the bd cytometric bead array system (cba) - bdj.co.jp · a multiplex bead system you can count on...

The BD™ Cytometric Bead Array System (CBA)

Table of Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

A Multiplex Bead System You Can Count On . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

BD™ Cytometric Bead Array (CBA) Assay Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Quality is Built in to Every BD CBA Product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Standardization of BD CBA Standards to International (NIBSC) Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

Non-human Primate Cross-reactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

BD FACSArray™ Bioanalyzer is Ideal for BD CBA Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

BD CBA Assay Troubleshooting Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

Product Listing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

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Microsoft and Windows are registered trademarks of Microsoft Corporation.For Research Use Only. Not for use in diagnostic or therapeutic procedures.All applications are either tested in-house or reported in the literature. See Technical Data Sheets for details.Purchase does not include or carry any 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 Becton Dickinson and Company is strictly prohibited.BD flow cytometers are class I (1) laser productsBD, BD Logo and all other trademarks are the property of Becton, Dickinson and Company. ©2004 BD

A Multiplex Bead System You Can Count On

BD Biosciences has added a new twist to counting with beads. Werecognize the value of precious samples and data reproducibility,so we’ve developed a technology to ensure both. The innovativeBD™ Cytometric Bead Array (CBA) technology allows forquantitative detection of multiple analytes in a single serum,plasma, tissue culture supernatant, or cell lysate sample. TheBD™ CBA System of assay kits, flow cytometers, and easy-to-usesoftware provides reproducible data and reliable performance thatyou can count on time and time again.

Introduction

Unless otherwise specified, all products are for Research Use Only.Not for use in diagnostic or therapeutic procedures. Not for resale.

• Get multiple results from a singlesmall-volume sample

• Run one standard mixture to generatestandard curves for all your analytes

• Avoid artifacts associated withenzyme-dependent signal generation

• Achieve quantitative results with lesstime and labor

• Combine versatile flow cytometerswith ready-to-use kits and analysissoftware

• Automate sample acquisition andincrease throughput with the plate-based BD FACSArray bioanalyzer orother BD flow cytometers equippedwith the high throughput sampler(HTS) option.

With the BD™ Cytometric Bead Array(CBA) System You Can:

Flow cytometry is an analytical tool that allows for thediscrimination of different particles on the basis of size andcolor. The BD™ CBA employs a series of particles with discretefluorescence intensities to simultaneously detect multiple solubleanalytes from a single serum, plasma, or tissue culturesupernatant sample. The BD CBA, combined with flow cytometry,creates a powerful multiple analyte (multiplex) assay system. TheBD CBA system uses the sensitivity of amplified fluorescencedetection by flow cytometry to measure soluble analytes with aparticle-based immunoassay. The combined advantages of thebroad dynamic range of fluorescence detection via flowcytometry, and the efficient capturing of analytes via suspendedparticles coated with distinct capture antibodies enable theBD CBA to use fewer sample dilutions to determine analyteconcentration in substantially less time (compared toconventional ELISA).

The specific capture beads are mixed with the phycoerythrin (PE)conjugated detection antibodies and then incubated withrecombinant protein standards or test samples to form sandwichcomplexes. Following acquisition of sample data using the flowcytometer, the sample results are generated in graphical andtabular format using the BD CBA Analysis Software.

BD CBA Assay Overview

4 www.bdbiosciences.comUnless otherwise specified, all products are for Research Use Only.

Not for use in diagnostic or therapeutic procedures. Not for resale.

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A C Q U I R E

BD CBAFigure 1. Typical BD CBA assay protocol. A singleanalyte is shown for representational purposes.

The powerful BD™ Cytometric BeadArray (CBA) assays enable multiplexanalysis of complex biological sampleson a flow cytometer. Each assay has beenstringently developed for ease-of-use,broad instrument compatibility and rapiddata analysis, sensitivity, reproducibility,and quality.

As with any complex assay, the ability ofthe novice user to become comfortablewith the assay is critical. In the BD CBAsystem, ease-of-use has been engineeredinto each and every kit. The standards foreach multiplex kit are provided togetherin a lyophilized format that yields a ready-to-use stock standard solution uponreconstitution. Each kit includes a pre-diluted detection reagent containing eachdetector antibody formulated at theiroptimal concentration. Even the assayprotocol has been designed to limithandling steps (Figure 1) and hands-ontime by allowing the researcher to add allkit reagents, samples, and/or standards totheir assay well or tube at the same time.The assay is incubated and thencompleted with a short wash stepfollowed by sample analysis on a flowcytometer.

Every BD CBA assay is compatible withany flow cytometer that is equipped witha 488 nm laser and capable of detectingand distinguishing fluorescence emissionsat 576 and 670 nm. In addition, analysissoftware capable of saving data in an FCS

2.0 file format is required. This includesall of the BD flow cytometry systems,however, it should be noted that stream-in-air instruments will yield lower assaysensitivities than other instruments. Thedata collected on a flow cytometer, oncesaved as an FCS 2.0 file, can be rapidlyanalyzed using the BD CBA Software.The BD CBA Software enables linearregression analysis of data files andextrapolation of sample values bycomparison against a known standardcurve. The software also has broadcompatibility as it is offered in both PCand Mac-compatible formats, requiringonly Microsoft Excel® to run. With theBD CBA Software, sample results areobtained within minutes of completing anexperiment.

Considerable effort has been made toensure that each BD CBA kit has thehighest possible sensitivity and bestreproducibility. Each antibody pair used inthe kits is evaluated for dynamic range,sensitivity, and parallel titration curves tonative biological samples (Figures 2and 3). In addition, the scientists atBD Biosciences have formulated the assaydiluent and wash buffers in each kit toreduce detrimental effects of serum andplasma proteins on assay performance.While this does not always yieldcomparable recovery results as singleassays (eg, ELISA), it does provide qualitymultiplex performance.

Possibly the most important aspect of theBD CBA kits is the quality performancethat is built into each one. Usingtechniques and methods developed atBD Biosciences, a worldwide leader in theproduction and conjugation of antibodies,the reagents in a BD CBA kit arestringently tested during the developmentprocess. Each capture antibody used for acapture bead has been tested both beforeand after it is coupled to the bead.Further, it is tested again when it iscombined with the other kit reagents tocomplete the manufacture of a batch ofkits. The same is true of the detectionantibodies, which are additionally testedbefore and after conjugated withR-phycoerythrin (R-PE) and again whenthey are formulated into a detectionreagent. Finally, they are tested with theother reagents in a given kit batch afterthey are bottled. All of this effort is madeto ensure that the kit that is delivered tothe researcher meets their expectationsand provides equal performance everytime. Further, the quality of the BD CBAkits does not end with the release of theproduct. We are continually looking formethods to enhance kit performancethrough improved antibody pairs, newstandards formulations and optimizedbuffers for serum samples.

Quality is Built into Every BD™ CBA Product

5Unless otherwise specified, all products are for Research Use Only.Not for use in diagnostic or therapeutic procedures. Not for resale.

IL-12p70

TNFIL-10 IL-6

IL-1β

IL-8

Figure 2a. Representative data generated using the BD CBA Human Inflammation Kit.Relative bead fluorescence intensities.




Figures 2c. Representative data generated using the BD CBA Human Inflammation Kit.Typical standard titration data.

Negative Control Standards 80 pg/ml Standards 625 pg/ml Standards 5000 pg/ml

Figures 2b. Representative data generated using the BD CBA Human Inflammation Kit.Sample data acquired using the BD CBA analysis template.

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Figures 2e. Representative data generated using the BD CBA Human Inflammation Kit.BD CellQuest™ data for the detection of individual proteins demonstrating assay specificity.

Figures 2d. Representative data generated using the BD CBA Human Inflammation Kit.Sample standard curves generated with the BD CBA software.





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Activated Human cell culture supernatant

Figure 4. Titration curve comparing the BD CBA Human CCL2/MCP-1 recombinantstandard to the NIBSC/WHO International standard.

Figure 3. Human Chemokine Kit II data for MCP-1. Representative data showingthe parallel titration curves of the BD CBA Human MCP-1 standard (20-5000pg/ml) and an activated human cell culture supernatant. For presentationpurposes, the first dilution of the supernatant, 1:2, was plotted at 5000 pg/ml.Each point thereafter (1:4, 1:16, 1:64, 1:256, and 1:1024) were similarly plotted.


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Laboratories throughout the world usedifferent bioassays and immunoassays tomeasure and report cytokine protein levelsthat are present in biological samples. Forthis reason, the availability ofinternational standard preparations ofproteins is essential to allow definitiveanalyses and comparison of results. Theseprimary (aka, gold) standards arefrequently used to calibrate biologicalactivities and protein concentrationsbetween different secondary assaystandards used by researchers.

The gold standards provide a means todetermine relative concentrations ofunknown samples and an ability tocompare results between experiments andlaboratories. In order to support thecomparison of protein measurementsobtained with BD CBA kits,BD Biosciences evaluates the assayperformance of the standards provided invarious BD CBA kits with gold standardsfrom the National Institute for BiologicalStandards and Control (NIBSC).

The NIBSC Human Protein Standards arerecognized by the World HealthOrganization (WHO) as InternationalBiological Standards. They meetestablished requirements for accuracy,consistency and stability.

The NIBSC/WHO standards are assignedpotency values in International Units (IU)of biological activity and nominal mass(ie, not absolute mass values) for purposesof bioassay and immunoassaydeterminations. These InternationalStandards are not intended to be used assamples of purified material.Consequently, they cannot be used toestablish absolute concentrations orspecific activities for cytokinepreparations. Rather, the standardsprovide a means to facilitate comparisonsof cytokine concentration valuesdetermined by experiments conductedwithin the same or different laboratories.

Summarized in the tables below arecomparisons between expectedconcentrations of the InternationalStandards relative to several BD CBAStandards. The resulting data, togetherwith the conversion factors between theBD CBA Standards and the InternationalStandards (ie, nominal mass values) aresummarized in Table 1. As demonstratedin Figures 4 and 5, the performance ofboth sets of standards in the exampleshown was found to be similar asmeasured by observed parallelism of thedose response slope.

This observed parallelism providedconfidence that comparisons of theconcentrations of the two standards(and subsequent quantitation of nativebiological samples) are valid. It isimportant to note that the standard’ssource (ie, insect cell, E. coli, etc.) cangreatly effect the measurement andperformance of a protein in a givenantibody-based immunoassay. Theconversion factors for the NIBSC/WHOstandards make it possible to determinethe equivalency of protein concentrationspresent in samples measured by differentimmunoassays that have beenstandardized to the same NIBSC/WHOstandards.

Standardization of BD™ CBA Standards toInternational NIBSC Standards


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Figure 5. Titration curve comparing the BD CBA Human IFN-γ recombinant standard to theNIBSC/WHO International standards

Standardization of BD™ CBA Standards toInternational NIBSC Standards



NIBSC 86/680 86/504 88/656 90/586 89/548 89/520 93/722Code Number I.U. 100,000 100 1,000 5,000 100,000Mass units per vial 1 µg 7.6 ng 100 ng 500 ng 1 µg

Nominal NIBSC concentration 5,000 5,000 5,000 5,000 5,000 5,000 5,000(pg/ml)

Calculated concentration using 6,420±579 4,108±250 5,155±1,070 4,953±2,334 5,199±842 3,874±1,242 2,617±305CBA (pg/ml)

CBA:NIBSC/WHO Mass 0.78 1.22 0.97 1.01 0.96 1.29 1.91Conversion Factor

HUMAN IL-1β HUMAN IL-2 HUMAN IL-4 HUMAN IL-5 HUMAN IL-6 HUMAN IL-8 HUMAN IL-10

Table 1. Conversion factors between the BD CBA Standards and the NIBSC Standards

NIBSC 95/544 87/650 87/586 92/520 92/794 92/518 01/424 90/712Code Number I.U. 10,000 40,000 250 100,000 200 1,600Mass units per vial 1 µg 1 µg 12.5 ng 1 µg 1 µg 1 µg 1 µg

Nominal NIBSC concentration 5,000 5,000 5,000 5,000 5,000 5,000 5,000 5,000(pg/ml)

Calculated concentration using 3,906±669 5,562±1,633 10,210±550 3,399±476 3,683±390 1,862±215 5,813±941 615±150CBA (pg/ml)

CBA:NIBSC/WHO Mass 1.28 0.90 0.49 1.47 1.36 2.69 0.86 8.12Conversion Factor

HUMAN IL-12P70 HUMAN TNF HUMAN IFN-γ HUMAN RANTES HUMAN MCP-1 HUMAN MIP-1α HUMAN VEGF HUMAN bFGF

NIBSC 93/566 91/656 88/532Code Number I.U. 10,000 10,000 200,000Mass units per vial 100 ng 1 µg 1 µg

Nominal NIBSC concentration 5,000 5,000 5,000(pg/ml)

Calculated concentration using 1,315±184 2,166±195 2,624±334CBA (pg/ml)

CBA:NIBSC/WHO Mass 3.80 2.31 1.91Conversion Factor

MOUSE IL-2 MOUSE IL-4 MOUSE TNF

Several of the antibody pairs used in the BD CBA kits are capableof detecting positive signals for rhesus and cynomolgus macaquesamples. The BD CBA results are confirmed by ELISA using theBD CBA antibody pairs with activated cell culture samples from

both rhesus and cynomolgus macaques. The cross-reactivity ofBD CBA Human assays with non-human primate (NHP) analyteshave not yet been normalized to native or recombinant NHPproteins, so direct quantitation is not yet available.

Non-Human Primate Cross-Reactivity

Human Chemokine Kit I CXCL8/IL-8, CCL5/RANTES, CCL2/MCP-1 552990

Human Th1/Th2 Cytokine Kit IL-4, IL-5, TNF, IFN-γ 550749

Human Th1/Th2 Cytokine Kit II IL-4, IL-6, TNF, IFN-γ 551809

Human Inflammation Kit I IL-8, IL-6, TNF (IL-1β and IL-12p70 not yet tested) 551811

Non-human Primate Th1/Th2 Kit IL-2, IL-4, IL-5, IL-6, TNF, IFN-γ 557800

DESCRIPTION RHESUS AND CYNOMOLGUS CROSS-REACTIVITY CAT. NO.

Table 2. BD CBA Cross-Reactivity with Non-Human Primate Cytokines and Chemokines

Unless otherwise specified, all products are for Research Use Only.Not for use in diagnostic or therapeutic procedures. Not for resale. 11

BD FACSArray™ Bioanalyzer is Ideal for BD™ CBA Applications



The BD FACSArray™ bioanalyzer, the latest instrument releasefrom BD Biosciences, provides researchers with a new andcompelling platform capable of analyzing cellular andBD™ Cytometric Bead Array assays. Supported by over 1,000BD Biosciences products, the bioanalyzer is designed for multi-parameter analysis of proteins in immunology and cell biologyapplications. The system is compact, easy to use, and particularlysuited for BD Cytometric Bead Array (CBA) applications.

Sampling speed is delivered by combining a new plate loadertechnology for sample input and digital electronics for acquisitionrates of up to 15,000 events per second. An entire 96-well platecan be turned around in less than 35 minutes, while saving 1000events per sample well. Each event contains information of up tosix parameters. Featuring a dual-laser system, the bioanalyzerallows the use of several extremely bright fluorophores in parallel,thus enabling applications with a wide dynamic range. TheBD FACSArray bioanalyzer offers a powerful, yet easy-to-usesolution for many avenues of life sciences research. Moreinformation can be found online atwww.bdbiosciences.com/bdfacsarray

Class 1 (I) Laser Product

Debris or non-bead events? Optimizing theinstrument settings.

If debris or non-bead events are apparent, switch the threshold toSSC or the dual threshold to FSC and SSC. If the appearance ofthe debris is not improved, change R1 to an acquisition gate andensure that the data file save only R1 gated events rather thancount R1 events and save all data using existing templateconfigured. CAUTION: An improperly drawn R1 gate can resultin loss of data. When detecting debris (FSC/SSC) during sampleacquisition, increase the FSC threshold or further wash or dilutesamples.

Care in Mixing

Inadequate mixing can lead to little or no beads in the assayreadout. We recommend vortexing beads briefly immediatelybefore mixing with standards or samples and again beforeanalysis using the flow cytometer. Vortex each sample tube for3-5 seconds at a relatively low setting before acquiring thesample. This will yield better discrimination of the beadpopulations in the FL3 channel.

Bead populations appear to overlap duringacquisition?

This may occur in samples with very high cytokineconcentrations. Ensure that compensation settings have beenoptimized using the Cytometer Setup Beads.

Samples have little or no FL2 fluorescence?

Check sample dilution, do not alter recommended assayincubation time, and protect sample tubes from light duringthe incubation step.

High background fluorescence or all samplesbrightly positive?

High background could be due to the sample being highlyconcentrated. Test various sample dilutions. If all samples arepositive or above the top standard’s median fluorescence intensity,dilute samples further as the samples may be too concentrated.

Recombinant Standards

Once reconstituted, do not use standards after 12 hours. Use afresh standard dilution set for each experiment.

Instrument setup flow cytometer on adual-laser BD FACSCalibur™.

While the fluorescently labeled particles in the BD CBA assays aredesigned to be excited by the 488nm laser common to all BD flowcytometers, they can also be excited by the red diode laser ondual-laser BD FACSCalibur flow cytometers. Use of the red diodelaser for exciting the CBA particles and detection of particleemission in the FL4 channel simplifies the instrument set upprocedure and reduces the need for fluorescence compensation.An instrument set up protocol and template for dual-laserBD FACSCalibur instruments as well as other instrument setuptemplates can be found on CBA page atwww.bdbiosciences.com/pharmingen/CBA/

What is the absolute lowest value that can bereliably detected in plasma or serum?

It is not recommended that researchers report values extrapolatedbelow the lowest standard curve point. This can addmathematical variation to results (due to extrapolation) and causeincreased CV’s. The optimal condition is for standard curves to berun with additional dilution points (ie, 10, 5, 2.5, 1.25 pg/ml)with the understanding that some proteins will have no signalabove background (0 pg/ml) and should be excluded from thefinal sample analysis. It is not uncommon for cytokines to bepresent in serum and plasma at levels below the limit of detectionfor conventional immunoassays. This is due to the fact that mostcytokines act locally in the environment around specific sites ofimmune activity, are highly potent, have short half-lives (in manycases), and act on a broad number of cell types. Thus, it isreasonable to assume that only in abnormal cases would they bemeasured in high amounts in systemic fluids like serum andplasma. This is not necessarily the case for broader acting proteinslike some of the chemokines, anaphylatoxins, hormones, growthfactors, etc., which may be produced in higher levels or stabilizedin system fluids (longer half-lives) and are more readily measured.

BD™ CBA Assay Troubleshooting Tips




BD™ Cytometric Bead Array Products

Human

Human Allergy/Asthma Mediator IL-3, IL-4 IL-7, IL-10, GM-CSF 50 tests Summer 2004

Human Allergy/Asthma Mediator Kit - II IL-3, IL-4, GM-CSF, G-CSF, Eotaxin 50 tests Summer 2004

Human Anaphylatoxin Kit C4a, C3a, C5a 50 tests 552363

Human Angiogenesis Kit IL-8, bFGF, VEGF, Angiogenin, TNF 50 tests 558014

Human Apoptosis Kit Cleaved PARP, Bcl-2, Active Caspase-3 50 tests 557816

Human B Cell Activation Kit (Qualitative) CD79b(Igβ), BLNK, Btk, Syk, PLCγ 50 tests Summer 2004

Human Chemokine Kit – I IL-8, RANTES, MIG, MCP-1, IP-10 50 tests 552990

Human Chemokine Kit – II IL-8, RANTES, MIP-1α, MIP1β, MCP-1 50 tests 558015

Human Inflammation Kit IL-8, IL-1β, IL-6, IL-10, TNF, IL-12p70 50 tests 551811

Human Th1/Th2 Cytokine Kit IL-2, IL-4, IL-5, IL-10, TNF, IFN-γ 50 tests 550749

Human Th1/Th2 Cytokine Kit – II IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ 50 tests 551809

Human T Cell Activation Kit (Qualitative) TCRz, SLP-76, ZAP70, Pyk2, Itk, PLCγ 50 tests Summer 2004

Human Quantitative Ig Isotyping IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgE 50 tests Summer 2004

Mouse

Mouse Allergy Kit IL-3, IL-4, IL-5, IL-9, IL-13, GM-CSF 50 tests Fall 2004

Mouse Chemokine Kit RANTES, MIG, KC, MCP-1, MIP-1α, MIP-1β 50 tests Fall 2004

Mouse Inflammation Kit IL-6, IL-10, MCP-1, IFN-γ, TNF, IL-12p70 50 tests 552364

Mouse Immunoglobulin Isotyping Kit Heavy and light chain isotypes of mouse IgG1, IgG2a , 100 tests 550026IgG2b ,IgG3 , IgA, IgM, IgE

Mouse Th1/Th2 Cytokine Kit IL-2, IL-4, IL-5, TNF, IFN-γ 50 tests 551287

Non-human Primate

Non-Human Primate Th1/Th2 Cytokine IL-2, IL-4, IL-5, IL-6, TNF, IFN-γ 50 tests 557800

Rat

Rat Th1/Th2 Cytokine IL-2, IL-4, IL-6, IL-10,TNF, IFN-γ 50 tests Fall 2004

Other

Human Inflammation Cytokine Standard IL-8, IL-1β, IL-6, IL-10, TNF, IL-12p70, lyophilized 1 vial 552932

Human Th1/Th2 Cytokine Standard IL-2, IL-4, IL-5, IL-6, IL-10, TNF, IFN-g, lyophilized 1 vial 551810

Mouse Inflammation Cytokine Standard IL-6, IL-10, MCP-1, IFN-γ, TNF, IL-12p70 1 vial 620280

Mouse Th1/Th2 Cytokine Standard IL-2, IL-4, IL-5, TNF, IFN-γ, lyophilized 1 vial 552967

Phosphorylated Stat 1 Kit Human or Mouse Phosphorylated Stat 1 100 tests 557740

BD CBA Software (v1.4) Mac and PC Compatible CD-Rom and User’s guide 1 CD 550065

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