BD Biosciences Techniques for Immune Function Analysis Application Handbook 1 st Edition For Research Use Only. Not for use in diagnostic or therapeutic procedures. 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. All applications are either tested in-house or reported in the literature. See Technical Data Sheets for details. BD, BD Logo and all other trademarks are the property of Becton, Dickinson and Company. ©2003 BD For additional information please access the Immune Function Homepage at www.bdbiosciences.com/immune_function Table of Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Chapter 1: Immunofluorescent Staining of Cell Surface Molecules for Flow Cytometric Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Chapter 2: BD™ Cytometric Bead Array (CBA) Multiplexing Assays . . . . . . 35 Chapter 3: BD™ DimerX MHC:Ig Proteins for the Analysis of Antigen-specific T Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Chapter 4: Immunofluorescent Staining of Intracellular Molecules for Flow Cytometric Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Chapter 5: BD FastImmune™ Cytokine Flow Cytometry. . . . . . . . . . . . . . . . 85 Chapter 6: BD™ ELISPOT Assays for Cells That Secrete Biological Response Modifiers . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Chapter 7: ELISA for Specifically Measuring the Levels of Cytokines, Chemokines, Inflammatory Mediators and their Receptors . . . . 125 Chapter 8: BD OptEIA™ ELISA Sets and Kits for Quantitation of Analytes in Serum, Plasma, and Cell Culture Supernatants. . . 143 Chapter 9: BrdU Staining and Multiparameter Flow Cytometric Analysis of the Cell Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Chapter 10: Cell-based Assays for Biological Response Modifiers . . . . . . . . . 177 Chapter 11: BD RiboQuant™ Multi-Probe RNase Protection Assay System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Chapter 12: Tools to Study the Complement System . . . . . . . . . . . . . . . . . . . 229 Chapter 13: Detection of In Vivo Cytokine Production with the In Vivo Capture Assays for Cytokines . . . . . . . . . . . . . 243 Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 Unless otherwise specified, all products are for Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. 4 www.bdbiosciences.com About the Cover A set of graphics was selected that represent the various technologies, applications, and reagents offered by BD Biosciences, which are useful for studying Immune Function. The graphics were mapped together onto the surface of a sphere, which symbolizes a cell, the fundamental biological unit involved in the generation and mediation of immunological and inflammatory responses. The composite graphic represents the integrated set of tools and solutions that are available for multiparameter, high-resolution analyses of the molecular and cellular mechanisms that underlie immune function. Preface The study of the immune system attracts large numbers of researchers from diverse scientific disciplines because of its central importance in providing immunological host defense and its intercommunication with other systems that maintain bodily homeostasis. The immune system is often studied in its intact form but it can also be readily disassembled into its cellular and molecular components (eg, lymphoid cell populations and effector molecules), recombined and modified in various ways, and analyzed in an in vitro or an in vivo setting. Due to the creative development and application of a wide variety of experimental protocols, often using new technological platforms and reagents, vast amounts of new information concerning immune function become available on a daily basis. Researchers busily scrutinize this information hoping to better define and understand the networks of cellular and molecular mechanisms that underlie immunity and inflammation in health and disease. BD Biosciences is pleased to introduce the new Techniques for Immune Function Analysis, Application Handbook 1 st Edition. This handbook grew out of the original Cytokine/ Chemokine Application Manual that was first published in 1997. The original manual was based on BD Biosciences technical publications and presentations and with a tremendous amount of input from customers dealing with immune function studies from a “Genes to Proteins to Cells” perspective. The new title for this publication reflects the enlarged scope of the book that served as a guide for applications and reagents designed to study the roles played by cells and the regulatory and effector molecules (ie, biological response modifiers including cytokines, chemokines, inflammatory mediators and their receptors) that mediate inflammation and natural and acquired immunity. New chapters dealing with the BD™ Cytometric Bead Array, the BD FastImmune™ System, the BD™ ELISPOT Assay, BD™ DimerX MHC:Ig Molecules, Immunofluorescent Staining of Cell Surfaces for Flow Cytometric Analysis, and Inflammatory Mediators have been added to this handbook. Previous chapters pertaining to the BD RiboQuant™ Multi-Probe RNase Protection Assay System, ELISA, BD OptEIA™ ELISA Sets and Kits, Immunofluorescent Staining of Intracellular Molecules for Flow Cytometric Analysis, and Bioassays have been revised as well with a presentation of new reagents and methods discussed therein. For additional information please access the new Immune Function Homepage at www.bdbiosciences.com/immune_function. Unless otherwise specified, all products are for Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. Preface 5US Orders: 877.232.8995 Abbreviations 7-AAD 7-aminoactinomycin ABTS 2, 2’-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) AEC 3-amino-9-ethyl-carbazole aka also known as APC allophycocyanin or antigen-presenting cell BrdU bromodeoxyuridine BRM biological response modifier BSA bovine serum albumin CO 2 carbon dioxide cpm counts per minute DAPI 4’,6-diamidino-2-phenylindole*2HCl ddH 2 O distilled deionized water DMF dimethyl formamide DMSO dimethyl sulfoxide DTT dithiothreitol ED 50 50% effective dose EDTA ethylenediamine tetraacetic acid ELISA enzyme-linked immunosorbent assay ELISPOT enzyme-linked immunospot assay FACS fluorescent activated cell sorting FcR immunoglobulin Fc receptors FBS fetal bovine serum FITC fluorescein isothiocyanate GM-CSF granulocyte-macrophage colony-stimulating factor H 2 O 2 hydrogen peroxide hr hour HRP horseradish peroxidase IFN interferon Ig immunoglobulin IL interleukin kDa kilodalton L liter LAL limulus amebocyte lysate LPS lipopolysaccharide MCP monocyte chemoattractant protein min minute MIP macrophage inhibitory protein mRNA messenger RNA NA/LE no azide/low endotoxin ND 50 50% neutralizing dose OD optical density NBCS newborn calf serum PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PBS-Tween PBS containing 0.05% Tween-20 PE phycoerythrin PerCP Peridinin chlorophyll protein pfu plaque forming units PBMC peripheral blood mononuclear cells PI propidium iodide Unless otherwise specified, all products are for Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. Preface 6 www.bdbiosciences.com Abbreviations (continued) PMA phorbol myristate acetate PMT photomultiplier tube PY pyronin Y RANTES Regulated upon Activation, Normal T Expressed and presumably Secreted rhIL recombinant human interleukin RNA ribonucleic acid RPA ribonuclease protection assay RT room temperature SDS sodium dodecyl sulfate [ 3 H]-TdR tritiated thymidine TBE Tris borate EDTA TCC terminal complement complex TCR T cell receptor TDS Technical Data Sheets TE Tris EDTA TMB tetramethylbenzidine TNF tumor necrosis factor U unit 1. BD Cy-Chrome™ is now listed as PE-Cy5. 2. TNF-α is now listed as TNF. Unless otherwise specified, all products are for Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. Preface [...]... cellular differentiation Unless otherwise specified, all products are for Research Use Only Not for use in diagnostic or therapeutic procedures Not for resale US Orders: 877.232.8995 11 Cell Surface Staining Multicolor flow cytometric analysis also enables analysis of complex cellular interactions in mixed cell populations For instance, analysis of the expression of cell surface markers or intracellular... manner For more details on data analysis please refer to unit 5.2 of Current Protocols in Immunology.19 24 www.bdbiosciences.com Unless otherwise specified, all products are for Research Use Only Not for use in diagnostic or therapeutic procedures Not for resale References US Orders: 877.232.8995 25 Cell Surface Staining Unless otherwise specified, all products are for Research Use Only Not for use... same species as the primary antibodies (25 µl of neat serum for 20 min) After blocking, add the other fluorescent antibodies and incubate for 20 – 30 min at 4°C Unless otherwise specified, all products are for Research Use Only Not for use in diagnostic or therapeutic procedures Not for resale US Orders: 877.232.8995 15 Cell Surface Staining 3 For staining in microwell plates, add 200 µl of staining buffer... specified, all products are for Research Use Only Not for use in diagnostic or therapeutic procedures Not for resale US Orders: 877.232.8995 17 Cell Surface Staining 6 For staining in microwell plates, add 200 µl of staining buffer to each well, transfer the contents to staining tubes and bring up the volume to 0.5 ml with staining buffer and keep them at 4°C until flow cytometric analysis For staining in tubes,... antibodies specific for cell surface and intracellular markers can be used to characterize cells within populations by multiparameter flow cytometric analysis In this way, it is possible to gather information regarding each cell’s state of activation and differentiation, lineage, migration potential, and functional responsiveness (Figures 1–4) For example, it is known that receptors for some cytokines... block Fc receptors 3 Stain for Receptors and Other Cell Surface Antigens a Direct immunofluorescent staining 1 Incubate ~106 cells in 100 µl of staining buffer (see Buffers for more information) containing a pre-titrated, optimal concentration (usually ≤ 1 µg) of a fluorescent monoclonal antibody specific for a receptor or with an immunoglobulin (Ig) isotype-matched control for 30 – 45 min at 4°C In... them at 4°C until flow cytometric analysis For staining in tubes, resuspend cell samples in 0.5 ml of staining buffer and keep them at 4°C until flow cytometric analysis If desired, cells may be fixed with BD Cytofix Buffer™ (Cat No 554655, 100 µl/test) prior to flow cytometric analysis After fixation, cells are washed as indicated in step 3.a.2 and stored at 4°C until analysis However, it should be noted... at 4°C until flow cytometric analysis For staining in tubes, resuspend cell samples in 0.5 ml of staining buffer in tubes and keep them at 4°C until flow cytometric analysis If desired, cells may be fixed with BD Cytofix Buffer (Cat No 554655, 100 µl/test) prior to flow cytometric analysis After fixation, cells are washed as indicated in step 3.b.2 and stored at 4°C until analysis c Indirect immunofluorescent... control antibody for 30 – 45 min at 4°C 2 After the incubation, add 100 – 200 µl of staining buffer and pellet the cells by centrifugation (250 × g for 5 min) Wash the cells 1× with 200 µl of staining buffer, pellet by centrifugation (250 × g for 5 min), and remove supernatant For staining in tubes, wash the cells 1× with 2 ml of staining buffer and pellet the cells by centrifugation (250 × g for 5 min),... www.bdbiosciences.com Unless otherwise specified, all products are for Research Use Only Not for use in diagnostic or therapeutic procedures Not for resale 2 After the incubation, add 100 – 200 µl of staining buffer and pellet the cells by centrifugation (250 × g for 5 min) Wash the cells 1× with 200 µl of staining buffer, pellet by centrifugation (250 × g for 5 min), and remove supernatant b Indirect immunofluorescent . BD Biosciences Techniques for Immune Function Analysis Application Handbook 1 st Edition For Research Use Only. Not for use in diagnostic or therapeutic procedures Immune Function Homepage at www.bdbiosciences.com /immune_ function. Unless otherwise specified, all products are for Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for. Surface Molecules for Flow Cytometric Analysis of Immune Function Introduction To understand immune responses, it is necessary to identify, isolate, and study a variety of cell types, cell functions,