CBA Kit Manual Human

BD Pharmingen Cytometric Bead Array (CBA) Kit Manual Human Th1/Th2 Cytokine CBA BD Biosciences

Section 1 1 Multiplexing BD Cytometric Bead Array CBA Multiplexing BD CBA CBA ELISA ELISA BD Human Th1/Th2 Cytokine CBA Kit 1 Interleukin-2 IL-2 Interleukin-4 IL-4 Interleukin-5 IL-5 Interleukin-10 IL-10 Tumor Necrosis Factor- TNF- Interferon- IFN- EDTA pg/ml BD CBA BD Biosciences BD Immunocytometry Systems BDIS BD Pharmingen BDIS BD Pharmingen 1

Section 2 6 IL-2 IL-4 IL-5 IL-10 TNF- IFN- 6 CBA BD FACS TM FL3 30 IL-5 25 Counts 20 15 10 IFN-γ TNF-α IL-10 IL-4 IL-2 5 0 10 0 FL3-H 10 4 Figure 1 PE BD CBA 50 2 2

2.1 CBA ELISA 1 6 ELISA 6 1 1 1 ELISA CBA 1 CBA ELISA 6 2.2 Human Th1/Th2 Cytokine CBA ELISA 12.1 12.5 BD CBA FACStar TM FACSVantage TM 3

Section 3 3.1 Human Cytokine Capture Beads A1 A6 Bead Brightest A1 A2 A3 A4 A5 Dimmest A6 Specificity IL-2 IL-4 IL-5 IL10 TNF- IFN- Capture Bead A1 A6 1 75 4 Cytometer Setup Beads D PMT 1 30 10 Cytometer Setup Beads 50 µl/ 4

3.2 Human Th1/Th2 PE Detection Reagent B PE IL-2 IL-4 IL-5 IL-10 TNF- IFN- 1 75 50 µl/ 4 PE Positive Control Detector E1 PE 1 10 50 µl/ Cytometer Setup Beads 4 FITC Positive Control Detector E2 FITC 1 10 50 µl/ Cytometer Setup Beads 4 Human Th1/Th2 Cytokine Standards C 2 1 0.2 ml Assay Diluent 10 10 IL-2 IL-4 IL-5 IL-10 TNF- IFN- 50 ng/ml 4 Human Th1/Th2 Cytokine Standards 2 8 12 551810 5

3.3 Buffer Assay Diluent G 1 Buffer 30mL 1 Human Th1/Th2 Cytokine Standards 4 Wash Buffer F 1 PBS 130mL 1 * 4 0.1 * Section 4 Human Th1/Th2 Cytokine CBA Kit CellQuest TM 488 nm 576 nm 670 nm BD FACScan TM FACSCalibur TM 12 75mm BD Falcon TM 352008 BD CBA Software BD Pharmingen 550065 CellQuest BD CBA Microsoft Excel Macintosh BD CBA Software BD CaliBRITE TM 3 Beads BD 340486 6

Section 5 Human Th1/Th2 Cytokine CBA 1. Human Th1/Th2 Cytokine Standards Assay Diluent 15 2. Assay Diluent Standards 3. Human Cytokine Capture Bead 10 µl/ 4. 50 µl 5. PE Detection Reagent 50 µl/ 6. Standards 50 µl/ 3 7. Wash Buffer 8. Wash Buffer 300 µl Cytometer Setup Beads 1. Cytometer Setup Beads A B C 50 µl/ 2. FITC 50 µl B PE 50 µl C 30 3. Wash Buffer 400 µl B C 4. Wash Buffer 450 µl A 5. A B C 7

Section 6 Human Th1/Th2 Cytokine Standards Human Th1/Th2 Cytokine Standards Capture Beads PE Detection Reagent 1. Human Th1/Th2 Cytokine Standards 1 Assay Diluent 0.2 ml 10 15 2. 12 75 mm BD Falcon 352008 Top Standard 1 2 1 4 1 8 1 16 1 32 1 64 1 128 1 256 3. 900 µl Assay Diluent Top Standard 4. 300 µl Assay Diluent 5. 100 µl 10 Top Standard 6. Top Standard 300 µl 1 2 1 2 300 µl 1 4 1 256 Figure 2 Assay Diluent 100 µl 300 µl 300 µl 300 µl 300 µl 300 µl 300 µl 300 µl 300 µl 10 Stock Standard Top Standard 1:2 Dilution Tube 1:4 Dilution Tube 1:8 Dilution Tube 1:16 Dilution Tube 1:32 Dilution Tube 1:64 Dilution Tube 1:128 Dilution Tube 1:256 Dilution Tube Figure 2 Human Th1/Th2 Cytokine Standard 8

pg/ml Table 1 Table 1 Human Th1/Th2 Cytokine Standard Ptotein Top 1 2 1 4 1 8 1 16 1 32 1 64 1 128 1 256 pg/ml Standard DilutionDilutionDilutionDilutionDilutionDilutionDilutionDilution Tube Tube Tube Tube Tube Tube Tube Tube Human IL-2 5000 2500 1250 625 312.5 156 80 40 20 Human IL-4 5000 2500 1250 625 312.5 156 80 40 20 Human IL-5 5000 2500 1250 625 312.5 156 80 40 20 Human IL-10 5000 2500 1250 625 312.5 156 80 40 20 Human TNF- 5000 2500 1250 625 312.5 156 80 40 20 Human IFN- 5000 2500 1250 625 312.5 156 80 40 20 Section 7 Human Th1/Th2 Cytokine Capture Capture Beads PE Detection Reagent A1 A6 1. 8 Cytokine Standards 9 1 18 2. Capture Bead 3. 10 µl Capture Beads " Capture " 10 µl IL-2 Capture Beads 18 180 µl Capture Beads 4. Capture Section 9 50 µl Capture / Capture Beads 9

Section 8 20 5000 pg/ml 1. Assay Diluent 1 2 1 10 1 100 2. Capture PE Detection Reagent Section 9 Human Th1/Th2 Cytokine CBA Capture Cytokine Standards Cytokine Setup Cytokine Standard Table 2 Section 10 1. 50 µl Capture Capture 2. 50 µl Human Th1/Th2 PE Detection Reagent 3. 50 µl Th1/Th2 Cytokine Standard 4. 50 µl 5. 3 10.1-10.3 6. 1mL Wash Buffer 200 x g 5 7. 8. 300 µl Wash Buffer 10

9. 3 5 CBA Table 2 50 µl 1 0 pg/ml Capture PE Detection Reagent Assay Diluent 2 20 pg/ml 3 40 pg/ml 4 80 pg/ml 5 156 pg/ml 6 312 pg/ml 7 625 pg/ml Capture PE Detection Reagent Cytokine Standards 1 256 Capture PE Detection Reagent Cytokine Standards 1 128 Capture PE Detection Reagent Cytokine Standards 1 64 Capture PE Detection Reagent Cytokine Standards 1 32 Capture PE Detection Reagent Cytokine Standards 1 16 Capture PE Detection Reagent Cytokine Standards 1 8 8 1250 pg/ml Capture PE Detection Reagent Cytokine Standards 1 4 9 2500 pg/ml Capture PE Detection Reagent Cytokine Standards 1 2 10 5000 pg/ml Capture PE Detection Reagent Cytokine Standards "Top Standard" 11

Section 10 BD FACScan FACSCalibur FACSComp CellQuest TM BD CBA 10.1 Cytometer Setup Beads 1. 50 µl Cytometer Setup Beads A B C 3 2. 50 µl FITC Positive Control Detector B 3. 50 µl PE Positive Control Detector C 4. A B C 30 5. 450 µl Wash Buffer A 400 µl Wash Buffer B C 6. 10.2 10.2 FACSComp CaliBRITE 1. 2. 3. CaliBRITE 4. FACSComp 5. Lyse/No Wash FACSComp 6. Section10.3 CaliBRITE Beads FACSComp FACSComp CaliBRITE FACSComp 4.2 Lyse/No Wash BD CBA BD CBA 10.3 3 5 FSC SSC Threshold PMT compensation Section10.3 6 8 12

10.3 Cytometer Setup Beads 1. CellQuest Template for Instrument Setup CBA instrument setup template BD CBA BD CBA CBA instrument setup template BD CBA version 1.1 FACStation HD BD Applications CBA instrument setup template CellQuest Version 3.2 OS 2. Acquisition BD CBA FSC SSC FL1 FL2 FL3 5 3. SSC FSC Log 4. SSC PMT FACSComp 100 5. Threshold FSC 650 6. Cytometer Setup Beads A Pause Restart Median 13

R1 Figure 3a SSC-H 10 0 10 4 R1 10 0 10 4 FSC-H Figure 3a FL3 R2 Median 5000 FL3 PMT Figure 3b R3 FL3 R3 Figure 3b R2 FL3-H 10 0 10 4 R2 Bright Beads (R2) Median: 5139.70 FL1 (Median) Median: 2.53 R3 10 0 10 4 FL1-H Figure 3b FL1 Median 2.0 2.5 FL1 PMT Figure 3b FL2 Median 2.0 2.5 FL2 PMT Figure 3c 14

FL3-H 10 0 10 4 10 0 10 4 FL2-H FL2 (Median) Median: 2.21 Figure 3c Cytometer Setup Beads B FL2- FL1 compensation R5 FL1 R5 Figure 3d FL2- FL1 R5 Median R4 Median Figure 3d FL2-H 10 0 10 4 R5 R4 10 0 10 4 FL1-H Figure 3d (R4) Median: 2.94 (R5) Median: 3.75 Cytometer Setup Beads C FL1- FL2 FL3- FL2 compensation 15

R7 FL2 R7 Figure 3e FL1- FL2 R7 Median R6 Median Figure 3e FL1-H 10 0 10 4 R9 FL2 R9 Figure 3f FL3- FL2 R9 Median R8 Median Figure 3f FL3-H 10 0 10 4 R7 (R6) Median: 2.81 (R7) Median: 3.08 R6 10 0 10 4 FL2-H Figure 3e R9 R8 (R8) Median: 27.88 (R9) Median: 28.13 10 0 10 4 FL2-H Figure 3f FL2- FL3 0.1 16

10.4 1. BD CBA FACStation HD BD Applications BD CBA Folder Sample Files Mouse Isotyping Files Instrument set up "Isotype Kit Acquire Template" Isotype Kit Acquire Template CellQuest Version 3.2 OS 2. Acquisition 10.3 3. Acquisition & Storage Resolution 1024 4. R1 gated events=1800 Capture Bead 300 5. "all events" 6. Set up #1 FSC vs SSC R1 Figure 3a 7. 8. "HIGH" 0 pg/ml Section9 Table 2 BD CBA 0 pg/ml #1 KT032598.001 1 17

18 Figure 4 Acquisition template example

10.5 BD CBA BD CBA 1. FACS BD CBA 2. 2 "Standards" "Samples" 3. 1 10 PE Detection Regent "Standards" "Samples" BD CBA Human Th1/ Th2 Cytokine CBA Analyte 1 INF- Analyte 2 TNF- Analyte 3 IL-10 Analyte 4 IL-5 Analyte 5 IL-4 Analyte 6 IL-2 19

Section 11 A. 10 4 Negative Control B. 10 4 Standards 80 pg/ml FL3-H FL3-H 10 0 10 0 FL2-H 10 4 10 0 10 0 FL2-H 10 4 C. 10 4 Standards 625 pg/ml D. 10 4 Standards 5000 pg/ml FL3-H FL3-H 10 0 10 0 FL2-H 10 4 10 0 10 0 FL2-H 10 4 Figure 5 CellQuest data examples for standards and detectors alone 20

10000 IFN-gamma 10000 TNF-alpha 1000 1000 100 100 10 10 1 1 00 1000 10000 Concentration (pg/ml) 1 1 00 1000 10000 Concentration (pg/ml) IL-10 IL-5 10000 10000 1000 1000 100 100 10 10 1 1 00 1000 10000 Concentration (pg/ml) 1 1 00 1000 10000 Concentration (pg/ml) IL-4 IL-2 10000 10000 1000 1000 100 100 10 10 1 1 00 1000 10000 Concentration (pg/ml) 1 1 00 1000 10000 Concentration (pg/ml) Figure 6 BD CBA Software example of standard curves 21

Figure 7 BD CBA Software example of sample results 22

Section 12 Human Th1/Th2 Cytokine CBA 12.1 Human Th1/Th2 Cytokine CBA 20 pg/ml 5000 pg/ml 4 Human Th1/Th2 Cytokine CBA 0 pg/ml 20 Median + 2SD Median Standard Assay Sensitivity Fluorescence Deviation pg/ml IL-2 3.3 0.2 2.6 IL-4 2.3 0.2 2.6 IL-5 2.6 0.2 2.4 IL-.4 0.2 2.8 TNF- 2.0 0.2 2.8 IFN- 2.1 0.3 7.1 12.2 3 3 Matrix Matrix EDTA Standard Diluent 23

12.2 Cytokine Matrix Standard spike concentration pg/ml Observed in given matrix pg/ml Recovery IL-2 Pooled Donor Sera 2500 1958 78 n 5 625 406 65 80 49 62 IL-2 Pooled Donor Plasma 2500 1656 78 n 5 625 427 68 80 54 67 IL-2 Cell culture supernatant 2500 2605 104 625 659 106 80 86 107 IL-4 Pooled Donor Sera 2500 2158 86 n 5 625 527 84 80 71 89 IL-4 Pooled Donor Plasma 2500 2197 88 n 5 625 512 82 80 63 79 IL-4 Cell culture supernatant 2500 2452 98 625 575 92 80 81 102 IL-5 Pooled Donor Sera 2500 1748 70 n 5 625 430 69 80 58 72 IL-5 Pooled Donor Plasma 2500 1710 68 n 5 625 380 61 80 54 67 IL-5 Cell culture supernatant 2500 2622 105 625 625 102 80 85 106 IL-10 Pooled Donor Sera 2500 2044 82 n 5 625 494 79 80 62 81 IL-10 Pooled Donor Plasma 2500 2034 81 n 5 625 445 71 80 62 78 IL-10 Cell culture supernatant 2500 2680 107 625 640 103 80 88 110 TNF- Pooled Donor Sera 2500 1846 74 n 5 625 447 72 80 59 73 TNF- Pooled Donor Plasma 2500 1731 69 n 5 625 407 65 80 54 67 TNF- Cell culture supernatant 2500 2856 114 625 669 107 80 91 113 IFN- Pooled Donor Sera 2500 1748 70 n 5 625 479 77 80 73 91 IFN- Pooled Donor Plasma 2500 1813 73 n 5 625 458 73 80 66 83 IFN- Cell culture supernatant 2500 2446 98 625 628 101 80 84 105 24

12.3 Matrix IL-2 IL-4 IL-5 IL-10 TNF- IFN- Assay Diluent 2 Matrix Dilution Observed IL-2 pg/ml Observed IL-4 pg/ml Observed IL-5 pg/ml Pooled Donor Sera Neat 3254 4079 3520 n=5 1 2 1747 2292 2083 1 4 863 1185 1150 1 8 388 600 529 1 16 187 277 275 1 64 48 70 61 1 256 12 19 17 Slope 1.01 1.02 1.02 Pooled Donor Plasma Neat 3785 4320 3750 n=5 1 2 1934 2345 2047 1 4 919 1181 1139 1 8 433 608 541 1 16 208 273 276 1 64 53 67 68 1 256 13 20 17 Slope 1.01 1.01 1.01 Cell Culture Medium Neat 4578 4546 4934 1 2 2345 2409 2509 1 4 1176 1105 1216 1 8 561 605 614 1 16 278 281 305 1 64 70 77 72 1 256 17 20 18 Slope 1.01 1.01 1.00 Matrix Dilution Observed IL-10 pg/ml Observed TNF- pg/ml Observed IFN- pg/ml Pooled Donor Sera Neat 3883 3502 3030 n=5 1 2 2094 1971 1607 1 4 1014 1054 892 1 8 513 534 441 1 16 244 270 227 1 64 61 67 55 1 256 17 18 20 Slope 1.01 1.01 1.00 Pooled Donor Plasma Neat 4245 3561 2743 n=5 1 2 2118 2061 1580 1 4 1032 1085 841 1 8 532 554 407 1 16 251 285 209 1 64 68 75 50 1 256 17 18 18 Slope 1.00 1.01 1.02 Cell Culture Medium Neat 4851 5500 4074 1 2 2570 2762 2142 1 4 1165 1222 1039 1 8 610 613 506 1 16 301 311 263 1 64 80 78 57 1 256 18 19 22 Slope 1.01 1.00 1.01 25

12.4 Human Th1/Th2 Cytokine CBA 1 Capture Bead A. 10 4 Human IL-2 B. 10 4 Human IL-4 FL3-H FL3-H 10 0 10 0 FL2-H 10 4 10 0 10 0 FL2-H 10 4 Human IL-5 Human IL-10 C. 10 4 D. 10 4 FL3-H FL3-H 10 0 10 0 FL2-H 10 4 10 0 10 0 FL2-H 10 4 Human TNF-α Human IFN-γ E. 10 4 F. 10 4 FL3-H FL3-H 10 0 10 0 FL2-H 10 4 10 0 10 0 FL2-H 10 4 Figure 8 CellQuest data for detection of individual cytokines 26

12.5 3 IL-2 IL-4 IL-5 IL-10 TNF- IFN- 10 Cytokine IL-2 IL-4 Actual Mean Conc. pg/ml : 71 534 2160 85 592 2488 SD 1 23 74 3 25 108 CV 2 4 3 4 4 4 Cytokine IL-5 IL-10 Actual Mean Conc. pg/ml : 78 587 2456 82 592 2486 SD 2 29 150 2 15 86 CV 3 5 6 2 2 3 Cytokine TNF- IFN- Actual Mean Conc. pg/ml : 81 592 2504 78 528 2194 SD 2 22 126 3 21 65 CV 3 4 5 4 4 3 27

3 IL-2 IL-4 IL-5 IL-10 TNF- IFN- 4 Cytokine IL-2 IL-4 Number of Replicates: Actual Mean Conc. pg/ml : 8 8 8 8 8 8 77 627 2509 74 601 2553 SD 7 29 247 5 40 147 CV 9 5 10 7 7 6 Cytokine IL-5 IL-10 Number of Replicates: Actual Mean Conc. pg/ml : 8 8 8 8 8 8 80 615 2560 81 631 2556 SD 4 39 165 5 45 189 CV 5 6 6 6 7 7 Cytokine TNF- IFN- Number of Replicates: Actual Mean Conc. pg/ml : 8 8 8 8 8 8 79 627 2564 69 6518 SD 4 31 131 6 40 139 CV 5 5 5 9 7 6 28

Section 13 Capture Bead Human Th1/Th2 PE Detection Reagent 6 Capture Bead Capture Bead FSC Threshold FSC/SSC Cytometer Setup Beads FL3 Cytometer Setup Beads 12 1 Human Th1/Th2 Cytokine CBA IL-4 IL-5 TNF- INF- 29

Section14 1. Bishop, J.E. and K.A. Davis. 1997. A flow cytometric immunoassay for 2-microglobulin in whole blood. J. Immunol. Methods 210:79-87. 2. Camilla C., J.P. Defoort, M. Delaage, R. Auer, J. Quintana, T. Lary, R. Hamelik, S. Prato, B. Casano, M. Martin and V. Fert. 1998. A flow cytometric immunoassay for b 2-microglobulin in whole blood.1.application to cytokine immunoassays. Cytometry Suppl. 8:132. 3. Carson, R., and D. Vignali. 1999. Simultaneous quantitation of fifteen cytokines using a multiplexed flow cytometric assay. J. Immunol. Methods 227: 41-52. 4. Chen, R., L. Lowe, J.D. Wilson, E. Crowther, K. Tzeggai, J.E. Bishop and R. Varro. 1999. Simultaneous quantification of six human cytokines in a single sample using microparticle-based flow cytometric technology. Clin. Chem. 9:1693-1694. 5. Collins, D. P., B.J. Luebering and D.M. Shaut. 1998. T-lymphocyte functionality assessed by analysis of cytokine receptor expression, intracellular cytokine expression, and femtomolar detection of cytokine secretion by quantitative flow cytometry. Cytometry 33:249-55. 6. Fulton, R., R. McDade, P. Smith, L. Kienker, J. Kettman, Jr. 1997. Advanced multiplexed analysis with the FlowMetrix system.clin. Chem. 43: 1749-1756. 7. Kricka, LJ. 1996. Simultaneous multianalyte immunoassays. In Immunoassay. Diamandis, E.P. and T.K. Christopoulos, eds. Academic Press. pp.389-404. 8. Lund-Johansen, F., K. Davis, J. Bishop and R. de W. Malefyt. 2000. Flow cytometric analysis of immunoprecipitates: High-throughput analysis of protein phosphorylation and protein-protein interactions. Cytometry 39:250-259. 9. McHugh, T.M. 1994. Flow microsphere immunoassay for the quantitative and simultaneous detection of multiple soluble analytes. Methods in Cell Biology 42:575-595. 10. Oliver, K.G., J.R. Kettman and R.J. Fulton. 1998. Multiplexed analysis of human cytokines by use of the FlowMetrix system. Clin. Chem. 44:2057-60. 11. Stall, A., Q. Sun, R. Varro, L. Lowe, E. Crowther, B. Abrams, J. Bishop, and K. Davis. 1998. A single tube flow cytometric multibead assay for isotyping mouse monoclonal antibodies. Abstract LB77. Experimental Biology Meeting 1998 (late-breaking abstracts). 30

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64-029-01 R0-0201-000.6-460