Methods for Studying Mononuclear Phagocytes

Methods for Studying Mononuclear Phagocytes is a practical guide to the study of mononuclear phagocytes that brings together various well-established and useful methods for examining these cells. The technical protocols have been made detailed, specific, practical, and inclusive of the necessary mystique for immediate and direct application in the laboratory. The book is divided into 11 parts arranged according to the sequence of steps that would generally be followed to study a given population of mononuclear phagocytes: (I) methods for obtaining and culturing populations of human and animal mononuclear phagocytes; (II) methods for separating populations of leukocytes to enrich or deplete their content of mononuclear phagocytes; (II) criteria and techniques for identifying mononuclear phagocytes; (IV) methods for quantifying the number of mononuclear phagocytes; (V) techniques for studying the morphology of mononuclear phagocytes; (VI) methods for quantifying the biochemical constituents of mononuclear phagocytes; (VII) methods of quantifying phagocytosis, pinocytosis, and chemotaxis; (VIII) methods for quantifying the secretory products of mononuclear phagocytes; (IX) procedures for quantifying the destruction of tumor cells and of microorganisms by mononuclear phagocytes; (X) methods for studying the cell biology of mononuclear phagocytes; and (XI) techniques for studying mononuclear phagocytes in vivo, including procedures for estimating their kinetics, accumulation, identification, and microbicidal properties.

Table of Contents:
Contributors Preface I. Obtaining and Culturing Mononuclear Phagocytes 1 The Culture of Mononuclear Phagocytes: A Brief Overview 2 Murine Mononuclear Phagocytes from Bone Marrow 3 Obtaining and Culturing Murine Monocytes 4 Obtaining and Culturing Human Monocytes 5 Isolation of Human Monocytes 6 Culture of Human Monocytes in Microplates and Enzymatic Assays for Following Their Maturation 7 Peritoneal Mononuclear Phagocytes from Small Animals 8 Obtaining and Culturing Human and Animal Alveolar Macrophages 9 Obtaining Human Mononuclear Phagocytes from Colostrum 10 Obtaining Adherent Cells from Spleen 11 Obtaining Kupffer Cells 12 Obtaining Mononuclear Phagocytes from Disaggregated Neoplasms 13 Obtaining Mononuclear Phagocytes from Granulomas 14 Teflon Film as a Substrate for the Culture of Mononuclear Phagocytes 15 Effects of Husbandry and Mouse Strains on Mononuclear Phagocytes 16 Endotoxin Contamination and in Vitro Monocyte-Macrophage Function: Methods of Detecting, Detoxifying, and Eliminating Endotoxin 17 Continuous Cell Lines with Properties of Mononuclear Phagocytes II. Separation of Mononuclear Phagocytes for Enrichment or Depletion 18 Separation of Mononuclear Phagocytes for Enrichment and for Depletion: An Overview 19 Separation of Murine Macrophages by Adherence to Solid Substrates 20 Separation of Human Monocytes and Guinea Pig Macrophages by Density Gradients of Metrizamide 21 Separation of Murine Mononuclear Phagocytes by Density Gradients of Percoll 22 Separation of Human Monocytes by Density Gradients of Percoll 23 Separation of Murine Mononuclear Phagocytes by Velocity Sedimentation at Unit Gravity 24 Use of Lidocaine for Detachment of Adherent Mononuclear Phagocytes 25 Use of Sephadex Columns to Deplete Mononuclear Phagocytes 26 Depletion of Mononuclear Phagocytes: Pitfalls in the Use of Carbonyl Iron, Carrageenan, Silica, Trypan Blue, or Anti-Mononuclear Phagocytes Serum III. Identification of Mononuclear Phagocytes 27 Identification of Mononuclear Phagocytes: Overview and Definitions 28 Characteristics of Mononuclear Phagocytes from Different Tissues 29 Fc and C3 Receptors 30 Identification of Mononuclear Phagocytes by Ingestion of Particulate Materials, such as Erythrocytes, Carbon, Zymosan, or Latex 31 Heteroantisera Raised against Mononuclear Phagocytes 32 Monoclonal Antibodies as Tools for the Study of Mononuclear Phagocytes 33 Antisera against Ia Antigens IV. Quantitation of Number of Mononuclear Phagocytes 34 Quantitation of Adherent Mononuclear Phagocytes by Inverted Phase Microscopy 35 Quantitation of DNA in Mononuclear Phagocytes 36 Lowry and Bradford Assays for Protein V. Morphology of Mononuclear Phagocytes 37 Use of Wrights Strain and Cytocentrifuge Preparations 38 Use of Phase Contrast Microscopy 39 Use of Peroxidase Stain by the Kaplow Method 40 Use of Nonspecific Esterase Stain 41 Histochemical Stains for Macrophages in Cell Smears and Tissue Sections: ß-Galactosidase, Acid Phosphatase, Nonspecific Esterase, Succinic Dehydrogenase, and Cytochrome Oxidase 42 Use of Transmission Electron Microscopy 43 Preparative Techniques for Scanning Electron Microscopy 44 Use of Ultrastructural Histochemistry VI. Biochemical Constituents of Mononuclear Phagocytes 45 Lysosomal Enzymes 46 Microsomal Heme Oxygenase 47 Histamine O-Methyltransferase 48 5'-Nucleotidase Assay 49 Alkaline Phosphodiesterase I 50 Quantitation of Leucine Aminopeptidase of Mononuclear Phagocytes 51 Hexose Monophosphate Shunt Activity and Oxygen Uptake 52 Secretion of Superoxide Anion 53 Release of Hydrogen Peroxide VII. General Functions of Mononuclear Phagocytes 54 Antibody-Dependent and Antibody-Independent Phagocytosis 55 Pinocytic Rate Using Horseradish Peroxidase 56 Chemotaxis of Human and Murine Mononuclear Phagocytes VIII. Secretion by Mononuclear Phagocytes 57 Secretory Functions of Mononuclear Phagocytes: Overview and Methods for Preparing Conditioned Supernatants 58 Characterization and Classification of Macrophage Proteinases and Proteinase Inhibitors 59 Growth of Macrophage on Collagen, Elastin, and Glycoprotein-Coated Plates as a Tool for Investigating Macrophage Proteinases 60 Neutral Proteases by 3H-Labeled Casein 61 Plasminogen Activators by Use of 3H-Labeled Casein Substrate 62 Elastinolytic Enzymes 63 Microtiter Assay for Antiviral Effects of Human and Murine Interferon Utilizing a Vertical Light Path Photometer for Quantitation 64 Endogenous Pyrogen 65 Prostaglandins 66 Quantitation of Selected Complement Components 67 Lysozyme IX. Destruction by Mononuclear Phagocytes 68 Destruction of Listeria monocytogenes in Vitro 69 Ingestion and Destruction of Candida albicans 70 Quantitation of Destruction of Toxoplasma 71 Destruction of Rickettsiae 72 Destruction of Leishmania 73 Destruction of Viruses 74 Cytostasis of Tumor and Nontumor Cells 75 Cytolysis of Tumor Cells by Release of [3H] Thymidine 76 Quantification of Cytolysis of Neoplastic Cells by Release of Chromium-51 77 Assessment of Cytolysis of Tumor Cells by Release of [125I] Iododeoxyuridine 78 Antibody-Dependent Cellular Cytotoxicity (ADCC) of Erythroid and Tumor Cells X. Mononuclear Phagocytes as Tools in Cell Biology 79 Overview: The Macrophage in Cell Biology 80 Biosynthetic Radiolabeling of Cellular and Secreted Proteins of Mononuclear Phagocytes 81 Extraction, Identification, and Quantitation of Lipids 82 Synthesis, Cellular Turnover, and Mass of Cholesterol 83 Solute Uptake and Membrane Transport by Mononuclear Phagocytes 84 Use of Lactoperoxidase for Labeling Membrane Proteins 85 Binding of Synthetic Chemotactic Peptides as a Model of Ligand-Receptor Interaction XI. Mononuclear Phagocytes In Vivo 86 Isolation of Phagosomes from Mouse Peritoneal Macrophages 87 Study of Mononuclear Macrophages in Vivo: Overview 88 Quantitation of the Inflammatory Accumulation of Mononuclear Phagocytes in Vivo 89 Systemic Labeling of Mononuclear Phagocytes 90 Labeling of Mononuclear Phagocytes in Granulomas and Inflammatory Sites 91 Identification of Fc and Complement Receptors in Tissue Sections 92 Determination of Macrophage-Mediated Antibacterial Resistance Supplement to Table of Contents Index