CBA Kit Manual Human

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

2 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

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

4 2.1 CBA ELISA 1 6 ELISA ELISA CBA 1 CBA ELISA Human Th1/Th2 Cytokine CBA ELISA BD CBA FACStar TM FACSVantage TM 3

5 Section 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 A Cytometer Setup Beads D PMT Cytometer Setup Beads 50 µl/ 4

6 3.2 Human Th1/Th2 PE Detection Reagent B PE IL-2 IL-4 IL-5 IL-10 TNF- IFN µl/ 4 PE Positive Control Detector E1 PE µl/ Cytometer Setup Beads 4 FITC Positive Control Detector E2 FITC µl/ Cytometer Setup Beads 4 Human Th1/Th2 Cytokine Standards C ml Assay Diluent IL-2 IL-4 IL-5 IL-10 TNF- IFN- 50 ng/ml 4 Human Th1/Th2 Cytokine Standards

7 3.3 Buffer Assay Diluent G 1 Buffer 30mL 1 Human Th1/Th2 Cytokine Standards 4 Wash Buffer F 1 PBS 130mL 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 BD CBA Software BD Pharmingen CellQuest BD CBA Microsoft Excel Macintosh BD CBA Software BD CaliBRITE TM 3 Beads BD

8 Section 5 Human Th1/Th2 Cytokine CBA 1. Human Th1/Th2 Cytokine Standards Assay Diluent Assay Diluent Standards 3. Human Cytokine Capture Bead 10 µl/ 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 Wash Buffer 400 µl B C 4. Wash Buffer 450 µl A 5. A B C 7

9 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 mm BD Falcon Top Standard µl Assay Diluent Top Standard µl Assay Diluent µl 10 Top Standard 6. Top Standard 300 µl µl 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

10 pg/ml Table 1 Table 1 Human Th1/Th2 Cytokine Standard Ptotein Top pg/ml Standard DilutionDilutionDilutionDilutionDilutionDilutionDilutionDilution Tube Tube Tube Tube Tube Tube Tube Tube Human IL Human IL Human IL Human IL Human TNF Human IFN Section 7 Human Th1/Th2 Cytokine Capture Capture Beads PE Detection Reagent A1 A Cytokine Standards Capture Bead µl Capture Beads " Capture " 10 µl IL-2 Capture Beads µl Capture Beads 4. Capture Section 9 50 µl Capture / Capture Beads 9

11 Section pg/ml 1. Assay Diluent Capture PE Detection Reagent Section 9 Human Th1/Th2 Cytokine CBA Capture Cytokine Standards Cytokine Setup Cytokine Standard Table 2 Section µl Capture Capture µl Human Th1/Th2 PE Detection Reagent µl Th1/Th2 Cytokine Standard µl mL Wash Buffer 200 x g µl Wash Buffer 10

12 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 pg/ml pg/ml pg/ml Capture PE Detection Reagent Cytokine Standards Capture PE Detection Reagent Cytokine Standards 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 pg/ml Capture PE Detection Reagent Cytokine Standards pg/ml Capture PE Detection Reagent Cytokine Standards pg/ml Capture PE Detection Reagent Cytokine Standards "Top Standard" 11

13 Section 10 BD FACScan FACSCalibur FACSComp CellQuest TM BD CBA 10.1 Cytometer Setup Beads µl Cytometer Setup Beads A B C µl FITC Positive Control Detector B µl PE Positive Control Detector C 4. A B C µl Wash Buffer A 400 µl Wash Buffer B C FACSComp CaliBRITE 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 FSC SSC Threshold PMT compensation Section

14 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 FL SSC FSC Log 4. SSC PMT FACSComp Threshold FSC Cytometer Setup Beads A Pause Restart Median 13

15 R1 Figure 3a SSC-H R FSC-H Figure 3a FL3 R2 Median 5000 FL3 PMT Figure 3b R3 FL3 R3 Figure 3b R2 FL3-H R2 Bright Beads (R2) Median: FL1 (Median) Median: 2.53 R FL1-H Figure 3b FL1 Median FL1 PMT Figure 3b FL2 Median FL2 PMT Figure 3c 14

16 FL3-H 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 R5 R FL1-H Figure 3d (R4) Median: 2.94 (R5) Median: 3.75 Cytometer Setup Beads C FL1- FL2 FL3- FL2 compensation 15

17 R7 FL2 R7 Figure 3e FL1- FL2 R7 Median R6 Median Figure 3e FL1-H R9 FL2 R9 Figure 3f FL3- FL2 R9 Median R8 Median Figure 3f FL3-H R7 (R6) Median: 2.81 (R7) Median: 3.08 R FL2-H Figure 3e R9 R8 (R8) Median: (R9) Median: FL2-H Figure 3f FL2- FL

18 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 Acquisition & Storage Resolution R1 gated events=1800 Capture Bead "all events" 6. Set up #1 FSC vs SSC R1 Figure 3a "HIGH" 0 pg/ml Section9 Table 2 BD CBA 0 pg/ml #1 KT

19 18 Figure 4 Acquisition template example

20 10.5 BD CBA BD CBA 1. FACS BD CBA 2. 2 "Standards" "Samples" 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

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

22 10000 IFN-gamma TNF-alpha Concentration (pg/ml) Concentration (pg/ml) IL-10 IL Concentration (pg/ml) Concentration (pg/ml) IL-4 IL Concentration (pg/ml) Concentration (pg/ml) Figure 6 BD CBA Software example of standard curves 21

23 Figure 7 BD CBA Software example of sample results 22

24 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 IL IL IL TNF IFN Matrix Matrix EDTA Standard Diluent 23

25 12.2 Cytokine Matrix Standard spike concentration pg/ml Observed in given matrix pg/ml Recovery IL-2 Pooled Donor Sera n IL-2 Pooled Donor Plasma n IL-2 Cell culture supernatant IL-4 Pooled Donor Sera n IL-4 Pooled Donor Plasma n IL-4 Cell culture supernatant IL-5 Pooled Donor Sera n IL-5 Pooled Donor Plasma n IL-5 Cell culture supernatant IL-10 Pooled Donor Sera n IL-10 Pooled Donor Plasma n IL-10 Cell culture supernatant TNF- Pooled Donor Sera n TNF- Pooled Donor Plasma n TNF- Cell culture supernatant IFN- Pooled Donor Sera n IFN- Pooled Donor Plasma n IFN- Cell culture supernatant

26 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 n= Slope Pooled Donor Plasma Neat n= Slope Cell Culture Medium Neat Slope Matrix Dilution Observed IL-10 pg/ml Observed TNF- pg/ml Observed IFN- pg/ml Pooled Donor Sera Neat n= Slope Pooled Donor Plasma Neat n= Slope Cell Culture Medium Neat Slope

27 12.4 Human Th1/Th2 Cytokine CBA 1 Capture Bead A Human IL-2 B Human IL-4 FL3-H FL3-H FL2-H FL2-H 10 4 Human IL-5 Human IL-10 C D FL3-H FL3-H FL2-H FL2-H 10 4 Human TNF-α Human IFN-γ E F FL3-H FL3-H FL2-H FL2-H 10 4 Figure 8 CellQuest data for detection of individual cytokines 26

28 IL-2 IL-4 IL-5 IL-10 TNF- IFN- 10 Cytokine IL-2 IL-4 Actual Mean Conc. pg/ml : SD CV Cytokine IL-5 IL-10 Actual Mean Conc. pg/ml : SD CV Cytokine TNF- IFN- Actual Mean Conc. pg/ml : SD CV

29 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 : SD CV Cytokine IL-5 IL-10 Number of Replicates: Actual Mean Conc. pg/ml : SD CV Cytokine TNF- IFN- Number of Replicates: Actual Mean Conc. pg/ml : SD CV

30 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

31 Section14 1. Bishop, J.E. and K.A. Davis A flow cytometric immunoassay for 2-microglobulin in whole blood. J. Immunol. Methods 210: Camilla C., J.P. Defoort, M. Delaage, R. Auer, J. Quintana, T. Lary, R. Hamelik, S. Prato, B. Casano, M. Martin and V. Fert A flow cytometric immunoassay for b 2-microglobulin in whole blood.1.application to cytokine immunoassays. Cytometry Suppl. 8: Carson, R., and D. Vignali Simultaneous quantitation of fifteen cytokines using a multiplexed flow cytometric assay. J. Immunol. Methods 227: Chen, R., L. Lowe, J.D. Wilson, E. Crowther, K. Tzeggai, J.E. Bishop and R. Varro Simultaneous quantification of six human cytokines in a single sample using microparticle-based flow cytometric technology. Clin. Chem. 9: Collins, D. P., B.J. Luebering and D.M. Shaut 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: Fulton, R., R. McDade, P. Smith, L. Kienker, J. Kettman, Jr Advanced multiplexed analysis with the FlowMetrix system.clin. Chem. 43: Kricka, LJ Simultaneous multianalyte immunoassays. In Immunoassay. Diamandis, E.P. and T.K. Christopoulos, eds. Academic Press. pp Lund-Johansen, F., K. Davis, J. Bishop and R. de W. Malefyt Flow cytometric analysis of immunoprecipitates: High-throughput analysis of protein phosphorylation and protein-protein interactions. Cytometry 39: McHugh, T.M Flow microsphere immunoassay for the quantitative and simultaneous detection of multiple soluble analytes. Methods in Cell Biology 42: Oliver, K.G., J.R. Kettman and R.J. Fulton Multiplexed analysis of human cytokines by use of the FlowMetrix system. Clin. Chem. 44: Stall, A., Q. Sun, R. Varro, L. Lowe, E. Crowther, B. Abrams, J. Bishop, and K. Davis 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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