NEW BD Cytometric Bead Array Flex Set System. The easiest way to multiplex with beads

Transcription

1 NEW BD Cytometric Bead Array Flex Set System The easiest way to multiplex with beads

2 Table of Contents Introduction BD CBA Flex Sets - The Most Flexible Multiplex Bead Array System Available Customize Your BD CBA Flex Set Assay in a Few Easy Steps BD FACSArray Bioanalyzer and the BD CBA Flex Set Assay System A erfect Combination for Superior Throughput NEW FCA Array Analysis Software BD CBA Flex Sets - Ideal for Multiplex Quantitation of hospho-roteins BD CBA Flex Set Assay erformance New Flexible System with Superior Quality and Reproducibility roduct List For Research Use Only. Not for use in diagnostic or therapeutic procedures. urchase 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. Macintosh and Mac are trademarks of Apple Computer, Inc., registered in the US and other countries. Microsoft and Windows are registered trademarks of Microsoft Corporation. BD flow cytometers are class I (1) laser products. FCA Array is a registered trademark of Softflow, Inc. BD, BD Logo and all other trademarks are the property of Becton, Dickinson and Company Becton, Dickinson and Company. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, stored in retrieval systems, or translated into any language or computer language, in any form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without prior written permission from BD Biosciences. 2

3 Introduction BD CBA Flex Sets - The Most Flexible Multiplex Bead Array System Available BD Biosciences has always provided the most reliable and complete multiplex bead array system. Now we ve taken the performance you trust and the ease-of-use you rely on and made it ultimately flexible. In addition to your need for a flexible assay system, we also recognize the value of your precious samples and the importance of data reproducibility, so we ve engineered the new BD Cytometric Bead Array (CBA) Flex Set system to maximize both. Visit for more information. With the BD CBA Flex Set System You Get: Reagents that are the easiest and most efficient to multiplex A multiplex system that requires no assay formulation regardless of plex size Quantitative results from a single, small-volume sample Superior performance requiring less total time and less hands-on time than competitive bead-based immunoassays Results in less time than competitor assays Simple assay protocols yielding superior data Automated sample acquisition and increased throughput with the plate-based BD FACSArray bioanalyzer A complete system backed by the service and reliability you expect from BD Biosciences 3

4 Introduction (continued) Flow cytometry is an analytical tool that allows for the discrimination of different particles on the basis of size and color. The BD CBA employs a series of particles with discrete fluorescence intensities to simultaneously detect multiple soluble analytes from a single serum, plasma, or tissue culture supernatant sample. The BD CBA, combined with flow cytometry, creates a powerful multiple analyte (multiplex) assay system. The BD CBA system uses the sensitivity of amplified fluorescence detection by flow cytometry to measure soluble analytes with a particle-based immunoassay. The combined advantages of the broad dynamic range of fluorescence detection via flow cytometry, and the efficient capturing of analytes via suspended particles coated with distinct capture antibodies enable the BD CBA to use fewer sample dilutions to determine analyte concentration in substantially less time (compared to conventional ELISA). The specific capture beads are mixed with recombinant protein standards or test samples, then incubated with phycoerythrin (E)- conjugated detection antibodies to form sandwich complexes. Following acquisition of sample data using the flow cytometer, the sample results are generated using the new FCA Array software. The new BD CBA Flex Set system provides an open and configurable menu of beadbased reagents designed to be the easiest method of creating multiplex assays. With our proprietary conjugation chemistry, pair optimization strategies, and direct E detection reagents, you can be assured of consistent and superior assay performance in complex biological samples. NIR-A Red-A Each antibody pair we develop is evaluated for dynamic range, sensitivity, and parallel titration to native biological samples. We have also specifically formulated the assay diluent and wash buffers for each assay type to reduce potentially detrimental effects on assay performance when using serum and plasma samples. By avoiding a streptavidin-biotin-e detection method employed by other assays, our direct E detection reagents minimize the risk of increased background often caused by endogenous biotin in serum and lysate samples. Thus, BD CBA Flex Sets provide a reliable and flexible method for quantitative detection of multiple analytes in a single serum, plasma, tissue culture supernatant, or cell lysate sample. For additional resources including instrument setup templates, please visit A B C D + Detector Antibodies Wash E F Beads Lysate, Serum or Supernatant Analyze Results Figure 1. BD CBA Flex Set Assay rotocol Schematic. Only a small representative sample of the available beads is shown. 4

5 Customize Your BD CBA Flex Assay in a Few Easy Steps 1) Choose from a menu of 100 test-size Human*, Mouse, Rat, and Cell Signaling BD CBA Flex Set Assays Each BD CBA Flex Set comes with antibody-conjugated capture beads, E-conjugated detection reagents, and one or two standards. The standards are provided as unit-dose pellets, which can be easily combined with any number of additional pelletized standards as you build larger and larger multiplexes. * lease see technical data sheets for non-human primate reactivity information. 2) Choose a 100 or 500 test size BD CBA Flex Set Master Buffer Kit Each BD CBA Flex Set Master Buffer Kit contains all the assay reagents and instrument setup beads necessary for 100 or 500 tests of any size multiplex configured from compatible BD CBA Flex Sets. Whether you are running a single-plex, a 10-plex, or larger, your buffer reagents are optimized to perform with your customized mixture and yield the correct number of assay tests. Human Rodent Cell Lysate 3) Install the NEW FCA Array multiplex analysis software on your computer workstation Using the intuitive, wizard-driven FCA Array software, you can design your BD CBA Flex Set assay at your workstation. Simply create a new template on demand, or quickly recall templates saved from previous experiments. For ease-of-use, you can download a complete list of BD CBA Flex Sets and their bead position identification labels (eg, A1, A2 ) from The list can be easily uploaded into the FCA Array software or you can add new bead positions on demand manually (more details about the FCA Array software available on page 7). 4) Acquire samples on a dual-laser BD flow cytometer, such as the BD FACSArray bioanalyzer, then analyze your data using the FCA Array software The BD CBA Flex Set reagents are compatible with dual-laser flow cytometers* equipped with a 488 nm and/or a 532 nm laser and a 635 laser capable of detecting and distinguishing fluorescence emission at 576 nm and 670 nm (off the 488 nm laser) and 660 nm (off the 635 nm laser) such as a BD FACSCalibur or 576 nm (off the 532 nm laser) and 660 nm and >680 nm (off the 635 nm laser) such as a BD FACSArray bioanalyzer. Additionally, acquisition software capable of saving data in an FCS 2.0 file format, such as BD CellQuest or BD FACSArray bioanalyzer software, is required. Assay throughput can be optimized by using the plate-based BD FACSArray bioanalyzer. Simply prepare your samples and standards in a 96-well filter-bottom plate, launch your previously designed FCA Array template, load your plate, and experience truly hands-free sample acquisition. * assays run on stream-in-air instruments may yield reduced sensitivities. Instrument setup information provided in the Master Buffer Kit instruction manuals. 5

6 BD FACSArray Bioanalyzer & the BD CBA Flex Set Assay System A erfect Combination for Superior Throughput Couple our BD CBA Flex Set assays with the plate-loaderequipped BD FACSArray bioanalyzer to significantly increase your lab s throughput. The BD FACSArray bioanalyzer is a new flow cytometry platform for fast and sensitive high-content analysis of cells and proteins for cell biology, immunology, and proteomics applications. The plate-based platform is perfect for multiplex bead assays, yet is also very flexible enabling complex cell-based applications. The BD FACSArray bioanalyzer is a uniquely flexible platform that is easy-to-use, yet provides high performance. The two-laser platform is a compact bench top unit that comes equipped with a fast microtiter-plate sampler and is capable of up to sixparameter detection. 6

7 New FCA Array Analysis Software With the flexible FCA Array software, you can easily recall a saved assay template or create a new one from scratch. After your template is designed, simply collect your BD CBA Flex Set data on a flow cytometer, export FCS 2.0 data files, then analyze data using the FCA Array software. The FCA Array software enables complete analysis of data files and extrapolation of sample values by comparison against a standard curve. Eight curve fitting models are offered including log-log or 4- and 5-parameter logistics. The software is compatible with both C (Windows 2000 and X) and Mac operating systems (OS9.2, OSX - coming summer 2005). With the FCA Array software, sample results are obtained within minutes of completing an experiment. 1) Create a new template or recall a saved one. 2) When creating a new template, customize your plex from a menu of BD CBA Flex Set specificities manually or upload a complete list from 7

8 New FCA Array Analysis Software (continued) 3) After your assay has been run and data acquired, use the drop-down menu to select the flow cytometer your data was collected on, then the software will display your bead cluster locations. Assign your bead labels to the bead clusters with a simple double-click. 4) Select your curve-fitting algorithm. 8

9 New FCA Array Analysis Software (continued) 5) View single standard curves or your raw data, and change curve-fitting models on demand. 6) Generate custom reports for every experiment. 9

10 BD CBA Flex Sets - Ideal for Multiplex Quantitation of hospho-roteins Unlike conventional assays used to detect or measure phosphorylated proteins such as immunoprecipitation and Western blotting, the BD CBA Flex Sets can provide sensitive, quantitative measurements of protein phosphorylation in considerably less time. In addition, the phosphorylation status of multiple proteins can be measured in the same sample in a quantitative fashion (Figure 3 and Figure 4). The assays are designed to measure either phosphorylated or total specificities from a cell lysate. The specificity of the assays has been validated by immunoprecipitation and Western blotting to ensure that only the protein of interest is detected (Figure 5). Test samples are run against a standard curve which is generated using dilutions of a phosphorylated recombinant standard. We have assessed the linearity of the assays by making dilutions of activated cell lysates and comparing them against the standard curve (Figure 2). In all cases the dilution curve of the sample is parallel to the standard curve. Reproducibility experiments demonstrated a low %CV for intra-assay and inter-assay reproducibility plus the sensitivity of the assay is comparable to or better than a conventional Western blot. CBA MFI Western Blot 1 Cell number 30 second exposure 10 minute exposure hospho-za UNITS/ml Recombinant Standard Jurkat + anti-cd3/cd28 Figure 2. Jurkat cells activated with anti-cd3 and anti-cd28 and crosslinked by anti-mouse Ig for 2 minutes. The reaction was stopped by the addition of SDS (1% final) whereupon the samples were placed in a boiling water bath. A phosphorylated recombinant standard was used to generate a standard curve. The upper panel shows the standard curve versus a titration of the activated Jurkat lysate. An important characteristic of the assay is that these two curves are parallel to each other. This ensures linearity of the readings so that twice as much lysate will have twice as many units/ml. The same concentrations of lysate were also run on an SDS-AGE gel followed by immunoblotting with an anti-phospho- ZA-70 antibody. Both a 30 second and a 10 minute exposure are shown. The BD CBA Flex Set assay is at least as sensitive as a Western blot. In this particular example, phosphorylation of ZA-70 can be detected in a BD CBA Flex Set assay using lysate from less than 1,000 cells. 12,500 25,000 50, , , Fold Increase in Units/ml 10 1 ERK JNK p38 LCγ ZA-70 Itk LAT Jun RSK Time (Minutes) Figure 3. Kinetic analysis of T cell activation by anti-cd3/cd28. Jurkat cells were activated with anti-cd3 and anti-cd28 for different lengths of time. Lysates were prepared as outlined in Figure 2. A 9-plex BD CBA assay using 10 µg of lysate was run measuring phosphorylated ERK, JNK, p38, LCγ, ZA-70, Itk, LAT, c-jun, and RSK. Using standard curves, units/ml were determined for each specificity and the fold increase in activity was plotted. 10

11 Multiplex Quantitation of hospho-roteins (continued) Control Lck MEK Inhibitor (D-98059) Lck Src Family Kinase Inhibitor (2) Lck Itk ZA-70 Itk ZA-70 Itk ZA-70 LC DAG LAT V A V SL-76 LAT S O Grb2 S LC DAG LAT V A V SL-76 LAT S O Grb2 S LC DAG LAT V A V SL-76 LAT S O Grb2 S KC Ras Rac KC Ras Rac KC Ras Rac MAKKK Ras MEKK1-4 MEKK4 MAKKK Ras MEKK1-4 MEKK4 MAKKK Ras MEKK1-4 MEKK4 IKK MEK MKK4,7 MKK3,6 IKK MEK MKK4,7 MKK3,6 IKK MEK MKK4,7 MKK3,6 NF-κB RSK ERK JNK p38 Elk Jun ATF2 NF-κB RSK ERK JNK p38 Elk Jun ATF2 NF-κB RSK ERK JNK p38 Elk Jun ATF2 Specificity Change % of Control ZA Itk LAT LCγ ERK JNK p c-jun RSK Specificity Change % of Control ZA Itk LAT LCγ ERK JNK p c-jun RSK 9 2 Specificity Change % of Control ZA Itk LAT -2-1 LCγ 19 7 ERK -2 0 JNK p c-jun RSK 22 6 Figure 4. Effect of inhibitors on T cell signaling. Jurkat cells were pre-incubated with either buffer, 200 µm D (MEK inhibitor), or 10 µm 2 (Src family tyrosine kinase inhibitor) for 20 minutes before being activated with anti-cd3/cd28 for 2 minutes. In the left panel, a T cell signaling pathway is shown. The 9 phospho-specificities that were tested simultaneously using BD CBA Flex Set beads are circled. The table at the bottom of the panel shows the median fluorescence intensity (MFI) units for each specificity. The middle panel shows the effects of pre-incubation with D Since this compound inhibits MEK, only ERK and RSK should be affected. This is shown in gray on the pathway. However, although ERK and RSK are almost completely inhibited, this compound does have effects on other signaling molecules. In the right panel, Jurkat cells were pre-incubated with 2 which inhibits Src family kinases such as Lck. This should shut down all of the signaling which is what is seen in the table. hospho-p38 hospho -ERK hospho-jnk CBA Control cells Activated cells Control cells Activated cells Control cells Activated cells A431 ±EGF HeLa±aniso U937±GMCSF Macro±IFN/LS 293±UV HeLa±TNF 3T3±osmotic A431±EGF HeLa±aniso U937±GMCSF Macro±IFN/LS 293±UV HeLa±TNF 3T3±osmotic A431±EGF HeLa±aniso U937±GMCSF Macro±IFN/LS 293±UV HeLa±TNF 3T3±osmotic Western Figure 5. Activation of MA kinases by different stimuli. Different cell types were activated with different stimuli. Cell lysates were prepared as described in Figure 2 and a 3-plex BD CBA Flex Set was run to measure phosphorylated MA kinases (ERK, JNK, p38). The bottom panel shows a Western blot of the same lysates using phospho-specific antibodies. Different stimuli have different patterns of activation and the results from the BD CBA Flex Set assays correlate well with the results from the Western blots. 11

12 BD CBA Flex Set Assay erformance New Flexible System with Superior Quality and Reproducibility The BD CBA Flex Set system has been developed to take the guesswork out of building multiplex assays. We ve designed and formulated all of the assay reagents so that you can easily build the multiplex assay of your choosing from pre-configured components all with the same superior assay performance you expect from a BD CBA product. The BD CBA Flex Set system provides the easiest-to-use multiplex assays with superior reproducibility ideal for large or long-term studies. In the tables below you will find performance data as well as representative comparisons to other, commercially available bead-based multiplex assay systems. Sensitivity The assay limits of detection are determined by averaging the average median fluorescence intensity of 20 replicates of the negative control (0 pg/ml), and the corresponding concentration at two standard deviations above the average. Representative assay sensitivity of select BD CBA Flex Sets is shown in Table 1 below. Table 1. Sensitivity BD CBA FLEX SET SENSITIVITY (UNITS/ML) hospho Btk(Y551) 0.6 hospho enos (S1177) 1.3 hospho ERK1/2 (T202/Y204) 6.6 hospho Itk (Y511) 3.7 hospho JNK1/2 (T183/Y185) 0.4 hospho p38 (T180/Y182) 0.7 hospho LCγ (Y783) 1.1 hospho Rsk (T573) 0.5 hospho Stat1 (Y701) 2.1 hospho Syk (Y352) 0.5 hospho ZA70 (Y319) 1.6 Total Stat1 0.6 Total Syk 2.3 Total ZA Recovery Individual protein was spiked into various matrices at three different levels within the assay range. The serum and plasma used in these experiments was diluted 1:4 before addition of the protein as recommended for each assay. The recovery at each level was averaged and the overall range of results is also shown. Each assay below (Table 2) is shown as an example of the recovery performance for human, mouse, and rat BD CBA Flex Set assays. Table 2. Recovery RECOVERY BD CBA FLEX SET SAMLE TYE AVERAGE (%) RANGE (%) Human IL-4 Media 97% % Human IL-4 Serum* 91% % Human IL-4 lasma** 82% 79-86% Human IFN-γ Media 60% 54-72% Human IFN-γ Serum* 63% 47-87% Human IFN-γ lasma** 49% 40-62% Mouse IL-4 Media 99% % Mouse IL-4 Serum*** 29% 25-37% Mouse IFN-γ Media 88% 84-95% Mouse IFN-γ Serum*** 83% 77-94% Rat IL-4 Media 79% % Rat IL-4 Serum*** 62% 48-79% Rat IFN-γ Media 42% 17-66% Rat IFN-γ Serum*** 22% 16-28% *Serum is pooled sera from 800 1,000 donors. **lasma is pooled EDTA-treated plasma from at least 20 donors. ***Mouse or rat serum was taken and tested individually. BD CBA FLEX SET SENSITIVITY (G/ML) Human IL Human IFN-γ 0.3 Mouse IL Mouse IFN-γ 0.3 Rat IL Rat IFN-γ

13 BD CBA Flex Set Assay erformance (continued) Reproducibility The precision of each assay was evaluated by testing the reproducibility of the assay to quantitate protein levels of samples containing three different levels of protein. The samples were tested either on a single experiment (10 replicates of each sample) to test the intra-assay reproducibility or on four separate experiments by four independent operators each running the samples in duplicate to test the inter-assay reproducibility. The reproducibility of selected BD CBA Flex Sets is shown below (Table 3) as well as compared to competitor assays (Table 4). Table 3. Reproducibility INTER-ASSAY RERODUCIBILITY INTRA-ASSAY RERODUCIBILITY BD CBA FLEX SET AVERAGE % CV RANGE (%) AVERAGE % CV RANGE (%) hospho Btk(Y551) 5.7% 5-7% 8.0% 5-13% hospho enos (S1177) 9.0% 4-18% 5.7% 3-9% hospho ERK1/2 (T202/Y204) 9.0% 3-14% 3.7% 3-5% hospho Itk (Y511) 7.0% 8-12% 5.3% 4-8% hospho JNK1/2 (T183/Y185) 7.7% 6-10% 4.0% 2-6% hospho p38 (T180/Y182) 7.7% 6-9% 7.3% 6-9% hospho LCγ (Y783) 12.3% 10-15% 4.3% 3-6% hospho Rsk (T573) 6.3% 5-9% 5.7% 3-7 % hospho Stat1 (Y701) 10.7% 7-16% 9.3% 6-15% hospho Syk (Y352) 7.7% 5-9% 9.0% 4-17% hospho ZA70 (Y319) 5.7% 5-6% 12.7% 10-18% Total Stat1 5.7% 4-8% 3.7% 2-6% Total Syk 7.7% 6-10% 7.0% 4-11% Total ZA % 7-12% 4.7% 3-6% Human IL-4 8.5% 7-10% 3.0% 3-3% Human IFN-γ 6% 5-7% 4.7% 3-7% Mouse IL-4 7% 5-10% 5.3% 2-8% Mouse IFN-γ 4.7% 3-6% 3.7% 2-6% Rat IL-4 5.3% 4-7% 3% 2-4% Rat IFN-γ 9.7% 7-13% 4.3% 4-5% Table 4. Reproducibility vs Competitor Assays BD CBA FLEX SETS COMETITOR A COMETITOR B COMETITOR C INTER-ASSAY INTRA-ASSAY INTER-ASSAY INTRA-ASSAY INTER-ASSAY INTRA-ASSAY INTER-ASSAY INTRA-ASSAY RERODUCIBILITY RERODUCIBILITY RERODUCIBILITY RERODUCIBILITY RERODUCIBILITY RERODUCIBILITY RERODUCIBILITY RERODUCIBILITY Avg. % CV Avg. % CV Avg. % CV Avg. % CV Avg. % CV Avg. % CV Avg. % CV Avg. % CV hospho ERK1/2 (T202/Y204) 9% 4% n/a n/a n/a n/a 35% 19% hospho JNK1/2 (T183/Y185) 8% 4% n/a n/a 12% 11% 78% 71% hospho p38 (T180/Y182) 8% 7% n/a n/a 18% 15% 52% 67% Human IL-4 9% 3% 13% 7% 9% 8% 10% 6% Human IFN-γ 6% 5% 12% 8% 17% 12% 16% 10% Mouse IL-4 7% 5% 15% 5% 11% 9% 15% 7% Mouse IFN-γ 5% 4% 20% 11% 34% 12% 12% 7% 13

14 BD CBA Flex Set Assay erformance (continued) Linearity Individual protein was spiked into various matrices, or cell lysates were prepared, then the samples were serially diluted and tested in the various assays. The recovery at each level was averaged and the overall range of results is also shown. Each assay below is shown as an example of the recovery performance of human, mouse, and rat BD CBA Flex Set assays. The linearity of selected BD CBA Flex Sets is shown below (Table 5) as well as compared to competitor assays (Table 6). Table 5. Linearity LINEARITY BD CBA FLEX SET SAMLE TYE AVG.(%) OF EXECTED RANGE (%) hospho Btk(Y551) Cell lysate 118.7% % hospho enos (S1177) Cell lysate 103.7% % hospho ERK1/2 (T202/Y204) Cell lysate 109.0% % hospho Itk (Y511) Cell lysate 91.3% 88-94% hospho JNK1/2 (T183/Y185) Cell lysate 109.0% % hospho p38 (T180/Y182) Cell lysate 85.0% 75-93% hospho LCγ (Y783) Cell lysate 116.0% % hospho Rsk (T573) Cell lysate 103.3% % hospho Stat1 (Y701) Cell lysate 86.3% 82-93% hospho Syk (Y352) Cell lysate 79.7% 72-86% hospho ZA70 (Y319) Cell lysate 99.3% % Total Stat1 Cell lysate 74.3% 67-81% Total Syk Cell lysate 121.7% % Total ZA-70 Cell lysate 90.7% % Human IL-4 Media 97% % Human IL-4 Serum* 91% % Human IL-4 lasma** 82% 79-86% Human IFN-γ Media 56% 53-60% Human IFN-γ Serum* 56% 53-58% Human IFN-γ lasma** 68% 55-82% Mouse IL-4 Media 94% % Mouse IL-4 Serum*** 68% 56-79% Mouse IFN-γ Media 90% 86-93% Mouse IFN-γ Serum*** 86% 82-90% Rat IL-4 Media 82% 78-90% Rat IL-4 Serum*** 78% 74-84% Rat IFN-γ Media 31% 28-34% Rat IFN-γ Serum*** 19% 17-20% Cell lysates were from cells treated to induce protein phosphorylation (or left untreated for the total protein assays). *Serum is pooled sera from 800 1,000 donors. **lasma is pooled EDTA-treated plasma from at least 20 donors. ***Mouse or rat serum was taken and tested individually. Table 6. Linearity vs Competitor Assays BD CBA FLEX SETS COMETITOR A COMETITOR B COMETITOR C AVG.% OF AVG.% OF AVG.% OF AVG.% OF SAMLE EXECTED RANGE (%) EXECTED RANGE (%) EXECTED RANGE (%) EXECTED RANGE (%) Human IL-4 Media 100% % 73% 72-73% 53% 50-55% 102% % Human IL-4 Serum 79% 79-79% 93% 90-97% 75% 74-75% 119% % Human IL-4 lasma 80% 77-82% 92% 89-95% 77% 73-83% 95% % Human IFN-γ Media 56% 53-60% 55% 54-56% 30% 26-37% 113% % Human IFN-γ Serum 56% 53-58% 67% 64-71% 41% 39-43% 129% % Human IFN-γ lasma 68% 55-82% 56% 55-57% 40% 38-44% 123% % Mouse IL-4 Media 94% % 241% % 241% % 301% % Mouse IL-4 Serum 68% 56-79% 367% % 372% % 57% 36-80% Mouse IFN-γ Media 90% 86-93% 135% % 62% % 751% % Mouse IFN-γ Serum 86% 82-90% 102% % 31% 22-45% 217% % rotein used in each spiked sample was from the BD CBA Flex Set assays, and the potency of that protein versus the competitor s standard may affect results. 14

15 BD CBA Flex Set Assay erformance (continued) Assay Range The range of each assay is determined by the levels of the assay standard used on each experiment. The standard curve ranges for each assay is listed below. It is possible that the assay will detect levels of protein above or below the range of the standard curve. To increase confidence in the result obtained for a sample, it is recommended that only sample dilutions that fall within the standard curve range be used for quantitation of unknowns. Table 7. Assay Range SECIFICITY ERK1/2 (T202/Y204) Itk (Y511), Btk(Y551) JNK1/2 (T183/Y185) p38/makinase (T180/Y182) LCγ (Y783) Stat1 (Y701) Syk (Y352) ZA-70 (Y319) Total Stat1 Total Syk Total ZA-70 Human IL-4 Human IFN-γ Mouse IL-4 Mouse IFN-γ Rat IL-4 Rat IFN-γ STANDARD CURVE RANGE 20 5,000 pg/ml 20 5,000 pg/ml 20 5,000 pg/ml 20 5,000 pg/ml 20 5,000 pg/ml 20 5,000 pg/ml Bead Compatibility Matrix* The table below shows the multiplexing compatibility of a representative sample of BD CBA Flex Sets. For additional specificities, please see individual datasheets or visit Table 8. BD CBA Flex Set Assay Compatibility BTK (Y551) ENOS ERK1/2 ITK (Y511) JNK1/2 38 LCγ (Y783) RSK (T573) STAT1 (Y701) SYK (Y352) ZA70 (Y319) BD CBA FLEX SET (S1177) (T202/Y204) (T183/Y185) (T180/Y182) hospho Btk (Y551) hospho enos (S1177) hospho ERK1/2 (T202/Y204) hospho Itk (Y511) hospho JNK1/2 (T183/Y185) hospho p38 (T180/Y182) hospho LCγ (Y783) hospho Rsk (T573) hospho Stat1 (Y701) hospho Syk (Y352) hospho ZA-70 (Y319) BD CBA FLEX SET TOTAL STAT1 TOTAL SYK TOTAL ZA70 Total Stat1 Total Syk Total ZA70 *Total signaling protein flex sets may not be run in the same well as phospho-specific flex sets. may be combined may not be combined 15

16 BD CBA Flex Set Assay erformance (continued) Time to Result The time to complete BD CBA Flex Set assays was tracked and compared to competitor assays. Results include preparation of reagents for the experiment, sample acquisition, and data analysis. Table 9. Soluble rotein Assays: rotocol Comparison and Time to Result BD CBA FLEX SETS COMETITOR A COMETITOR B COMETITOR C No. Washes No. Aspirations No. Incubations 2/1* Time to Result 4-5 hrs 10 hrs 10 hrs 4 hrs * 2 for Human assays and 1 for Mouse assays Table 10. Cell Signaling Assays: rotocol Comparison and Time to Result BD CBA FLEX SETS COMETITOR A COMETITOR B COMETITOR C No. Washes 0 n/a 9 11 No. Aspirations 1 n/a No. Incubations 1 n/a 3 3 Time to Result 6 hrs n/a 10 hrs hrs BD CBA Flex Set 15-plex Multiplex MFI Concentration (pg/ml) hil-2 hil-4 hil-5 hil-6 hil-8 hil-1b hi-10 hil-10 hlt-α hmc-1 htnf hmi-1β hil-12p70 hifnγ hmig Figure 6. Representation Standard curves generated using a 15-plex of BD CBA Flex Sets. 16

17 roduct List Instrumentation DESCRITION CONTENTS CLONE AS SIZE CAT. NO. RICE BD FACSArray Bioanalyzer BD Cytometric Bead Array Flex Sets Software DESCRITION CONTENTS SIZE CAT. NO. RICE FCA Array Software C Compatible Software and User's Guide. Required for use with CD BD CBA Flex Sets. Compatible with BD CBA kits. BD CBA Kit Software Mac and C compatible software and user s guide. Compatible only CD with BD CBA kit format hospho-specific and Total Signaling roteins Supporting Reagents* DESCRITION CONTENTS CLONE AS SIZE CAT. NO. RICE Cell Signaling Master Buffer Kit Buffers and setup reagents required for use with BD CBA Cell Sufficient for 100 tests of any Signaling Flex Sets size BD CBA Flex Set Cell Signaling Master Buffer Kit Buffers and setup reagents required for use with BD CBA Cell Sufficient for 500 tests of any Signaling Flex Sets size BD CBA Flex Set hospho-specific Flex Sets* DESCRITION REACT CONTENTS BEAD LOCATION SIZE CAT. NO. RICE Btk (Y551) Hu Capture Bead and Detection Reagents plus 1 standard D5 100 tests ERK1/2 (T202/Y204) Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard C4 100 tests Itk (Y511) Hu, Ms Capture Bead and Detection Reagents plus 1 standard C6 100 tests JNK1/2 (T183/Y185) Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard B5 100 tests enos (S1177) Hu Capture Bead and Detection Reagents plus 1 standard C7 100 tests p38/makinase (T180/Y182) Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard B6 100 tests LCγ (Y783) Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard B7 100 tests RSK (T573) Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard D7 100 tests Stat1 (Y701) Hu, Ms Capture Bead and Detection Reagents plus 1 standard C5 100 tests Syk (Y352) Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard B9 100 tests ZA-70 (Y319) Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard B8 100 tests Total Signaling rotein Flex Sets* DESCRITION REACT CONTENTS BEAD LOCATION SIZE CAT. NO. RICE Stat1 Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard D4 100 tests Syk Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard B9 100 tests ZA-70 Hu, Ms, Rt Capture Bead and Detection Reagents plus 1 standard B8 100 tests Human Supporting Reagents DESCRITION CONTENTS CLONE BEAD LOCATION SIZE CAT. NO. RICE Human Soluble rotein Master Buffer Kit Buffers and setup reagents required for use with BD CBA Human Soluble rotein Flex Sets Sufficient for 100 tests of any size BD CBA Flex Set Human Soluble rotein Master Buffer Kit Buffers and setup reagents required for use with BD CBA Human Soluble rotein Flex Sets Sufficient for 500 tests of any size BD CBA Flex Set * Total signaling protein flex sets may not be run in the same assay well as phospho-specific flex sets. 17

18 roduct List (continued) BD Cytometric Bead Array Flex Sets (continued) Human Flex Sets DESCRITION CONTENTS CLONE BEAD LOCATION SIZE CAT. NO. RICE Angiogenin Capture Bead and Detection Reagents plus 2 standards C4 100 tests Spring 2005 Soluble CD178 (sfas Ligand) Capture Bead and Detection Reagents plus 2 standards C6 100 tests Spring 2005 Eotaxin Capture Bead and Detection Reagents plus 2 standards C7 100 tests Spring 2005 basic FGF Capture Bead and Detection Reagents plus 2 standards C5 100 tests Spring 2005 G-CSF Capture Bead and Detection Reagents plus 2 standards C8 100 tests Spring 2005 GM-CSF Capture Bead and Detection Reagents plus 2 standards C9 100 tests Spring 2005 IFN-γ Capture Bead and Detection Reagents plus 2 standards E7 100 tests /Spring 2005 IL-1β Capture Bead and Detection Reagents plus 2 standards B4 100 tests /Spring 2005 IL-2 Capture Bead and Detection Reagents plus 2 standards A4 100 tests /Spring 2005 IL-3 Capture Bead and Detection Reagents plus 2 standards D5 100 tests Spring 2005 IL-4 Capture Bead and Detection Reagents plus 2 standards A5 100 tests /Spring 2005 IL-5 Capture Bead and Detection Reagents plus 2 standards A6 100 tests /Spring 2005 IL-6 Capture Bead and Detection Reagents plus 2 standards A7 100 tests /Spring 2005 IL-7 Capture Bead and Detection Reagents plus 2 standards A8 100 tests Spring 2005 IL-8 Capture Bead and Detection Reagents plus 2 standards A9 100 tests /Spring 2005 IL-9 Capture Bead and Detection Reagents plus 2 standards B6 100 tests Spring 2005 IL-10 Capture Bead and Detection Reagents plus 2 standards B7 100 tests /Spring 2005 IL-11 Capture Bead and Detection Reagents plus 2 standards E6 100 tests Spring 2005 IL-12p70 Capture Bead and Detection Reagents plus 2 standards E5 100 tests /Spring 2005 I-10 Capture Bead and Detection Reagents plus 2 standards B5 100 tests /Spring 2005 LIF Capture Bead and Detection Reagents plus 2 standards D6 100 tests Spring 2005 LT-α Capture Bead and Detection Reagents plus 2 standards D7 100 tests /Spring 2005 MC-1 Capture Bead and Detection Reagents plus 2 standards D8 100 tests /Spring 2005 MIG Capture Bead and Detection Reagents plus 2 standards E8 100 tests /Spring 2005 MI-1α Capture Bead and Detection Reagents plus 2 standards B9 100 tests Spring 2005 MI-1β Capture Bead and Detection Reagents plus 2 standards E4 100 tests /Spring 2005 Oncostatin M (OSM) Capture Bead and Detection Reagents plus 2 standards E6 100 tests Spring 2005 RANTES Capture Bead and Detection Reagents plus 2 standards D4 100 tests Spring 2005 TNF Capture Bead and Detection Reagents plus 2 standards D9 100 tests /Spring 2005 VEGF Capture Bead and Detection Reagents plus 2 standards B8 100 tests Spring 2005 Some specificities are reactive with Rhesus, Cynomolgus and Macaque non-human primate species Mouse/Rat Supporting Reagents DESCRITION CONTENTS CLONE BEAD LOCATION SIZE CAT. NO. RICE Mouse/Rat Soluble rotein Master Buffer Kit Buffers and setup reagents required for use with BD CBA Mouse/ Sufficient for 100 tests of Rat Soluble rotein Flex Sets any size BD CBA Flex Set Mouse/Rat Soluble rotein Master Buffer Kit Buffers and setup reagents required for use with BD CBA Mouse/ Sufficient for 500 tests of Rat Soluble rotein Flex Sets any size BD CBA Flex Set Mouse Flex Sets DESCRITION CONTENTS CLONE BEAD LOCATION SIZE CAT. NO. RICE GM-CSF Capture Bead and Detection Reagents plus 2 standards B9 100 tests Spring 2005 IFN-γ Capture Bead and Detection Reagents plus 2 standards A4 100 tests Spring 2005 IL-2 Capture Bead and Detection Reagents plus 2 standards A5 100 tests Spring 2005 IL-3 Capture Bead and Detection Reagents plus 2 standards A6 100 tests Spring 2005 IL-4 Capture Bead and Detection Reagents plus 2 standards A7 100 tests Spring 2005 IL-5 Capture Bead and Detection Reagents plus 2 standards A8 100 tests Spring 2005 IL-6 Capture Bead and Detection Reagents plus 2 standards B4 100 tests Spring 2005 IL-9 Capture Bead and Detection Reagents plus 2 standards B5 100 tests Spring 2005 IL-10 Capture Bead and Detection Reagents plus 2 standards C4 100 tests Spring 2005 IL-12p70 Capture Bead and Detection Reagents plus 2 standards D7 100 tests Spring 2005 IL-13 Capture Bead and Detection Reagents plus 2 standards B8 100 tests Spring

19 roduct List (continued) BD Cytometric Bead Array Flex Sets (continued) Mouse Flex Sets DESCRITION CONTENTS CLONE BEAD LOCATION SIZE CAT. NO. RICE KC Capture Bead and Detection Reagents plus 2 standards A9 100 tests Spring 2005 MC-1 Capture Bead and Detection Reagents plus 2 standards B7 100 tests Spring 2005 MIG Capture Bead and Detection Reagents plus 2 standards D9 100 tests Spring 2005 MI-1α Capture Bead and Detection Reagents plus 2 standards C7 100 tests Spring 2005 MI-1β Capture Bead and Detection Reagents plus 2 standards C9 100 tests Spring 2005 RANTES Capture Bead and Detection Reagents plus 2 standards D8 100 tests Spring 2005 TNF Capture Bead and Detection Reagents plus 2 standards C8 100 tests Spring 2005 Rat Flex Sets DESCRITION CONTENTS CLONE BEAD LOCATION SIZE CAT. NO. RICE IFN-γ Capture Bead and Detection Reagents plus 2 standards A6 100 tests Spring 2005 IL-2 Capture Bead and Detection Reagents plus 2 standards E5 100 tests Spring 2005 IL-4 Capture Bead and Detection Reagents plus 2 standards B9 100 tests Spring 2005 IL-6 Capture Bead and Detection Reagents plus 2 standards A9 100 tests Spring 2005 IL-10 Capture Bead and Detection Reagents plus 2 standards A8 100 tests Spring 2005 TNF Capture Bead and Detection Reagents plus 2 standards C8 100 tests Spring 2005 BD Cytometric Bead Array Ready-to-Use Kits** Human DESCRITION CONTENTS CLONE AS SIZE CAT. NO. RICE Allergy/Asthma Mediator Kit I IL-3, IL-4, IL-5, IL-7, IL-10, GM-CSF Appx 80 tests (samples plus standards) Allergy/Asthma Mediator Kit II IL-3, IL-4, GM-CSF, G-CSF, Eotaxin Appx 80 tests (samples plus standards) Angiogenesis Kit IL-8, Basic FGF, VEGF, TNF, Angiogenin Appx 80 tests (samples plus standards) Apoptosis Kit Cleaved AR, Bcl-2, Active Caspase-3 Appx 80 tests (samples plus standards) Chemokine Kit I IL-8, RANTES, MIG, MC-1, I-10 Appx 80 tests (samples plus standards) Chemokine Kit II IL-8, RANTES, MI-1α, MI-1β, MC-1 Appx 80 tests (samples plus standards) Inflammation Kit IL-8, IL-1β, IL-6, IL-10, TNF, IL-12p70 Appx 80 tests (samples plus standards) Th1/Th2 Cytokine Kit I IL-2, IL-4, IL-5, IL-10, TNF, IFN-γ Appx 80 tests (samples plus standards) Th1/Th2 Cytokine Kit II IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ Appx 80 tests (samples plus standards) Non-Human rimate DESCRITION CONTENTS CLONE AS SIZE CAT. NO. RICE Non-Human rimate Th1/Th2 Kit IL-2, IL-4, IL-5, IL-6, TNF, IFN-γ Appx 80 tests (samples plus standards) Mouse DESCRITION CONTENTS CLONE AS SIZE CAT. NO. RICE Inflammation Kit IL-6, IL-10, MC-1, IFN-γ, TNF, IL-12p70 Appx 80 tests (samples plus standards) Immunoglobulin Isotyping Kit Heavy and light chain isotypes of mouse IgG1, IgG2a, 100 tests IgG2b,IgG3, IgA, IgM, IgE Th1/Th2 Cytokine Kit IL-2, IL-4, IL-5, TNF, IFN-γ 50 tests Supporting Reagents DESCRITION CONTENTS CLONE AS SIZE CAT. NO. RICE Human Inflammation Cytokine Standard IL-8, IL-1, IL-6, IL-10, TNF, IL-12p70 Lyophilized, 1 vial Human Th1/Th2 Cytokine Standard IL-2, IL-4, IL-5, IL-6, IL-10, TNF, IFN-γ Lyophilized, 1 vial Mouse Inflammation Cytokine Standard IL-6, IL-10, MC-1, IFN-γ, TNF, IL-12p70 Lyophilized, 1 vial Mouse Th1/Th2 Cytokine Standard IL-2, IL-4, IL-5, TNF, IFN-γ Lyophilized, 1 vial **IMORTANT - re-configured Kits and Flex Sets MAY NOT be combined. 19

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