on the vacuum manifold and apply vacuum. Donotexceed10”Hgofvacuum.Vacuumuntilwellsaredrained(5-10seconds).BlotexcessWashBufferfromthebottomoftheplatebypress-ing the plate on a stack of clean paper towels. Place the plate on top of the inverted plate cover.
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LEGENDplex™ NHP Adipokine Panel
For measuring cell culture supernatant and urine samples:
• Add25µLofAssayBuffertoallwells.• Add 25 µL of each standard to the standard wells. • Add25µLofeachsampletothesamplewells(SeeSampleDilution)
2. Vortexthepre-mixedbeadsbottlefor30seconds.Add25μLofthepre-mixedbeadstoeachwell.Thevolumeshouldbe75μLineachwellafterbeadsaddition.(Note:Duringadditionofthebeads,shakethepre-mixedbeadsbottleintermittentlytoavoidbeadsettling).
3. Seal the plate with a plate sealer. Toavoidplateleaking,donotapplypositivepressuretothesealerwhensealingtheplate.Wraptheentireplate, including the inverted plate cover, with aluminum foil. Place the plate on a plate shaker, secure it and shake at approximate 500 rpm 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 washing step once more.
5. Add25µLofDetectionAntibodiestoeachwell.
6. Sealtheplatewithafreshplatesealer.Wraptheentireplate,includingtheinverted plate cover, with aluminum foil. Place the plate on a plate shaker and shake at approximately 500 rpm for 1 hour at room temperature.
7. Do not vacuum! Add 25 µL of SA-PE to each well directly.
8. Sealtheplatewithafreshplatesealer.Wraptheentireplate,includingtheinverted plate cover, with aluminum foil. Place the plate on a plate shaker and shake at approximate 500 rpm for 30 minutes at room temperature.
9. Repeatstep4above.
10. Add200µLof1XWashBuffertoeachwell(or150µLifplateistobereadbyanautosampler.Highervolumemayresultinleakingofthefilterplateduring reading. Besuretosetthesamplevolumetobeanalyzedto70uLor less so that sample can be read again if needed).Resuspendthebeadson a plate shaker for 1 minute.
11. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay (Note:Prolongedsamplestoragecanleadtoreducedsignal)
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Iftheflowcytometerisequippedwithanautosampler,readtheplatedirectly using the autosampler. The probe height may need to be adjusted when using an autosampler.
If an autosampler is not available, the samples need to be transferred from thefilterplatetoFACStubesandreadmanually.
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/urine 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 pre-mixed beads to all wells
Wash 2 times using vacuum �ltration unitAdd 200 μL (or 150µL) of 1x Wash Bu�er Read on a �ow cytometer
BA
C
A B C
A B C
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PerformingtheAssayUsingMicrotubesora96-WellV-orU-bot-tom Microplate
• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.
• Determine the number of assays to run. Standards and samples should be runinduplicateandarrangedintheorderconvenientfordataacquisitionand analysis (see below). Ifanautomationdeviceisusedforreading,theorientationandreadingsequenceshouldbecarefullyplanned.
Ifusingtubes,labelalltubesandarrangetubesonarack(asshowninat-tached RACK MAP, assuming using micro FACS tubes and a 96-microtube rack,e.g.,NationalScientificSupplyCo.,Catalog#:TN0946-01R).A96-mi-crotuberackisrecommendedbecauseitallowspipettingwithamultichan-nelpipette.
If using a microplate, arrange the standard and samples according to the PLATEMAPattached.
1. For measuring cell culture supernatant and urine samples:
• Add25µLofAssayBuffertoalltubes/wells.• Add25µLofeachstandardtothestandardtubes/wells.• Add25µLofeachsampletothesampletubes/wells(See Sample Dilu-tion).• Add25µLofthepre-mixedbeadstoalltubes/wells.• Add25µLDetectionAntibodiestoalltubes/wells.
Note: Thevolumeshouldbe100μLineachtube/well.Shakebeadsbottleintermittentlyduringtheadditiontoavoidbeadsettling.
2. Covertheentirerack/platewithaluminumfoiltoprotectthetubes/platefrom light. Shake(approximate1,000rpmforin-tubeassayand600rpmforin-plateassay) on a plate shaker for 2 hours at room temperature.
3. Withoutwashingthetubes/plate,add25µLSA-PEtoeachtube/wells.
4. Covertheentirerack/platewithaluminumfoiltoprotectthetubes/platefrom light. Shake(approximate1,000rpmforin-tubeassayand600rpmforin-plateassay)on a plate shaker for 30 minutes at room temperature.
5. Centrifugethetubes/plateat1,000xg for 5 minutes, using a swinging bucketrotorwithmicroplateadaptor(Please refer to Materials to be Pro-videdbytheend-user,Page7).
6. Removethesupernatantusingamicrochannelpipette.Becarefulnottoremove beads, but to remove as much liquid as possible.
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7. Add200µLof1XWashBuffertoalltubes/well.Resuspendthebeadsbyvortexing(forin-tubeassay)orshakingonaplateshakerfor1minute(forin-plateassay).Centrifugethetubes/plateat1,000xg for 5 minutes, using a swinging bucket rotor with microplate adaptor. Remove the supernatant.
8. Add200µLof1XWashBuffertoalltube/well(or 150 µL if plate is to be readwithanautosampler.Highervolumemayresultinleakingofthefil-terplateduringreading.Besuretosetthesamplevolumetobeanalyzedto 70 uL or less so that sample can be read again if needed).Resuspendthebeadsbyvortexing(forin-tubeassay)orpipetting(forin-plateassay).
9. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay(Note:Prolongedsamplestoragecanleadtoreducedsignal).
Iftheflowcytometerisequippedwithanautosampler,thesamplescanbereadeitherdirectly(forin-plateassay)orafterbeingtransfferedtoa96-wellplate(forin-tubeassay).The probe height may need to be adjusted when using an autosampler.
If an autosampler is not available, the samples can be read directly in FACS tubesorafterbeingtransferredfromthemicroplatetomicroFACStubes.
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LEGENDplex™ NHP Adipokine Panel
Assay Procedure Summary for Tubes (or V-Bottom Plate)
Arrange the number of tubes needed on a rack (or set up the plate)
Incubate 2 hours, RT, shaking
Without washing, add 25 μL SA-PEIncubate 30 min, RT, shaking
For cell culture supernatant/urine samples, Add to the tubes/wells:25 μL Assay Bu�er to all tubes/wells25 μL diluted standard to standard tubes/wells25 μL sample to sample tubes/wells25 μL pre-mixed beads to all tubes/wells25 μL Detection Antibodies to all tubes/wells
Spin down beadsWash one timeAdd 200 μL (or 150µL) of 1x Wash Bu�erRead on a �ow cytometer
A B
C
A
B
C
Capture beads
Detection Antibody
Analytes
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Chapter 4: FLOW CYTOMETER SETUP
Inordertogeneratereliabledata,theflowcytometermustbesetupproperlybeforedataacquisition.Thefollowingsectionswilladdressmachinesetupfortheflowcytometerslistedbelow.
ListofFlowCytometersandPossibleConfigurations
Flow CytometerReporterChannel
Channel Emission
ClassificationChannel
ChannelEmission
Compensationneeded?
BD FACSCaliburTM (singlelaser) FL2 575 nm FL3 670 nm Yes
BD FACSCaliburTM (duallaser) FL2 575 nm FL4 660 nm No*
BD FACSCantoTM,BD FACSCantoTM II PE 575 nm APC 660 nm No*
BDTM LSR, LSRII,BD LSRFortessaTM PE
575-585 nm APC 660 nm No*
BD FACSAriaTM PE 575 nm APC 660 nm No*
*Compensationisnotrequiredforthespecifiedflowcytometerswhensetupproperly,butisrecommendedforconsistentresults.
Forflowcytometersnotlistedhere,theend-userneedstosetupthemachinefollowing similar guidelines. Please refer to Setup Procedure for Other Flow Cytometerssectioninthischapter.
The setup process typically includes the following steps. Please see the detailed setupprocedurethatfollows,regardingyourspecificinstrument.
1).Startuptheinstrumentfollowingthemanufacturer’srecommendations.
2).Createatemplatefordataacquisitionusingyourinstrument’sdata acquisitionsoftware.Atemplateisadocumentorworksheetwithdensityplotsthatallowstheusertoperformmachinesetupanddataacquisition.
3).SetupthePMTvoltagesofeachchanneltobeusedfordataacquisition using the Setup Beads provided in the kit.
4).DeterminewhethercompensationisneededbasedontheconfigurationofyoursystemasshowninTable1.Ifcompensationisneeded,perform compensationusingtheSetupBeadsprovidedinthekit.
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LEGENDplex™ NHP Adipokine Panel
Setup Procedure for FACSCaliburTM with Dual Lasers
For a dual laser FACSCaliburTM,useFL2forreporterandFL4forbeadsclassifica-tion.Ingeneral,thereisnoneedforcompensationbetweenthesechannelsifthe machine is set up properly, following the setup procedure described below.
1. Start up the Instrument
Performinstrumentstartupandverificationcheckfollowingthemanufac-turer’srecommendations.
2. ObtainaTemplateforDataAcquisition
A template for FACSCaliburTM is a document with density plot that allows theusertoperformmachinesetupanddataacquisition.
Ifyouhavealreadycreatedatemplatefortheflowcytometer,openthattemplate and proceed to Step 3.
If a template is not yet available, create a new template by following the instructionsbelow:
2.1 FromtheBDCellQuestdataacquisitionsoftware,gotoFile→new document.
2.2 CreateadotplotwithFSC(forwardscatter)forX-axisandSSC(side scatter)forY-axis.Be sure to set FSC and SSC to linear mode. Create twogatesandlabelthemBeadsAandBeadsB(Figure4).
Figure 4.
2.3 Create4fluorescentdotplotsasshownbelow(Figure5)withFL2for X-axis,FL4orFL1forY-axis.Forthefluorescentdotplots,gateon BeadsA(dotplotsontheleftpanelbelow)andBeadsB(dotplotson therightpanelbelow).The dot plots should be in log mode.
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2.4 Savethenewdocumentas“LEGENDplexTemplateforFACSCalibur Dual Laser” and proceed to the next step of the setup.
Figure 5.
3. Set up the PMT Voltages
The Setup Beads 3: Raw Beads are used to set up the PMT voltage of the classificationchannel(FL4/APC)andchannelFL1.TheSetupBeads2:PE
BeadsareusedtosetupthePMTvoltageofthereporterchannel(FL2/PE)The Setup Beads 1: FITC Beads are not needed for this setup.
FollowtheinstructionsbelowforsettingupthePMTsettings:
3.1 Vortex the vial of the Raw Beads for 30 seconds to resuspend the beads.
3.2 Transfer400μLoftheRawBeadstoafreshFACStube.
3.3 Settheflowcytometerflowratetolow.Insetupmode,runthe RawBeads.AdjustthesettingsforFSCandSSCsothatbothbead populationsarevisible(Figure6).
Pauseandrestartacquisitionfrequentlyduringthesetupprocedure torefreshthebeadpopulationsafteradjustingsettings.
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LEGENDplex™ NHP Adipokine Panel Figure 6.
3.4 ContinueadjustingthesettingssothatBeadsAandBeadsBarewell separatedandtheFSCandSSCreadingsare>200.
3.5 Move the gates for Beads A and Beads B so that the smaller beads fall into Beads A gate and the larger beads fall into Beads B gate
(Figure6).
3.6 AdjusttheFL1settingsothattheFL1signalsforallbeadsare between 1x100 and 1x101(Note:Thisstepisnotrequired,butis
recommended).
3.7 AdjusttheFL4settingsothattheFL4signalsforallbeadsarebe- tween 1x101 and 5x103(Figure7).
Figure 7.
3.8 Vortex the vial of Setup Beads 2: PE Beads for 30 seconds to resus- pend the beads.
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3.9 Transfer400μLofthePEBeadstoafreshFACStube.
3.10ReplacetheRawBeadstubefromtheflowcytometerwiththe PE beads tube.
3.11Settheflowcytometerflowratetolow.Insetupmode,runthePE Beads.Note:PEbeadsareonlyofsmallsize,fallingintheBeadsA
gate(Figure8).
3.12AdjusttheFL2settingsothatthemedianfluorescenceintensityof thePEbeadsfallsbetweenthelot-specificrangelabeledonthePE Beadsvial(Figure8,gateR3).
Figure 8.
3.13 Save the document again for future use.
3.14Theflowcytometerisnowreadyforsampleacquisition.
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LEGENDplex™ NHP Adipokine Panel
Setup Procedure for FACSCaliburTM with a Single Laser
For a single laser FACSCaliburTM,useFL2forreporterandFL3forbeadsclassifi-cation.Compensationisneededtoproperlysetuptheinstrument.
1. Start up the Instrument
Performinstrumentstartupandverificationcheckfollowingthemanufac-turer’srecommendations.
2. ObtainaTemplateforDataAcquisition
A template for FACSCaliburTM is a document with density plot that allows theusertoperformmachinesetupanddataacquisition.
Ifyouhavealreadycreatedatemplatefortheflowcytometer,openthattemplate and proceed to Step 3.
If a template is not yet available, create a new template by following the instructionsbelow:
2.1 FromtheBDCellQuestdataacquisitionsoftware,gotoFile→new document.
2.2 CreateadotplotwithFSC(forwardscatter)forX-axisandSSC(side scatter)forY-axis.Be sure to set FSC and SSC to linear mode. Create twogatesandlabelthemBeadsAandBeadsB(Figure9).
Figure9.
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Gated on Beads BGated on Beads A2.3 Create4fluorescentdotplotsasshownbelow(Figure10)withFL2 forX-axis,andFL3orFL1forY-axis.Forthefluorescentdotplots,gate onBeadsA(dotplotsontheleftpanelbelow)andBeadsB(dotplots ontherightpanelbelow).The dot plots should be in log mode.
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Figure 10.
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2.4 Setallcompensationstozero.
2.5 Savethenewdocumentas“LEGENDplexTemplateforFACSCalibur Single Laser” and proceed to the next step of the setup.
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LEGENDplex™ NHP Adipokine Panel3. SetupPMTVoltagesandCompensation
The Setup Beads 3: Raw Beads are used to set up the PMT voltage of the classificationchannelFL3andchannelFL1.TheSetupBeads2:PEBeadsare used to set up the PMT voltage of the reporter channel FL2. All three setupbeadsareneededforsettingupcompensation.
FollowtheinstructionsbelowforsettingupthePMTsettingsandcompen-sation:
3.1 Vortex the vial of the Raw Beads for 30 seconds to resuspend the beads.
3.2 Transfer400μLoftheRawBeadstoafreshFACStube.
3.3 Settheflowcytometerflowratetolow.Insetupmode,runtheRaw Beads.AdjustthesettingsforFSCandSSCsothatbothbeads populationsarevisible(Figure11).
Pauseandrestartacquisitionfrequentlyduringthesetupprocedure torefreshthebeadpopulations,afteradjustingsettings.
Figure 11.
3.4 ContinueadjustingthesettingssothatBeadsAandBeadsBarewell separatedandtheFSCandSSCreadingsare>200.
3.5 Move the gates for Beads A and Beads B so that the smaller beads fall into Beads A gate and the larger beads fall into Beads B gate (Figure11).
3.6 AdjusttheFL1settingsothattheFL1signalsforallbeadsarebe- tween 1x100 and 1x101 (Figure12).
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Figure 12.
3.7 AdjusttheFL2settingssothattheFL2signalsforallbeadsarebe- tween 1x100 and 1x101(Figure12).
3.8 AdjusttheFL3settingsothattheFL3signalsforallbeadsarebe- tween 1x101 and 5 x 103(Figure12).
3.9 Vortex the vial of the Setup Beads 1: FITC Beads for 30 seconds to resuspend the beads.
3.10Transfer200μLoftheFITCBeadstoafreshFACStube.Add200μL ofRawBeadsandmixwell(thiswillgenerateFITC-positiveand FITC-negativepopulationsofbeadsandisneededforproper compensation).
3.11 In setup mode, run the mixed FITC and Raw Beads.
3.12OntheFL1vsFL2dotplot(Figure13),thebeadswilldisplayasFITC- negativeandFITC-positivepopulations(indicatedbyanarrow).
Note:FITCbeadsareonlyofsmallsize,fallingintheBeadsAgate.
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LEGENDplex™ NHP Adipokine Panel Figure 13.
3.13AdjusttheFL2-%FL1compensationsetting(e.g.,FL2-FL1=20%)so thattheFITC-negativeandFITC-positivepopulationshavesimilar meanFL2fluorescenceintensities(Figure14).
Figure 14.
3.14VortexthevialofPEBeadsfor30secondstoresuspendthebeads.
3.15Transfer200μLofthePEBeadstoafreshFACStube.Add200μLof RawBeadsandmixwell(ThiswillgeneratePE-positiveand PE-negativepopulationsofbeadsandisneededforpropercompen- sation).
3.16 In setup mode, run the mixed PE and Raw Beads.
3.17OntheFL1vsFL2dotplot(Figure15),thebeadswilldisplayasPE- negativeandPE-positivepopulations(indicatedbyanarrow).Note: PEbeadsareonlyofsmallsize,fallingintheBeadsAgate.
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Figure 15.
3.18AdjusttheFL2settingsothatthemedianfluorescenceintensityofthe PEbeadsfallsbetweenthelot-specificrangelabeledonthePEBeads vial(Figure16).
3.19AdjusttheFL1-%FL2compensationsetting(e.g.,FL1-FL2=1.5%)so thatthePE-negativeandPE-positivepopulationshavesimilarmean FL1fluorescenceintensities(Figure16).
Figure 16.
3.20OntheFL3vsFL2dotplot(Figure17),thebeadswilldisplayasPE- negativeandPE-positivepopulations(indicatedbyanarrow).
3.21 AdjusttheFL3-%FL2compensationsetting(e.g.,FL3-%FL2=40%)so thattheFL3-lowpopulationandthePE-positivepopulationhave similarmedianFL3signal(Figure18).
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LEGENDplex™ NHP Adipokine Panel Figure 17.
Figure 18.
3.22 Save the document again for future use.
3.23Theflowcytometerisnowcompensatedandreadyforsample analysis.
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Setup Procedure for BD FACSAriaTM, FACSCantoTM and LSR Series
ThispartoftheguideappliestoBDdigitalflowcytometersusingFACSDivaTM softwareversion6.0andabove.
For the BD FACS machines running FACSDivaTM, use the PE channel for reporter andtheAPCchannelforbeadsclassification.Ingeneral,thereisnoneedforcompensationbetweenthesechannelsifthemachineissetupproperlyfollow-ing the setup procedure described below.
Thissetupprocedureisrequiredunderthefollowingsituations:
• YouarerunningtheLEGENDplexkitforthefirsttime.
• It has been over a month since the procedure was last performed.
• Yourflowcytometerhasbeenservicedsinceyoulastperformedthis procedure.
This setup process is not needed if you have run this experiment before and haveaccesstoasavedexperimenttemplate(Thesettingswillbesavedinthefinalstepofthissetupprocedureandanysettingssavedcanbeimportedtoanewexperiment.PleaserefertoStep2andStep3.9below).
1. Start up the Instrument
Performinstrumentstartupandverificationcheckfollowingthemanufac-turer’srecommendations.
2. ObtainaTemplateforDataAcquisition
A template for FACSDivaTM is a worksheet with density plots that allows the usertoperformmachinesetupanddataacquisition.
If a template is not yet available, create a new template by following the instructions.Afteratemplateiscreated,savethefileinD:\BDExport\Tem-plates\Experiment.DonotchangethenameoftheTemplatesfolder.
Ifyouhavealreadycreatedatemplatefortheflowcytometer,openthattemplateandproceedtoStep3.Toopenanexistingtemplate,selectEx-periment>NewExperiment.AlistoftemplatessavedinD:\BDExport\Tem-plates\Experimentwillpopup.Selectthedesiredtemplatefromthelist.
Tocreateanewtemplate,followtheinstructionsbelow:
2.1 From the BD FACSDivaTMsoftware,gotoExperiment>NewExperi- ment.
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LEGENDplex™ NHP Adipokine Panel 2.2 In the global worksheet, open the worksheet. Create a dot plot with
FSC(forwardscatter)forX-axisandSSC(sidescatter)forY-axis.Be sure to set FSC and SSC to linear mode. Create two gates and label themBeadsAandBeadsB(Figure19).
Figure19.
2.3 CreatetwodotplotswithPEforX-axis,APCforY-axis(Figure20), gatedonBeadsA(leftpanelbelow)andBeadsB(rightpanelbelow), respectively.CreateonedotplotwithFITCforX-axis,APCforY-axis, gatedonBeadsAandBeadsB(graphnotshown).The plots should all be in log mode.
Figure 20.
2.4 Savethedocumentas“LEGENDplexTemplateforFACSDiva” inD:\BDExport\Templates\Experimentandproceedtothenextstep of setup.
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3. Set up PMT Voltages
The Setup Beads 3: Raw Beads are used to set up the PMT voltage of the classificationchannelAPC,reporterchannelPE,andFITCchannel.TheSetup Beads 1: FITC Beads and 2: PE Beads are not needed for this setup becausenocompensationisrequiredifthesetupproceduredescribedhereis closely followed.
FollowtheinstructionsbelowforsettingupthePMTsettings:
3.1 Vortex the vial of Raw Beads for 30 seconds to resuspend the beads.
3.2 Transfer400μLoftheRawBeadstoafreshFACStube.
3.3 Settheflowcytometerflowratetolow.RuntheRawBeads.Adjust thesettingsforFSCandSSCsothatbothbeadpopulationsarevisible (Figure21).
Pauseandrestartacquisitionfrequentlyduringthesetupprocedure torefreshthebeadspopulationsafteradjustingsettings.
Figure 21.
3.4 ContinueadjustingthesettingssothatBeadsAandBeadsBarewell separatedandtheFSCandSSCreadingsare>50(x1000).
3.5 Move the gates for Beads A and Beads B so that the smaller beads fall into Beads A gate and the larger beads fall into Beads B gate (Figure21).
3.6 AdjusttheFITCsettingsothattheFITCsignalforthemajorityof beads is between 1x101 and 1x102 (Figure22).
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LEGENDplex™ NHP Adipokine Panel Figure 22.
Beads Beads A+ Beads B
3.7 AdjustthePEsettingsothatthePEsignalforthemajorityofbeadsis between 1x101 and 1x102(Figure23).
Figure 23.
3.8 AdjusttheAPCsettingssothatthetheAPCfluorescenceintensitiesof allbeadpopulationsarebetween1x102 and 5 x 104(Figure23).
3.9 Save the document again for future use.
Tosaveyourassay-specificsettings,inthebrowser,right-click CytometerSettingsandselectSavetoCatalog.Namethefile,and thenclickOK.Toimportthesavedsettingforanewexperiment,right clickoncytometersettingsandselectimportsettings.
3.10Theflowcytometerisnowreadyforsampleacquisition.
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Setup Procedure for Other Flow Cytometers
Forflowcytometersnotaddressedabove,thesetupprocedurewilldifferfromone to another. It is very important for the end-user to set up the machine following similar guidelines. Inthiscase,machinecompensationbetweenthereporterandbeadsclassificationchannelsisstronglyrecommended.
Theflowcytometersetupinstructionsarealsopostedonourwebsite:www.biolegend.com/legendplex. Newflowcytometersetupinstructionswillbeadded to on our website when they become available.
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LEGENDplex™ NHP Adipokine Panel
Chapter 5: DATA ACQUISITION AND ANALYSIS
DataAcquisition
1. Beforereadingsamples,makesurethattheflowcytometerissetupprop-erly.Forflowcytometersetup,pleasefollowtheFlowCytometerSetupguide in this manual or visit: www.biolegend.com/legendplex.
2. Createanewtemplateoropenanexistingtemplate(fordetailsonhowtocreateacytometer-specifictemplate,pleaserefertotheFlowCytometerSetupGuide).
3. Vortex each sample for 5 seconds before analysis.
4.Settheflowratetolow.Setthenumberofbeadstobeacquiredto2000-2500 on Beads A gate or Beads B gate. Do not acquire too many events (e.g.>10,000totalevents).
Note: Do not acquire too few or too many beads. Too few beads acquired may result in high CVs and too many beads acquired may result in slow data analysis later.
Whenreadingsamples,settheflowcytometertosetupmodefirstandwaituntilbeadpopulationisstabilizedbeforeswitchingtoacquisitionmode.
To simplify data analysis using the LEGENDplexTMDataAnalysisSoftware,read samples in the same order as shown on the PLATE MAP or RACK MAP attachedattheendofthemanual.Foranin-plateassay,readcolumnbycolumn(A1,B1,C1...A2,B2,C2...).Foranin-tubeassay,readrowbyrow(A1,A2,A3,...B1,B2,B3...).
Whennamingdatafiles,trytousesimplenameswithaconsecutivenum-beringforeasydataanalysis(e.g.forstandards,C0.001,C0.002,C1.003,C1.004,C2.005,C2.006,C3.007,C3.008,...C7.015,C7.016;forsamples,S1.017,S1.018,S2.019,S2.020,S3.021,S3.022…)
StoreallFCSfilesinthesamefolderforeachassay.Ifrunningmultipleas-says, create a separate folder for each assay.
5. Proceed to data analysis using LEGENDplexTMDataAnalysisSoftwarewhendataacquisitioniscompleted.
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Data Analysis
• TheFCSfilegeneratedonaflowcytometershouldbeanalyzedusingBioLegend’s LEGENDplexTMDataAnalysisSoftwareorothercompatibledataanalysissoftware.TheLEGENDplexTMDataAnalysisSoftwarecanbedownloaded here: www.biolegend.com/legendplex.
• Afterdownloading,installitonaPC(runningWindows7orWindows8)anduseitinconjunctionwiththeDataAnalysisSoftwareDongleincludedin this kit. The dongle has a license key stored in it and is needed to run the software.Tousethedongle,simplyplugitintheUSBportofthecomputeronwhichthedataanalysissoftwareisinstalled,priortolaunchingthesoftware.
• TheSoftwareDonglehasafixednumberofpoints.Eachanalysiswillconsume a certain number of points. The number of points consumed dependsonassayplexsizeandnumberofsamples.Afterthepointsona dongle are consumed, a new dongle will be needed to run more data analysis.Althoughthesoftwarewillcontinuetobefunctional,thedatawillnotbesaveduntilanewdongleisavailable.Anewdongleisprovidedineach LEGENDplexTMkit.Asavedanalysiscanalwaysbere-analyzedusingthesoftwareregardlessofdonglestatus.
• Follow the LEGENDplexTMDataAnalysisSoftwareUserGuideandOnlineHelptousethesoftware(www.bioLegend.com/legendplex; or press F1 for online help at any step of the data analysis).
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LEGENDplex™ NHP Adipokine Panel
Chapter 6: FREQUENTLY ASKED QUESTIONS
Q. WhatisthedifferencebetweenLEGENDplexTM and Luminex®Assays?
BioLegend’s LEGENDplexTM assays and Luminex®-basedmultiplexassaysare both bead-based immunoassays using the same basic principle of sandwichimmunoassays.Bothsystemsusefluorescence-codedbeadstoachievemultiplexing.Themajordifferenceishowthedataisacquired.LEGENDplexTMassaysusecommonlabflowcytometersandtheirrespec-tivesoftwarefordataacquisition,whereasLuminex®-based assays use dedicatedmachinesandsoftwarefordataacquisition.Therefore,oneofthe advantages of LEGENDplexTM assays is that they can be run on common flowcytometersandnospecializedmachineisneeded.
Q. Can I use LEGENDplexTM kits on Luminex®machines?
No. LEGENDplexTMkitsshouldbeusedonaregularflowcytometerandthedatacanbeanalyzedusingtheuser-friendlydataanalysissoftwareavail-ablefordownloadatwww.biolegend.com/legendplex.
Q. WhatarethecompatibleflowcytometersfortheLEGENDplex™assays?
In general, LEGENDplexTMassayscanbeusedonmostcommonflowcytom-eters, such as:
BD FACSCalibur™ BD FACSCanto™ BD FACSCanto™ II BD TM LSR I BD TM LSR II BD LSRFortessaTM BD FACSAria™ BD FACSArrayTM
PleaserefertotheMATERIALSTOBEPROVIDEDBYEND-USERsectionfordetailsonthechannelconfigurationsofthecytometer.
Forflowcytometersnotlistedabove,theenduserneedstomakesure
thatthemachineissetupproperlybeforeuse(refertoSetup Procedure for Other Flow CytometerssectioninChapter4).Inthiscase,machinecompensationbetweenthereporterandbeadsclassificationchannelsisstrongly recommended.
TheFCSorListmodefilesfromthefollowinginstrumentsarecompatiblewith the LEGENDplex TMDataAnalysisSoftware.
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Vendor Instrument Model VendorInstrument
Model
BD
FACScan™ Sony iCyt Eclipse™
FACSCalibur™ Life Technologies Attune®
FACSCanto™ II Miltenyi Biotec MACSQuant®
LSRFortessa™ Partec PAS
LSR II Stratedigm S1400
FACSAria™
Beckman Coulter
CyAn™ ADP
FACSAria™ II FC 500
Accuri™ C6 Gallios™
ORFLO Moxi Flow™ MoFlo®Astrios™
CyteK DxP10™ MoFlo®XDP
Q. Whatistherightprocedureforrunningtheassay?
1. Perform the assay as instructed in this kit manual.
2. Determinethetypeofflowcytometeryouhaveandperformmachinesetup as instructed on Flow Cytometer Setup guide in this manual or visit: www.biolegend.com/legendplex.Openanexistingorcreateamachine-specifictemplatefordataacquisition.
3. AnalyzethesamplesontheflowcytometerandsavetheFCSfilesina new folder.
4. Install the LEGENDplexTM DataAnalysisSoftwarealongwiththesoft-waredongleonaPCwithoperatingsystemWindows7orWindows8(32bitor64bit).Makesuretoinstallthecorrectversion(32bitor64bitversion),dependingonyourcomputer’soperatingsystem.
5. TransferthefoldercontainingtheFCSfilesofyourexperimenttothecomputerwherethedataanalysissoftwareisinstalled.
6. PerformdataanalysisasinstructedintheDataAnalysisSoftwareUserGuide.
Q. WhendoIneedtodomachinecompensation?
Ifaflowcytometerequippedwithasinglelaserisusedforbothreporter(FL2/PE)andclassification(FL3),thencompensationisabsolutelyrequiredto compensate the signal spill over between the two channels, especially from FL2 to FL3. Please use the Setup Beads provided and follow the FLOW CYTOMETER SETUP guide in this manual.
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LEGENDplex™ NHP Adipokine Panel Ifaflowcytometerislistedinthecompatibleflowcytometerlistinthis
manualandisequippedwithtwolasers,oneforreporter(FL2/PE)andtheotherforclassification(FL4/APC),thencompensationisnotrequirediftheFLOW CYTOMETER SETUP guide in this manual is closely followed. How-ever,compensationisrecommendedforconsistentresults.
Forotherflowcytometersnotlistedinthecompatibleflowcytometerlist, the end-user needs to set up the machine following similar guidelines (refertomanufacturer’smanualforproperinstrumentsetup).Inthiscase,machinecompensationbetweenthereporterandbeadsclassificationchannels is strongly recommended.
Q. Isthereaspecialsoftwarerequiredfordataanalysis?
Theflowcytometerrawdatafiles(FCS2.0,3.0)canbeanalyzedusingtheLEGENDplexTMDataAnalysisSoftwareorotherequivalentanalysissoft-ware. Download the LEGENDplexTM DataAnalysisSoftwareforfreehere:www.biolegend.com/legendplex.Asoftwaredonglewithlicensekeyisprovided in the kit.
Fordataacquisition,nospecialsoftwareisneeded.Justusethedataac-quisitionsoftwarethatcomeswiththeflowcytometer,aslongasthedatageneratedisinFCSformat,whichmeetsFCSconvention2.0,3.0and3.1.
Q. CanIselecttomeasureonlysomeanalyteswithinapanel?
Yes.Thetargetswithinapanelarefullycustomizable.Thecustomercanselectanycombinationoftargetswithinapanelandorderacustomizedproduct.Pleaseuseourwebsitetargetselectiontoolfororderingcustom-izedproduct(www.biolegend.com/legendplex).
Q. CanIselecttomeasureanalytesacrosspanels?
Cross-panelcustomizationisalsopossible,aslongasthetotalplexsizeisno more than 13 and there is no overlapping beads region among targets selected.Forcross-panelcustomization,[email protected].
Q. I have run out of a component from the kit. Can I use a similar compo-nentfromadifferentkit?
TheLEGENDplex™Beads,Standards,DetectionAntibodiesandSA-PEarelot-specificandmustbeusedincombinationwitheachother.Donotmixthesecomponentsfromdifferentkitsorlots.
Othercomponents,suchasAssayBuffer,WashBuffer,Plate,andPlateSealerarenotlot-specificandcanthereforebeexchangedbetweendiffer-ent kits and lots.
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Q. My standard curve is not linear; can I use the non-linear part of the stan-dardcurveduringanalysis?
Yes. It is possible to use the non-linear part of the standard curve for calculatingtheresults.TheLEGENDplexTMDataAnalysisSoftwareusesafive-parametercurvefittingalgorithm,whichdeterminestheminimumandmaximumdetectionconcentrationsofeachtargetandreportsthem.Ifsampleconcentrationsfallabovethemaximumdetectableconcentration,thesamplewillhavetobedilutedandreanalyzed.Ifsampleconcentra-tionsfallbelowtheminimumdetectionconcentration,itisconsideredNotDetectablebytheparticularassay.
Q. Duringdataacquisition,whydothebeadpopulations(definedbyFSCandSSC)sometimesappeartobedispersedorshifted?
Thisisusuallycausedbyafastflowrateorasuddenchangeinflowrate.Therearethreeflow-throughsettings:low,medium,andhigh.Ifyouareusingalowflowrateandthenchangetomediumandhighflowrate,thesuddenchangeofflowratemaysometimesresultinadispersedorshiftedbeadpopulation.Therefore,itisnotrecommendedtochangeflowrateduringdataacquisition.Thebestwayistorunthesampleinsetupmodeusinganidealflowrate,andoncethepopulationisstable,thenchangeittoacquisitionmode.
Q. DoestheLEGENDplex™DataAnalysisSoftwarerunonAppleMacintosh?
Notyet.ThecurrentversionofthesoftwarehastorunonaPCwithoperatingsystemWindows7orWindows8(32bitor64bit).IfyourflowcytometerisconnectedtoaMac,afterdataacquisition,theentirefoldercontainingthedata(FCSfiles)shouldbetransferredtoaPCandanalyzed.
Q. Whydoeseachkitincludeasoftwaredongle?
The dongle allows you to use our LEGENDplexTMsoftwarefordataanalysisand each dongle contains 1 million points.
Q. Iranoutofpointsonmydongle.CanIgetanewoneseparately?Or,canIuseadonglefromonekitforanotherkit?
Dongle is not sold separately. Contact BioLegend if you need a new dongle. The dongle can be used across kits.
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biolegend.com 41
LEGENDplex™ NHP Adipokine Panel
Chapter 7: ASSAY CHARACTERIZATION
Standard Curve
This standard curve was generated using the LEGENDplexTM NHP Adipokine Panelfordemonstrationpurposeonly.Astandardcurvemustberunwitheach assay.
AssaySensitivity
Theassaysensitivityorminimumdetectableconcentration(MDC)isthetheoreticallimitofdetectioncalculatedusingtheLEGENDplexTM Data AnalysisSoftwarebyapplyinga5-parametercurvefittingalgorithm.
Analyte MDC (pg/mL)NHPAdiponectin 39.0
NHP Adipsin 4.3
NHPRBP4 3.4
NHP MCP-1 1.3
NHPIL-1β 1.3
NHP IP-10 0.7
NHP IL-10 2.2
NHP IL-8 1.2
NHPLeptin 2.4
NHP IL-6 2.4
1
10
100
1000
10000
1 100 10000
MFI
Concentration (pg/mL)
Adiponectin Adipsin RBP4 MCP-1 IL-1b IP-10 IL-10 IL-8 Leptin IL-6 IFN-r Resistin TNF-a
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LEGENDplex™ NHP Adipokine Panel
42
NHPIFN-γ 1.9
NHPResistin 2.2
NHPTNF-α 0.6
Cross-Reactivity
Therewasnoornegligiblecross-reactivityfoundfortheanalytesinthispanel.
Accuracy (Spike Recovery)
Forspikerecoveryincellculturemedium(CCM),RPMIorDMEMwith10%FCSwasfirstdilutedtwo-foldwithAssayBufferandspikedwithtargetpro-teinsatthreedifferentlevelswithintheassayrange.Thespikedsampleswerethenassayed,andthemeasuredconcentrationswerecomparedwiththe expected values.
For spike recovery in urine, Rhesus and Cynomolgus monkey urine samples (n=16)werefirstdilutedtwo-foldwithAssayBufferandspikedwithtargetproteinsatthreedifferentlevelswithintheassayrange.Thespikedsam-pleswerethenassayed,andthemeasuredconcentrationswerecomparedwith the expected values.
Analyte Recovery in CCM Recovery in Urine NHPAdiponectin 96% 96%NHP Adipsin 94% 69%NHPRBP4 70% NANHP MCP-1 92% 109%NHPIL-1β 91% 62%NHP IP-10 89% 91%NHP IL-10 78% 106%NHP IL-8 108% 116%NHPLeptin 94% 94%NHP IL-6 89% 115%
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LEGENDplex™ NHP Adipokine Panel
NHPIFN-γ 110% 131%NHPResistin 84% 103%NHPTNF-α 87% 105%
NA=NotApplicableduetohighendogenouslevels
LinearityofDilution
ForspikelinearityinCCM,RPMIorDMEMwith10%FCSwasfirstdilutedtwo-foldwithAssayBufferandspikedwithaknownconcentrationoftargetproteins.Thespikedsampleswereseriallydiluted1:2,1:4,1:8withAssaybufferandassayed.Themeasuredconcentrationsofseriallydilutedsamples were compared with that of the spiked samples.
For sprike linearity in urine, Rhesus and Cynomolgus monkey urine samples (n=16)weredilutedtwo-foldwithAssayBufferandspikedwithaknownconcentrationoftargetproteins.Thespikedsampleswereseriallydiluted1:2,1:4,1:8withAssayBufferandassayed.Themeasuredconcentrationsof serially diluted samples were compared with that of the spiked samples.
Analyte Linearity in CCMLinearity in
Urine
NHPAdiponectin 95% 102%
NHP Adipsin 115% 113%NHPRBP4 117% 100%NHP MCP-1 114% 110%NHPIL-1β 114% 105%NHP IP-10 111% 114%NHP IL-10 121% 103%NHP IL-8 119% 108%NHPLeptin 113% 109%NHP IL-6 111% 103%NHPIFN-γ 120% 96%NHPResistin 125% 110%NHPTNF-α 114% 103%
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LEGENDplex™ NHP Adipokine Panel
44
Intra-Assay Precision
Twosampleswithdifferentconcentrationsoftargetproteinswereanalyzedin one assay with 16 replicates for each sample. The intra-assay precision was calculated as below.
Analyte Sample Mean (pg/mL) STDEV %CV
NHP Adiponectin
Sample 1 923.9 108.8 12%Sample 2 3639.8 313.3 9%
NHP Adipsin
Sample 1 204.8 8.5 4%Sample 2 787.0 29.0 4%
NHPRBP4Sample 1 256.5 13.9 5%Sample 2 1024.5 41.9 4%
NHP MCP-1Sample 1 48.0 2.5 5%Sample 2 199.3 9.1 5%
NHPIL-1βSample 1 50.4 3.2 6%Sample 2 202.1 11.2 6%
NHP IP-10Sample 1 65.8 3.7 6%Sample 2 265.7 12.1 5%
NHP IL-10Sample 1 40.4 4.2 10%Sample 2 143.6 7.8 5%
NHP IL-8Sample 1 49.4 3.2 7%Sample 2 195.3 10.1 5%
NHPLeptinSample 1 52.8 3.0 6%Sample 2 201.7 8.8 4%
NHP IL-6Sample 1 51.7 2.9 6%Sample 2 180.5 8.8 5%
NHPIFN-γSample 1 47.5 4.7 10%Sample 2 182.5 9.9 5%
NHPResistinSample 1 52.2 2.4 5%Sample 2 194.7 7.1 4%
NHPTNF-αSample 1 54.2 3.9 7%Sample 2 206.9 10.4 5%
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biolegend.com 45
LEGENDplex™ NHP Adipokine Panel
Inter-Assay Precision
Twosampleswithdifferentconcentrationsoftargetproteinswereanalyzedin three independent assays with 3 replicates for each sample. The inter-assay precision was calculated as below.
Analyte Sample Mean (pg/mL) STDEV %CV
NHP Adiponectin
Sample 1 987.4 131.7 13%Sample 2 3554.8 336.5 9%
NHP Adipsin
Sample 1 214.9 17.5 8%Sample 2 775.2 45.8 6%
NHPRBP4Sample 1 274.3 30.7 11%Sample 2 1020.1 80.5 8%
NHP MCP-1Sample 1 50.9 4.5 9%Sample 2 195.6 14.5 7%
NHPIL-1βSample 1 52.6 4.9 9%Sample 2 205.2 16.4 8%
NHP IP-10Sample 1 76.0 10.6 14%Sample 2 290.3 34.0 12%
NHP IL-10Sample 1 42.4 4.7 11%Sample 2 140.8 10.0 7%
NHP IL-8Sample 1 58.2 8.1 14%Sample 2 201.1 19.1 9%
NHPLeptinSample 1 59.5 7.6 13%Sample 2 212.4 18.2 9%
NHP IL-6Sample 1 55.5 6.2 11%Sample 2 180.2 13.3 7%
NHPIFN-γSample 1 53.4 7.8 15%Sample 2 185.1 17.6 10%
NHPResistinSample 1 55.3 5.0 9%Sample 2 193.9 10.6 5%
NHPTNF-αSample 1 58.6 7.0 12%Sample 2 202.5 16.1 8%
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LEGENDplex™ NHP Adipokine Panel
46
Biological Samples
Urine
NormalRhesusmonkeyurinesamples(n=8)weretestedforendogenouslevelsofadipokines.Theconcentrationsmeasuredareshownbelow:
Analyte Range (pg/mL)% of
Detectable
Mean of detectable
(pg/mL)RhesusAdiponectin 3,244-714,352 100% 187,980
Rhesus Adipsin 104.8-1,159.6 100% 564.0
RhesusRBP4 665.0 - 201,520 100% 93,739
Rhesus MCP-1 3.3-13,484 100% 3,594
RhesusIL-1β 7.4-19.1 100% 11.4
Rhesus IP-10 3.5 - 226 100% 72.7
Rhesus IL-10 2.5 - 325.6 100% 60.9
Rhesus IL-8 ND - 923.9 75% 167.9
RhesusLeptin ND - 12.8 25% 10.8
Rhesus IL-6 ND - 190.1 38% 68.9
RhesusIFN-γ 2.8-31.7 100% 8.5
RhesusResistin 5.6-24,524 100% 7,185
RhesusTNF-α ND -7.7 25% 5.4 ND=NotDetectable
NormalCynomolgusmonkeyurinesamples(n=8)weretestedforendogenouslevelsofadipokines.Theconcentrationsmeasuredareshownbelow:
Analyte Range (pg/mL)% of
Detectable
Mean of detectable
(pg/mL)Cynomolgus Adiponectin 3,996-979,288 100% 302,385
Cynomolgus Adipsin 125.4-6,091 100% 1,767
CynomolgusRBP4 6,013-166,038 100% 106,457
Cynomolgus MCP-1 228.1-11,386 100% 2,396CynomolgusIL-1β 1.8-9.7 100% 5.6
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biolegend.com 47
LEGENDplex™ NHP Adipokine Panel
Cynomolgus IP-10 2.3 - 189.6 100% 40.5
Cynomolgus IL-10 19.7 - 270.5 100% 120.4
Cynomolgus IL-8 ND - 18.0 25% 7.7
CynomolgusLeptin ND - 31.0 38% 16.5
Cynomolgus IL-6 ND 0% ND
CynomolgusIFN-γ ND - 72.0 75% 18.4
CynomolgusResistin 228.8-13,629 100% 6,818
CynomolgusTNF-α ND - 9.9 38% 4.9 ND=NotDetectable
Cell Culture Supernatant
RhesusPBMC(1x106cells/mL)wereculturedundervariousconditions(PHA,5µg/mL;PMA,20ng/mL;LPS, 1 µg/mL;Ionomycin(I),500ng/mL;IFN-γ,100ng/mL).Supernatantswerecollectedafter3days.Allsamplesweredilutedtwo-foldwithAssayBufferandassayedwiththeLEGEND-plexTMNHPAdipokinePanel.Theresults(allinpg/mL)aresummarizedbelow.
Analyte (-) PHA PMA+I LPS IFN-γ + LPS
Rhesus Adiponectin 26.6 ND 24.6 18.8 47.2
Rhesus Adipsin 4,673 897.8 455.3 935.6 1,045
RhesusRBP4 21.2 13.1 20.6 19.0 19.8Rhesus MCP-1 1,964 10,259 1,375 12,832 11,026
RhesusIL-1β ND 54.9 24.7 72.2 91.8Rhesus IP-10 917.9 5,624 1,951 600.3 5,015Rhesus IL-10 7.3 79.2 247.8 75.0 38.9Rhesus IL-8 3,249 12,492 12,907 12,907 12,907RhesusLeptin ND ND ND ND ND
Rhesus IL-6 ND 2146 54.8 2,225 6,191RhesusIFN-γ ND 1,037 8,374 3.3 >13,977RhesusResistin 106.7 89.6 89.0 103.6 107.4RhesusTNF-α 7.2 70.3 454.9 62.4 888.4ND=NotDetectable
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LEGENDplex™ NHP Adipokine Panel
48
CynomolgusPBMC(1x106cells/mL)wereculturedundervariouscondi-tions(PHA,5µg/mL;PMA,20ng/mL;LPS, 1 µg/mL;Ionomycin(I),500ng/mL;IFN-γ,100ng/mL).Supernatantswerecollectedafter3days.Allsamplesweredilutedtwo-foldwithAssayBufferandassayedwiththeLEGENDplexTMNHPAdipokinePanel.Theresults(allinpg/mL)aresumma-rizedbelow.
Analyte (-) PHA PMA+I LPS IFN-γ + LPS
Cynomolgus Adiponectin 47.2 15.4 13.8 31.2 17.0
Cynomolgus Adipsin 7,914 1,506 608.8 1,349 1,648
Cynomolgus RBP4 18.6 20.6 26.3 21.8 22.5
Cynomolgus MCP-1 663.4 11,880 2,465 13,350 11,442
Cynomolgus IL-1β ND 106.7 21.0 100.0 175.9
Cynomolgus IP-10 2,338 6,778 2,247 522.4 6,548
Cynomolgus IL-10 10.2 135.9 458.5 170.4 31.7
Cynomolgus IL-8 1,776 12,907 12,907 12,907 12,907
Cynomolgus Leptin ND ND ND ND ND
Cynomolgus IL-6 2.3 3,080 53.1 2,397 6,191
Cynomolgus IFN-γ ND 126.0 8,909 3.0 >13,977
Cynomolgus Resistin 129.2 126.3 112.3 125.4 134.1
Cynomolgus TNF-α 6.3 151.9 728.1 140.9 3,401
ND=NotDetectable
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biolegend.com 49
LEGENDplex™ NHP Adipokine Panel
TROUBLESHOOTING
Problem Possible Cause Solution
Bead popula-tionshiftingupward or downward dur-ingacquisition
The strong PE signal from high concentra-tionsamplesorstan-dards may spill over to classificationChannel(e.g.,FL3/FL4/APC)and mess up the bead separation.
OptimizeinstrumentsettingsusingKitSetup Beads, and make appropriate com-pensationbetweenchannels.
Filter plate will not vacuum or some wells clogged
Vacuum pressure is insufficientorvacuummanifold does not seal properly.
Increase vacuum pressure such that 0.2mLbuffercanbesuctionedin3-5seconds. Clean the vacuum manifold and make sure no debris on the manifold. Press down the plate on the manifold to make a good seal.
Samples have insoluble particlesorsampleistooviscous(e.g.,serumandplasmasamples)
Centrifuge samples just prior to assay setup and use supernatant. If high lipid contentispresent,removelipidlayeraftercentrifugation.Samplemayneeddilutionif too viscous.
Ifsomewellsarestillcloggedduringwashing, try the following:
1).Addbuffertoallthewells,pipetteupand down the clogged wells and vacuum again.
2).Useapieceofcleanwipe,wipetheun-der side of the clogged wells and vacuum again.
3).Takeathinneedle(e.g.,insulinnee-dle),whileholdingtheplateupward,pokethelittleholeundereachofthecloggedwells and vacuum again. Do not poke too hard or too deep as it may damage the filterandcauseleaking.
Filter plate was used without pre-wet.
Pre-wetplatewithwashbufferbeforerunning the assay.
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Tel: 858-768-5800
LEGENDplex™ NHP Adipokine Panel
50
Insufficientbead count or slow reading
Beads inappropriately prepared
Sonicate bead vials and vortex just prior toaddition.Agitatethepre-mixedbeadsintermittentlyinreservoirwhilepipettingthis into the plate.
Samples cause beads aggregationduetoparticulatematterorviscosity
Centrifuge samples just prior to assay setup and use supernatant. If high lipid contentispresent,removelipidlayeraftercentrifugation.Samplemayneeddilutionif too viscous.
Beads were lost during washing for in-tube assay
Make sure beads are spun down by visu-allycheckthepellet(beadsareinlightblueorbluecolor).Beverycarefulwhenremoving supernatant during washing.
Probemaybepartiallyclogged
Sample probe may need to be cleaned, or if needed, probe should be removed and sonicated.
Plate leaked
Vacuum pressure set too high
Adjust vacuum pressure such that 0.2 mL buffercanbesuctionedin3-5seconds.Do not exceed 10” Hg of vacuum.
Plate set directly on table or absorbent tow-elsduringincubationsorreagentadditions
Set plate on plate holder or raised edge sobottomoffilterisnottouchinganysurface.
Liquid present on the under side of the plate aftervacuum
Afterwashing,pressdownplatefirmlyona stack of clean paper towels to dry the underside of the plate.
Pipettetouchinganddamagedplatefilterduringadditions
Pipettetothesideofwells.
High back-ground
Background wells were contaminated
Avoidcross-wellcontaminationbychang-ingtipsbetweenpipettingwhenperform-ingtheassayusingamultichannelpipette.
InsufficientwashesThe background may be due to non-spe-cificbindingofSA-PE.Increasenumberofwashes.
Debris(FSC/SSC)duringsample acquisi-tion
Debris or platelet may existinsamplesolution
Centrifugesamplesbeforeanalyzingsamples. Remove platelet as much as possible.
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biolegend.com 51
LEGENDplex™ NHP Adipokine Panel
Variationbe-tweenduplicate samples
Beadsaggregation Sonicate and vortex the Beads prior to use.
Multichannelpipettemay not be calibrated or inconsistent pipet-ting
CalibratePipette.Ensuregoodpipettingpractice.Primepipettebeforeusemayhelp.
Plate washing was not uniform
Make sure all reagents are vacuumed out completely in all wash steps.
Samples may contain particulatematters.
Centrifuge samples just prior to assay setup and use supernatant. If high lipid contentispresent,removelipidlayeraftercentrifugation.Samplemayneeddilutionif too viscous.
Low or poor standard curve signal
The standard was in-correctlyreconstituted,stored or diluted
Followtheprotocoltoreconstitute,storeand dilute standard. Double check your calculation.
Wrong or short incuba-tiontime
Ensurethetimeofallincubationswasappropriate.
Signals too high, standard curves satu-rated
PMTvalueforFL2/PEset too high
MakesurethePMTsettingforthere-porter channel is appropriate
Plateincubationtimewas too long Useshorterincubationtime.
Sample read-ings are out of range
Samples contain no or below detectable levels of analyte
Make sure the experiment to generate thesamplesworked.Useproperpositivecontrols.
Samplesconcentrationshigher than highest standard point.
Dilutesamplesandanalyzeagain.
Standard curve was saturated at higher end of curve.
MakesurethePMTsettingforthere-porter channel is appropriate. Use shorter incubationtimeifincubationtimewastoo long
Missed beads populationsduring reading, ordistributionis unequal
Sample may cause some beads to ag-gregate.
Centrifuge samples just prior to assay setup and use supernatant. If high lipid contentispresent,removelipidlayeraftercentrifugation.Samplemayneeddilutionif too viscous.
Beadspopulationsarenot mixed properly
Makesureallbeadpopulationsaremixed.and in similar numbers.
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LEGENDplex™ NHP Adipokine Panel
52
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LEGENDplex™ NHP Adipokine Panel
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LEGENDplex™ Kits are manufactured by BioLegend Inc. 9727 Pacific Heights Blvd.San Diego, CA 92121Tel: 1.858.768.5800Tel: US & Canada Toll-Free: 1.877.Bio-Legend (1.877.246.5343)Fax: 1.877.455.9587Email: [email protected]
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