Human Acute Phase Panel 1 Mix and Match Subpanel · LEGENDplex™ Human Acute Phase Panel 1 Mix and...

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

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

Enabling Legendary Discovery™

750000670_V01

LEGENDplex™Mul�-Analyte Flow Assay Kit


Human Acute Phase Panel 1 Mix and Match Subpanel

Please read the entire manual before running the assay.

BioLegend.com

For Research Purposes Only. Not for use in diagnostic or therapeutic procedures. Purchase does not include or carry the right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of BioLegend is strictly prohibited.

It is highly recommended that this manual be read in its entirety before using this product. Do not use this kit beyond the expiration date.

biolegend.com1

LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel

Table of Contents Page

Chapter 1: KIT DESCRIPTION..................................................

Introduction……………………………………………..........................

PrincipleoftheAssay……………………....……………....….…......

BeadsUsage...........................................………..……………...

StorageInformation…………………………………….......…..........

MaterialsSupplied………………….....……………….................…

MaterialstobeProvidedbytheEnd-User……...........……...

Precautions.................................……………………................

Chapter 2: ASSAY PREPARATION..............................................

SampleCollectionandHandling…………………………............

ReagentPreparation………………..………………………...............

StandardPreparation.........................................................

SampleDilution……...........……............................................

Chapter 3: ASSAY PROCEDURE..................................................

PerformingtheAssayUsingaFilterPlate……………….........

PerformingtheAssayUsingaV-bottomPlate……….........

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

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

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

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

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

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

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

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

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

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

3

3

3

4

6

6

8

9

10

10

10

11

12

13

13

16

19

19

19

20

21

21

21

21

22

23

Tel: 858-768-5800


2

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

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

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

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

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

24

24

25

27

33

biolegend.com3


Chapter 1: KIT DESCRIPTIONIntroduction

Theacutephaseresponseisacomplexsystemicreactiontriggeredbytheonsetofvariouscriticalconditionsincludingtrauma,infection,tissuedamage,inflammation,stress,orneoplasia.Themostimportantchangeofthisresponseisthehighlyincreasedproductionofalargefamilyofproteinsfromtheliver,collectivelyknownasacutephaseproteins.Consideredasapartofinnateimmunity,acutephaseproteinsareresponsibleformanysystemiceffectssuchasanti-proteaseactivities,leukocytosis,complementcascade,increasedcortisol,andmanyothers.Measuringandmonitoringacutephaseproteinsprovideresearchvalueinunderstandingthemechanisms,therapeutics,andprognosis of many diseases.

The LEGENDplexTMHumanAcutePhasePanel1(8-plex)containsfluorescence-encodedbeadssuitableforuseoncommonflowcytometers.Itallowsforthesimultaneousquantificationof8keyacute phase molecules includingα2-microglobulin,α1-AcidGlycoprotein(α1-AGP),Haptoglobin,α1-antitrypsin,Ceruloplasmin,Fibrinogen,Prothrombin,andSerumAmyloidPComponent(SAP).ThisassaypanelprovideshigherdetectionsensitivityandbroaderdynamicrangethantraditionalELISAmethods.Thepanelhasbeenvalidatedforuseoncellculturesupernatant,serum,andplasmasamples.

The Human Acute PhasePanel1isdesignedtoallowflexiblecustomizationwithinthepanel.Pleasevisitwww.biolegend.com/legendplex for more informationonpaneldesignandhowtomixandmatchwithinthepanel.

This assay is for research use only

Principle of the Assay

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

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

Tel: 858-768-5800


4

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

Beads Usage

The Human Acute Phase Panel 1usestwosetsofbeads.Eachsethasauniquesizethatcanbeidentifiedbasedontheirforwardscatter(FSC)andsidescat-ter(SSC)profiles(BeadsAandBeadsB,Figure1).Eachbeadsetcanbefurtherresolvedbasedontheirinternalfluorescenceintensities.TheinternaldyecanbedetectedusingeithertheFL3,FL4,orAPCchannels,dependingonthetypeofflowcytometerused.ThesmallerABeadsconsistsof6beadpopulationsandthelargerBBeadsconsistsof7beadpopulations(Figure2-3).FourPopulationsof each set of beads are used for this panel (indicated as Beads ID in Table 1).

Usingatotalof8beadpopulationsdistinguishedbysizeandinternalfluores-centdye,theHuman Acute Phase Panel 1allowssimultaneousdetectionof8proteinsinasinglesample.Eachanalyteisassociatedwithaparticularbeadsetas indicated (Figures 2-3 and Table 1).

Figure 1. Beads Differentiated by Size

Beads A = smaller beads

Beads B = larger beads

Figure 2. Beads A Classification by FL4

A5 A7 A8

A4

A6

A10

biolegend.com5

LEGENDplex™ Human Acute Phase Panel 1 Mix and Match SubpanelFigure 3. Beads B Classification by FL4

ForBeadsusageinthefullpanel,pleaserefertoTable1below.

Table 1. Panel Targets and Bead ID*

Target Bead ID

Human Acute Phase Panel 1 (8-plex) Mix and

MatchTop Standard

ConcentrationsCat. # 740999 or 741000

α2-macroglobulin A4 √The top standard

concentrationofeachtarget

mayvaryandmaysubject

to change from lot to lot.

Pleaserefertothelot-specific

CertificateofAnalysis

for this

information.

α1-AGP A5 √

Haptoglobin A7 √

α1-antitrypsin A10 √

Ceruloplasmin B3 √

Fibrinogen B4 √

Prothrombin B5 √

SAP B7 √

*BeadIDisusedtoassociateabeadpopulationtoaparticularanalyteintheLEGENDplexTMDataAnalysisSoftware.TheassociationofanalyteandbeadIDwillbedefinedduringthegatingstepofthedataanalysis.

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

B4 B5

B6 B7

B3

B9

B2

Tel: 858-768-5800


6

Storage Information

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

• Oncethestandardshavebeensufficientlyreconstituted,immediatelytransfercontentsintopolypropylenevials.DONOTSTORERECONSTITUT-ED STANDARDS IN GLASS VIALS.

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

Materials Supplied

The LEGENDplexTMkitcontainsreagentsfor100tests,listedinthetablebelow.Whenassayedinduplicate,thisisenoughforan8-pointstandardcurveand40samples.

TheBufferSetcontainsSetupBeads,allBuffers,PlateSealers,SA-PEandDataAnalysisSoftwareDongle.AmanualisalsoprovidedforeachMixandMatchsubpanel.

Table 2: The LEGENDplexTM Kit Human Acute Phase Panel 1 Mix & Match Sub-panel Kit Components

Kit Components Quantity Volume Cat #Capture Beads (see below Table 3 for more information) Varies Varies Varies

HumanAcutePhasePanel1DetectionAntibodies 1bottle 3.5 mL 741001

Human Acute Phase Panel 1 Standard 1 vial Lyophilized 741002

LEGENDplex™BufferSetN(seebelowTable4formoreinformation)

1 -- 741003

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

740379**

Human Acute Phase Panel 1 Mix/Match Sub Manual 1 -- 750000670

*Forkitwithfilterplate.**ForkitwithV-bottomplate.Onlyoneplateispro-videdforeachkit.

biolegend.com7


Table 3: Capture Beads for Mix and Match Subpanels***

Kit Components Quantity Volume Cat.#Humanα2-macroglobulinCaptureBeadA4,13X 1 vial 270 µL 741004

Humanα1-AGPCaptureBeadA5,13X 1 vial 270 µL 741005

HumanHaptoglobinCaptureBeadA7,13X 1 vial 270 µL 741006

Humanα1-antitrypsinCaptureBeadA10,13X 1 vial 270 µL 741007

HumanCeruloplasminCaptureBeadB3,13X 1 vial 270 µL 741008

HumanFibrinogenCaptureBeadB4,13X 1 vial 270 µL 741009

HumanProthrombinCaptureBeadB5,13X 1 vial 270 µL 741010

HumanSAPCaptureBead,B7,13X 1 vial 270 µL 741011 *** Please refer to Panel Targets and Bead ID (Table 1, page 5), toseewhichcapture beads are selected in each panel.

Table 4: LEGENDplex™ Buffer Set N (Cat# 741003 )

Components Quantity Volume Part #Setup Beads 1: FITC Beads 1 vial 1 mL 77840Setup Beads 2: PE Beads 1 vial 1 mL 77842SetupBeads3:RawBeads 1 vial 2 mL 77844LEGENDplexTM SA-PE 1bottle 3.5 mL 77743LEGENDplexTMAssayBuffer 3bottles 75 mL 77562Lyophilized Standard ReconstitutionBuffer 1 vial 1 mL 75241

LEGENDplexTMWashBuffer,20X 1bottle 25 mL 77564DataAnalysisSoftwareDongle 1 -- 21217Plate Sealers 4 sheets -- 78101NoplateisincludedinBufferSetN.Plateneedstobeorderedseparately.Please order the correct type of plate based on the preferred assay protocol (Cat# 740377 or 740378 forFilterPlateandCat#740379forV-bottomPlate).

Tel: 858-768-5800


8

Materials to be Provided by the End-User

• Aflowcytometerequippedwithtwolasers(e.g.,a488nmbluelaseror532nmgreenlaseranda633-635nmredlaser)capableofdistinguishing575 nm and 660 nm oraflowcytometerequippedwithonelaser(e.g.,488nmbluelaser)capableofdistinguishing575nmand670nm.

Partial list of compatible flow cytometers:

Flow Cytometer

Reporter Channel

ReporterEmission

Classification Channel

Channel Emission

Compen-sation

needed?

BD FACSCaliburTM FL2 575 nm FL4 660 nm No*

BD AccuriTM C6 FL2 585 nm FL4 675 nm No*

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

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

GalliosTM PE 575 nm APC 660 nm No*

CytoFLEX PE 585 nm APC 660 nm No*

NovoCyte PE 572 nm APC 660 nm No*

AttuneTM NxT PE 574 nm APC 670 nm No**Compensation is not required for the specified flow cytometers when set up properly.

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

• Multichannelpipettescapableofdispensing5μLto200μL

• Reagentreservoirsformultichannelpipette

• Polypropylene microfuge tubes (1.5 mL)

• MicroFACStubes,1.1mL(iftheflowcytometerdoesnotcontainanautos-ampler)

• Laboratory vortex mixer

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

• Aluminum foil

• Absorbentpadsorpapertowels

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

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

biolegend.com9


If the assay is performed in a filter plate:

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

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

• Ifneeded,additionalFilterplatescanbeorderedfromBioLegend(Cat#740377 or 740378).

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

• Centrifugewithaswingingbucketadaptorformicrotiterplates(e.g.,Beck-man Coulter AllegraTM6RCentrifugewithMICROPLUSCARRIERadaptorforGH3.8 and JS4.3 Rotors) .

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

Precautions

• All blood components and biological materials should be handled as potentiallyhazardous.FollowuniversalprecautionsasestablishedbytheCenterforDiseaseControlandPreventionandbytheOccupationalSafetyandHealthAdministrationwhenhandlinganddisposingofinfectiousagents.

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

• Donotmixorsubstitutereagentsfromdifferentkitsorlots.Reagentsfromdifferentmanufacturersshouldnotbeusedwiththiskit.

• Donotusethiskitbeyonditsexpirationdate.

• SA-PEandbeadsarelight-sensitive.Minimizelightexposure.

Tel: 858-768-5800


10

Chapter 2: ASSAY PREPARATION

Sample Collection and Handling

Preparation of Serum Samples:

• Allowthebloodtoclotforatleast30minutesandcentrifugefor20min-utesat1,000xg.

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

• Whenusingfrozensamples,itisrecommendedthatsamplesbethawedcompletely,mixedandcentrifugedtoremoveparticulatespriortouse.

Preparation of Plasma Samples:

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

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

• Whenusingfrozensamples,itisrecommendedthatsamplesbethawedcompletely,mixedwellandcentrifugedtoremoveparticulates.

Preparation of Cell Culture Supernatant:

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

Reagent Preparation

Preparation of Antibody-Immobilized Beads

1. Theindividualbeads(13X)needtobecombinedwithoneanotheranddilutedwithAssayBuffertocreatea1Xworkingsolutionofbeadspriorto use.

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

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

A. Total volume (µL) = 30x(numberofreactions)

B.Volumeneededfromeach13Xbeadsvial(µL)=2.3 x (number of

biolegend.com11


reactions)

C.AssayBufferneeded(µL)=A–Bx(numberofindividualbeadsvialsto be mixed)

Note:calculationsfortotalvolumeincludea20%excesstoaccountforanylossduringpipetting.

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

A. Total volume (µL) = 30 x 50 = 1500 µL

B. Volume per beads vial needed (µL) = 2.3 x 50 = 115 µL

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

Combine115µLofeachbeadsvial(5vials)with925µLofassaybuffertogetthedesiredfinalvolumeof1500µLof1Xworkingsolutionofbeads.

4. Sonicatepre-mixedBeadsbottlefor1minuteinasonicatorbathandthenvortexfor30secondspriortouse.Ifnosonicatorbathisavailable,increasethevortexingtimeto1minutetocompletelyresuspendthebeads.

Preparation of Wash Buffer

• Bringthe20XWashBuffertoroomtemperatureandmixtobringallsaltsintosolution.

• Dilute25mLof20XWashBufferwith475mLdeionizedwater.Storeun-usedportionsbetween2°Cand8°Cforuptoonemonth.

Standard Preparation

1. Priortouse,reconstitutethelyophilizedHumanAcute Phase Panel 1 Stan-dardwith250μLLyophilizedStandardReconstitutionBuffer

2. Mixandallowthevialtositatroomtemperaturefor10minutes,andthentransfer the standard to an appropriately labeled polypropylene microcen-trifugetube.ThiswillbeusedasthetopstandardC7.

Note: The top standard concentrations of analytes in this panel were set at various concentrations, but may be subject to change from lot to lot (see lot-specific Certificate of Analysis provided in the kit box for details).

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

4. Add75µLofAssayBuffertoeachofthesixtubes.Prepare1:4dilutionofthe top standard by transferring 25 µL of the top standard C7 to the C6

Tel: 858-768-5800


12

tubeandmixwell.ThiswillbetheC6standard.

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

Tube/Standard ID

Serial Dilution

Assay Buffer to add (µL)

Standard to add

Final Conc. (pg/mL)

C7 -- -- -- 10,000

C6 1:4 75 25 µL of C7 2,500

C5 1:16 75 25 µL of C6 625

C4 1:64 75 25 µL of C5 156.25

C3 1:256 75 25 µL of C4 39.01

C2 1:1024 75 25 µL of C3 9.77

C1 1:4096 75 25 µL of C2 2.44

C0 -- 75 -- 0

Sample Dilution

• Serumorplasmasamplesmustbediluted20,000-foldwithAssayBufferasdescribedinthetabelbelow.

Sample 1st Diluton(1:200)

2ndDilution(1 : 100) FinalDilutionFold

Serum,plasma

2 µL + 398 µLAssayBuffer

2µL1stDilution+198µLAssayBuffer 20,000

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

• Forcellculturesupernatantsamples,thelevelsofanalytecanvarygreatlyfrom sample to sample. To test cell culture supernatant samples,a prelimi-naryexperimentmayberequiredtodeterminetheappropriatedilutionfactor.Iffurtherdilutionisdesired,dilutionshouldbedonewithcorre-spondingfreshcellculturemediumorAssayBufferasadiluenttoensureaccurate measurement.

biolegend.com13


Chapter 3: ASSAY PROCEDURE

The LEGENDplexTM assaycanbeperformedinafilterplate,orinaV-bottomplate.

Performing the Assay Using a Filter Plate

• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.

• Setthefilterplateonaninvertedplatecoveratalltimesduringassaysetupandincubationsteps,sothatthebottomoftheplatedoesnottouchanysurface.Touchingasurfacemaycauseleakage.

• Keeptheplateuprightduringtheentireassayprocedure,includingthewashingsteps,toavoidlosingbeads.

• Theplateshouldbeplacedinthedarkorwrappedwithaluminumfoilforallincubationsteps.

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

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

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

Cell Culture Medium orAssayBuffer Standard Sample*

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

For measuring serum or plasma samples:AssayBuffer Standard Sample*

StandardWells 25 µL 25 µL ---

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

Tel: 858-768-5800


14

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

Add to the plate:25 μL Assay Bu�er to all wells25 μL diluted standard to standard wells or 25 μL sample to sample wells25 μL pre-mixed beads to all wells

BA

C

A B C

A B C

biolegend.com15

LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanel3. Vortexmixedbeadsbottlefor30seconds.Add25μLofmixedbeadsto

eachwell.Thevolumeshouldbe75μLineachwellafterbeadsaddition.(Note:Duringadditionofthebeads,shakemixedbeadsbottleintermit-tentlytoavoidbeadsettling).

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

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

6. Add25µLofDetectionAntibodiestoeachwell.

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

8. Do not vacuum!Add25µLofSA-PEtoeachwelldirectly.

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

10. Repeat step 5 above.

11. Add150µLof1XWashBuffertoeachwell.Resuspendthebeadsonaplateshakerfor1minute.

12. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay (Note: Prolonged sample storage can lead to reduced signal).

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

Ifanautosamplerisnotavailable,thesamplescanbetransferredfromthefilterplateto micro FACS (or FACS) tubes and read manually.

Tel: 858-768-5800


16

Performing the Assay Using a V-bottom Plate

• Allowallreagentstowarmtoroomtemperature(20-25°C)beforeuse.

• Keeptheplateuprightduringtheentireassayprocedure,includingthewashingsteps,toavoidlosingbeads.

• Theplateshouldbeplacedinthedarkorwrappedwithaluminumfoilforallincubationsteps.

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

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

Cell Culture Medium orAssayBuffer Standard Sample*

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

For measuring serum or plasma samples:

AssayBuffer Standard Sample*

StandardWells 25 µL 25 µL ---

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

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

3. Sealtheplatewithaplatesealer.Covertheentireplatewithaluminumfoil to protect the plate from light. Shakeat800rpmonaplateshakerfor2 hours at room temperature (Depending on the shaker, the speed may need to be adjusted. The optimal speed is one that is high enough to keep beads in suspension during incubation, but not too high that it may cause sample to spill from the wells).

4. Centrifugetheplateat1050rpm(~250g)for5minutes,usingaswingingbucketrotor(G.H3.8)withmicroplateadaptor(Please refer to Materials to be Provided by the End-User, page 8). Donotuseexcessivecentrifugationspeedasitmaymakeithardertoresuspendbeadsinlatersteps.Make sure the timer of the centrifuge works properly and standby to make sure

biolegend.com17

LEGENDplex™ Human Acute Phase Panel 1 Mix and Match Subpanelthe centrifuge reaches preset speed.

5. Immediatelyaftercentrifugation,dumpthesupernatantintoabiohazardwastecontainerbyquicklyinvertingandflickingtheplatein one continu-ous and forceful motion.Thebeadspelletmayormaynotbevisibleafterdumping the supernatant. Loss of beads should not be a concern as the beadswillstayinthetipofthewellnicely.Blottheplateonastackofcleanpapertowelanddraintheremainingliquidfromthewellasmuchaspos-sible. Be careful not to disturb the bead pellet.

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

6. Washtheplatebydispensing200μLof1XWashBufferintoeachwellandincubate for one minute. Repeatstep4and5above.Asecondwashisoptional,butmayhelpreducebackground.

7. Add25µLofDetectionAntibodiestoeachwell.

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

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

10. Sealtheplatewithanewplatesealer.Wraptheentireplatewithaluminumfoilandshaketheplateonaplateshakeratapproximate800rpmfor30minutes at room temperature.

11. Repeatstep4,and5.

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

13. Add150µLof1XWashBuffertoeachwell.Resuspendthebeadsbypipet-ting.

14. Readsamplesonaflowcytometer,preferablywithinthesamedayoftheassay (Note: Prolonged sample storage can lead to reduced signal).

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.

Tel: 858-768-5800


18

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 time Add 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 to sample wells25 μL diluted standard to standard wells or 25 μL sample to sample wells25 μL mixed beads to all wells

BA

C

A B C

A B C

biolegend.com19


Chapter 4: FLOW CYTOMETER SETUP

Inordertogeneratereliabledata,theflowcytometermustbesetupproperlybeforedataacquisition.

Thesetupinstructionshavebeenremovedfromthismanualanduploadedontoourwebsitetosavepaper.

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

Chapter 5: DATA ACQUISITION AND ANALYSIS

Data Acquisition

1. Beforereadingsamples,makesurethattheflowcytometerissetupprop-erly.

2. Createanewtemplateoropenanexistingtemplate(fordetailsonhowtocreateacytometer-specifictemplate,pleaserefertotheFlowCytometerSetup Guide).

3. Vortex each sample for 5 seconds before analysis.

4. Settheflowratetolow.Setthenumberofbeadstobeacquiredtoabout300peranalyte(e.g.,acquire2,100beadsfora7-plexassayor3,000beadsfor a 13-plex assay). Do not set to acquire total events as samples may containlargeamountsofdebris.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. Read samples.

Whenreadingsamples,settheflowcytometertosetupmodefirstandwaituntilbeadpopulationisstabilizedbeforerecordingorswitchingtoacquisi-tionmode.

To simplify data analysis using the LEGENDplexTMDataAnalysisSoftware,readsamplesinthesameorderasshownonthePLATEMAPattachedattheendofthemanual.Foranin-plateassay,readcolumnbycolumn(A1,B1,C1...A2,B2,C2...).

Tel: 858-768-5800


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. Proceed to data analysis using LEGENDplexTMDataAnalysisSoftwarewhendataacquisitioniscompleted.

Data Analysis

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

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

• ForMacusers,installonaMacOSXversion10.7(Lion)orlaterandyouwillbepromotedtorequestasoftwarelicensekeyafterthesoftwareinstallation.

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

biolegend.com21


Chapter 6: ASSAY CHARACTERIZATIONRepresentative Standard Curve

ThisstandardcurvewasgeneratedusingtheLEGENDplexTM Human Acute PhasePanel1fordemonstrationpurposesonly.Astandardcurvemustberunwitheachassay.

5.0

50.0

500.0

5000.0

1 10 100 1000 10000 100000 1000000 10000000

MFI

Concentration (pg/ml)

A4.α2-macroglobulin

A5.α1-AGP

A8. Haptoglobin

A10.α1-antitrypsin

B3.Ceruloplasmin

B4.Fibrinogen

B5.Prothrombin

B7.SAP

Assay SensitivityTheassaysensitivityorminimumdetectableconcentration(MDC)isthetheoreticallimitofdetectioncalculatedusingtheLEGENDplexTM Data AnalysisSoftwarebyapplyinga5-parametercurvefittingalgorithm.

AnalyteMDC (pg/mL) (n=12)

Mean STDEV

α2-macroglobulin 232.7 89.9

α1-AGP 31.2 12.4

Haptoglobin 6.1 2.7

α1-antitrypsin 26.1 15.4

Ceruloplasmin 107.6 47.2

Fibrinogen 27.7 15.6

Prothrombin 105.4 34.0

SAP 1.4 0.5

Cross-ReactivityTargethumanproteinsweretestedindividuallyattheindicatedconcen-trationsbelowusingtheLEGENDplexTM Human Acute Phase Panel 1,withnegligiblecross-reactivityobservedfornon-intendedtargets.

Tel: 858-768-5800


22

Analyte Conc. (ng/mL)

α2-macroglobulin 40,000

α1-AGP 4,000

Haptoglobin 1,000

α1-antitrypsin 4,000

Ceruloplasmin 16,000

Fibrinogen 4,000

Prothrombin 10,000

SAP 200

Thefollowingrecombinantproteinsweretestedindividuallyatatleast50ng/mL.Noornegligiblecross-reactivitywasfound.

Human

PAI-1 SP-D Adiponectin MCP-1

Ferritin BPI Adipsin IL-1β

CRP LBP NGAL IL-6

Properdin CD14 MMP-2 IL-8

tPA Procalcitonin OPN IL-10

SAA MIF MPO IL-12 (p70)

α1-antichymotrypsin CD141 IGFBP-4 IL-17A

β2-microglobulin α1-microglobulin ICAM-1 IL-18

Myoglobin suPAR VCAM-1 IL-23

Antithrombin Leptin MMP-9 IL-33

Plasminogen Proinsulin CystatinC TNF-α

FactorXIII RBP4 Myoglobin IFNγ

PTX3 Resistin MRP8/14 IFNα2

Accuracy (Spike Recovery)

Forspikerecoveryincellculturemedium(n=2),RPMIorDMEMwith10%FBSwerespikedwithtargetrecombinantproteinsatthreedifferentlevelswithintheassayrange.Thespikedsampleswerethenassayed,andmea-suredconcentrationswerecomparedwiththeexpectedvalues.

biolegend.com23


Analyte % of Spike Recovery

α2-macroglobulin 102%

α1-AGP 95%

Haptoglobin 96%

α1-antitrypsin 94%

Ceruloplasmin 92%

Fibrinogen 97%

Prothrombin 95%

SAP 94%

Linearity of Dilution

Serum(n=10)andplasma(n=28)sampleswereinitiallydiluted20,000-foldwithAssayBuffer,thenseriallydiluted2,4,and8foldwithAssayBufferand assayed.

Cellculturesamples(n=2)werespikedwithtargetproteinswithknownconcentrationsintheassayrange,thenseriallydiluted2,4,and8foldwithAssayBufferandassayed.

Themeasuredconcentrationsofseriallydilutedsampleswerethencomparedwiththeconcentrationofthelowestdilutionbasedonserialdilutionfactorused.

Analyte% Linearity

Serum Plasma Cell Culture

α2-macroglobulin 102% 101% 86%

α1-AGP 97% 102% 111%

Haptoglobin 92% 96% 101%

α1-antitrypsin 104% 108% 107%

Ceruloplasmin 112% 93% 128%

Fibrinogen NA* 92% 90%

Prothrombin NA* 82% 92%

SAP 109% 105% 101%*Serum is not claimed as a sample type for Fibrinogen and Prothrombin

Tel: 858-768-5800


24

Intra-Assay Precision

Twosampleswithdifferentconcentrationsofeachtargetproteinwereanalyzedinoneassaywith16replicatespersample.Theintra-assayprecisionisshownbelow.

Analyte Sample Mean (pg/mL) STDEV %CV

α2-macroglobulinSample 1 8,744.5 370.8 4%

Sample 2 35,240.5 1,235.5 4%

α1-AGPSample 1 870.4 53.1 6%

Sample 2 3,636.5 129.8 4%

HaptoglobinSample 1 252.0 8.5 3%

Sample 2 946.1 29.2 3%

α1-antitrypsinSample 1 945.2 48.5 5%

Sample 2 3,668.5 137.8 4%

CeruloplasminSample 1 3,598.6 169.9 5%

Sample 2 13,861.8 637.2 5%

FibrinogenSample 1 1,168.4 50.7 4%

Sample 2 4,474.9 188.4 4%

ProthrombinSample 1 2,509.0 144.5 6%

Sample 2 10,141.4 497.5 5%

SAPSample 1 58.7 4.2 7%

Sample 2 224.8 14.3 6%

Inter-Assay Precision

Twosampleswithdifferentconcentrationsofeachtargetproteinwereanalyzedinsixindependentassayswithfourreplicatespersample.Theinter-assayprecisionisshownbelow.

Analyte Sample Mean (pg/mL) STDEV %CV

α2-macroglobulinSample 1 12,363.9 2,196.6 18%

Sample 2 46,666.9 8,082.5 17%

α1-AGPSample 1 1,190.7 221.4 19%

Sample 2 4,676.7 666.0 14%

biolegend.com25


HaptoglobinSample 1 318.9 50.8 16%

Sample 2 1,119.4 166.7 15%

α1-antitrypsinSample 1 1,284.2 218.3 17%

Sample 2 4,671.0 722.9 15%

CeruloplasminSample 1 4,816.7 982.5 20%

Sample 2 17,926.3 3,221.1 18%

FibrinogenSample 1 1,525.9 298.5 20%

Sample 2 5,420.4 1,074.9 20%

ProthrombinSample 1 3,783.0 855.6 23%

Sample 2 13,069.8 2,469.0 19%

SAPSample 1 85.7 23.7 28%

Sample 2 291.5 63.8 22%

Biological Samples

Thevaluesinthissectionareprovidedforreferenceonly.Theassayspro-videdinthiskitareintendedforresearchuseonly.

Serum and plasma (samples are paired)

Normalhumanserumsamples(n=20)weretestedforendogenouslevelsoftheproteins.Theconcentrationsareshownbelow.

Analyte Range (ng/mL) % Detectable Mean (ng/mL)

α2-macroglobulin 1029.6-7730.9. 100% 2466.7

α1-AGP 384.3-2489.0 100% 925.9

Haptoglobin 77.3-3061.1 100% 1200.5

α1-antitrypsin 577.5-4823.8 100% 1386.8

Ceruloplasmin 209.0-848.9 100% 416.9

SAP 6.8-40.2 100% 20.3

Serum is not claimed as a sample type for Fibrinogen and Prothrombin

Tel: 858-768-5800


26

Normalhumanplasma(Heparin,Citrate,andEDTA)samples(n=60)weretestedforendogenouslevelsoftheproteins.Theconcentrationsareshownbelow.

Analyte Range (μg/mL) % Detectable Mean (μg/mL)

α2-macroglobulin 615.9-4761.8 100% 2100.4

α1-AGP 168.6-2129.4 100% 726.5

Haptoglobin 48.7-3644.1 100% 1175.5

α1-antitrypsin 300.7-3022.8 100% 1088.5

Ceruloplasmin 75.6-1388.1 100% 417.7

Fibrinogen 176.4-7101.6 100% 2431.3

Prothrombin 20.6-256.3 100% 120.7

SAP 2.8-47.2 100% 16.6

Cell Culture Supernatant

Human HepG2 cells (1x106cells/mL)wereculturedunderLPS(1µg/mL),IL-6(10ng/mL),IL-1β(10ng/mL),andTNF-α(25ng/mL)stimulationswithunstimulatedcellsasacontrol.CellCulturesupernatantswerecollected 24hourafterstimulationand assayed. The results (all ng/mL) are sum-marizedbelow.

Analyte Control LPS IL-6 IL-1β TNF-α

α2-macroglobulin 170.0 199.9 266.6 523.3 264.2

α1-AGP 552.3 465.6 620.8 973.4 637.8

Haptoglobin 259.5 224.7 1,286.0 827.9 403.2

α1-antitrypsin 1,298.0 1,165.0 1,286.0 1,346.0 1,241.0

Ceruloplasmin 194.6 167.0 248.1 437.1 273.8

Fibrinogen 330.8 259.5 1,055.0 820.5 271.4

Prothrombin 465.6 371.8 449.1 1,165.0 518.6

SAP 7.3 6.9 7.5 7.8 7.2

biolegend.com27


TROUBLESHOOTING

Problem Possible Cause Solution

Bead popula-tionshiftingupwardordownwarddur-ingacquisition

The strong PE signal from high concentra-tionsamplesorstan-dards may spill over to classificationChannel(e.g.,FL3/FL4/APC)and mess up the bead separation.

OptimizeinstrumentsettingsusingKitSetupBeads,andmakeappropriatecom-pensationbetweenchannels.

Filterplatewillnot vacuum orsomewellsclogged

Vacuum pressure is insufficientorvacuummanifold does not seal properly.

Increase vacuum pressure such that 0.2 mLbuffercanbesuctionedin3-5seconds.Cleanthevacuummanifoldandmakesurenodebrisonthemanifold.Pressdowntheplateonthemanifoldtomakeagoodseal.

Samples have insoluble particlesorsampleistooviscous(e.g.,serumand plasma samples)

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

Ifsomewellsarestillcloggedduringwash-ing,trythefollowing:

1).Addbuffertoallthewells,pipetteupanddownthecloggedwellsandvacuumagain.

2).Useapieceofcleanwipe,wipetheun-dersideofthecloggedwellsandvacuumagain.

3).Takeathinneedle(e.g.,insulinneedle),whileholdingtheplateupward,pokethelittleholeundereachofthecloggedwellsandvacuumagain.Donotpoketoohardortoodeepasitmaydamagethefilterandcauseleaking.

Filterplatewasusedwithoutpre-wet.

Pre-wetplatewithwashbufferbeforerun-ning the assay.

Tel: 858-768-5800


28

Insufficientbead count or slowreading

Beads inappropriately prepared

Sonicatebeadvialsandvortexjustpriortoaddition.Agitatemixedbeadsintermit-tentlyinreservoirwhilepipettingthisintothe plate.

Samples cause beads aggregationduetoparticulatematterorviscosity.


Beadswerelostduringwashingforin-tubeassay

Makesurebeadsarespundownbyvisu-allycheckthepellet(beadsareinlightblueorbluecolor).Beverycarefulwhenremovingsupernatantduringwashing.

Probe might be par-tiallyclogged.

Sampleprobemayneedtobecleaned,orifneeded,probeshouldberemovedandsonicated.

Plateleaked

Vacuum pressure set too high

Adjustvacuumpressuresuchthat0.2mLbuffercanbesuctionedin3-5seconds.Donot exceed 10” Hg of vacuum.

Plate set directly on tableorabsorbenttow-elsduringincubationsorreagentadditions

Set plate on plate holder or raised edge sobottomoffilterisnottouchinganysurface.

Liquid present on the under side of the plate aftervacuum

Afterwashing,pressdownplatefirmlyonastackofcleanpapertowelstodrytheunderside of the plate.

Pipettetouchinganddamagedplatefilterduringadditions.

Pipettetothesideofwells.

HighBack-ground

Backgroundwellswerecontaminated

Avoidcross-wellcontaminationbychang-ingtipsbetweenpipettingwhenperform-ingtheassayusingamultichannelpipette.

InsufficientwashesThebackgroundmaybeduetonon-specificbindingofSA-PE.Increasenumberofwashes.

Debris (FSC/SSC) during sample acquisi-tion

Debris or platelet may exist in sample solu-tion.

Centrifugesamplesbeforeanalyzingsamples. Remove platelet as much as possible.

biolegend.com29


Variationbe-tweenduplicate samples

Beadsaggregation Sonicate and vortex the Beads prior to use.

Multichannelpipettemay not be calibrated or inconsistent pipet-ting

CalibratePipette.Ensuregoodpipettingpractice.Primepipettebeforeusemayhelp.

Platewashingwasnotuniform

Makesureallreagentsarevacuumedoutcompletelyinallwashsteps.

Samples may contain particulatematters.


Loworpoorstandard curve signal

Thestandardwasin-correctlyreconstituted,stored or diluted

Followtheprotocoltoreconstitute,storeanddilutestandard.Doublecheckyourcalculation.

Wrongorshortincuba-tiontime

Ensurethetimeofallincubationswasappropriate.

Signals too high,standardcurves satu-rated

PMT value for FL2/PE set too high

MakesurethePMTsettingforthere-porter channel is appropriate

Plateincubationtimewastoolong Useshorterincubationtime.

Sample read-ings are out of range

Samples contain no or belowdetectablelevelsof analyte

Makesuretheexperimenttogeneratethesamplesworked.Useproperpositivecontrols.

Samplesconcentrationshigher than highest standard point.

Dilutesamplesandanalyzeagain.

Standardcurvewassaturated at higher end of curve.

MakesurethePMTsettingforthere-porter channel is appropriate. Use shorter incubationtimeifincubationtimewastoolong

Missed beads populationsduringreading,ordistributionis unequal

Sample may cause some beads to ag-gregate.


Beadspopulationsarenot mixed properly

Makesureallbeadpopulationsaremixed.and in similar numbers.

Tel: 858-768-5800


30

Notes

biolegend.com31


Tel: 858-768-5800


32

Notes

biolegend.com33


PLA

TE M

AP

(for

in-p

late

ass

ay)

1

2 3

4 5

6 7

8 9

10

11

12

A

C0

C4

Sa

mpl

e1

Sa

mpl

e5

Sa

mpl

e9

Sa

mpl

e 13

Sa

mpl

e17

Sa

mpl

e21

Sa

mpl

e25

Sa

mpl

e29

Sa

mpl

e33

Sa

mpl

e 37

B

C0

C4

Sa

mpl

e1

Sa

mpl

e5

Sa

mpl

e9

Sa

mpl

e 13

Sa

mpl

e17

Sa

mpl

e21

Sa

mpl

e25

Sa

mpl

e29

Sa

mpl

e33

Sa

mpl

e37

C

C1

C5

Sa

mpl

e2

Sa

mpl

e6

Sa

mpl

e10

Sa

mpl

e 14

Sa

mpl

e18

Sa

mpl

e22

Sa

mpl

e26

Sa

mpl

e30

Sa

mpl

e34

Sa

mpl

e38

D

C1

C5

Sa

mpl

e2

Sa

mpl

e6

Sa

mpl

e10

Sa

mpl

e 14

Sa

mpl

e18

Sa

mpl

e22

Sa

mpl

e26

Sa

mpl

e30

Sa

mpl

e34

Sa

mpl

e38

E

C2

C6

Sa

mpl

e3

Sa

mpl

e7

Sa

mpl

e11

Sa

mpl

e 15

Sa

mpl

e19

Sa

mpl

e23

Sa

mpl

e27

Sa

mpl

e31

Sa

mpl

e35

Sa

mpl

e39

F

C2

C6

Sa

mpl

e3

Sa

mpl

e7

Sa

mpl

e11

Sa

mpl

e 15

Sa

mpl

e19

Sa

mpl

e23

Sa

mpl

e27

Sa

mpl

e31

Sa

mpl

e35

Sa

mpl

e39

G

C3

C7

Sa

mpl

e4

Sa

mpl

e8

Sa

mpl

e12

Sa

mpl

e 16

Sa

mpl

e20

Sa

mpl

e24

Sa

mpl

e28

Sa

mpl

e32

Sa

mpl

e36

Sa

mpl

e40

H

C3

C7

Sa

mpl

e4

Sa

mpl

e8

Sa

mpl

e12

Sa

mpl

e 16

Sa

mpl

e20

Sa

mpl

e24

Sa

mpl

e28

Sa

mpl

e32

Sa

mpl

e36

Sa

mpl

e40

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

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


750000670_V01

Human Acute Phase Panel 1 Mix and Match Subpanel · LEGENDplex™ Human Acute Phase Panel 1 Mix and...

Documents

Transcript of Human Acute Phase Panel 1 Mix and Match Subpanel · LEGENDplex™ Human Acute Phase Panel 1 Mix and...