Contents

Preface page v List of protocols xi Abbreviations xv

1 Isolation of macrophages from tissues, fluids, and immune response sites 1

Mary Ellen Handel-Fernandez and Diana M. Lopez

1 Introduction 1

2 The heterogeneity of macrophages 1

3 Isolation of free mononuclear phagocytes 2

Murine peritoneal cells 2 Blood monocytes 6 Alveolar macrophages 7

4 Macrophages in haematopoietic tissues 10

5 Fixed tissue macrophages 14

Mechanical and enzymatic digestion of tissue 15

6 Macrophages in immune response sites 25

Macrophages in infection 25 Tumour-associated macrophages 26 References 28

2 Purification of macrophages 31

Sandra Gessanl, Laura Fantuzzi, Patrizia Puddu, and FHippo Belardelli

1 Introduction 31

2 Purification of macrophages by adherence-based methods 32

Adhesion properties of macrophages 32

Macrophage adhesion molecules 33

Effect of adherence on macrophage gene expression 33

Effect of adherence on macrophage functional activities 35

3 Technical approaches for macrophage purification by adherence-based methods 35 Adherence to uncoated plastic or glass surfaces 36

Adherence to gelatin-coated surfaces 36 Adherence to microexudate-coated surfaces 38 Adherence to collagen matrices 39

Methods for detachment of adherent macrophages 40 Mechanical detachment 40 Recovery of adherent cells by EDTA treatment 41 Recovery of adherent macrophages by lignocaine treatment 42

General considerations on the adherence-based methods 43

Effects of different surfaces on macrophage morphology and functions 43 Effects of different detachment procedures on macrophage physiology 44 Physical methods of macrophage purification 45

Purification of macrophages by isopycnic gradient centrifugation 46 Isolation of whole mononuclear cells from peripheral blood by the

Ficoll-Hypaque gradient 46 Purification of monocytes by Percoll gradient 48 Importance of the control of experimental conditions in the preparation of a gradient 49 Counterflow centrifugal elutriation 50 Additional issues to be considered in monocytes/macrophages purification 54 Macrophage heterogeneity 54

Comparison of the efficacy of different purification techniques with respect to the source of macrophages 55 Non-adherent versus adherent culture of macrophages 56 Problems caused by LPS contamination during the course of macrophage purification and culture 56

Conclusions 56

Acknowledgements 57

References 57

3 Characterization of macrophage antigens and receptors by immunochemistry and fluorescent analysis: expression, endocytosis, and phagocytosis 61

Leanne Peiser, Peter J. Gough, Elizabeth Barky, and Siamon Gordon

1 Introduction 61

2 Immunochemical labelling of monocytes and macrophages 62 Introduction 62

Practical considerations for immunofluorescent staining of macrophage populations in vitro 64 Preparation of in vitro macrophage cultures for immunochemical staining

3 Detection techniques for fluorescent analysis of macrophages 70 Flow cytometry 70 Fluorescent microscopy 71

4 Immunohistochemical staining of macrophages in mouse tissues 71 Preparation of tissue 72 Immunochemical staining of tissue 76

5 Fluorescent analysis of macrophage endocytic function 78 Introduction to the endocytic pathway 78

Practical considerations for testing macrophage endocjrtic function 81 Practical considerations for testing macrophage phagocytic function 86

6 Conclusion 89

Acknowledgements 90 References 90

4 Analysis of antigen processing and presentation 93

1 Introduction 93

2 Preliminary considerations in study design 93

A definition of antigen processing and presentation 93

T cell choice restricts functional interpretation 94

'In vivo Veritas' 95

The dendritic cell issue 95

Pathogens are not simple antigens 96

3 Analysis of class I and II antigen processing 96

Pathways of processing 96

Modified protocols for use with pathogens 105

Cell biology of antigen processing 110

4 Analysing antigen presenting function of macrophages 110

Correlative studies of cell phenotype 110 Functional assays of co-stimulation 111 References 112

5 Macrophage secretory products lis

Paola Allavena, Giancarlo Bianchi, Walter Luini, Andrea Doni, Pietro Transidico, Silvano Sozzani, and Alberto Mantovani

1 Introduction 115

2 Cytokines and chemokines: chemotaxis 116

3 Leukocyte transmigration 118

4 Reverse transmigration 121

5 Soluble cytokine receptors 122

6 Cross-linking of soluble receptors 124 References 125

6 Analysis of macrophage lytic functions 127

Maria Carla Bosco, Tiziana Musso, hxca Carta, and Luigi Varesio

1 Introduction 127

Biological perspective 127 Technical notes 129

2 Macrophage-mediated anti-tumour activity 130

Morphological tumour cell counting assay 130 Macrophage-mediated cytolysis: release of radioisotopes 133 Macrophage-mediated cytostasis of tumour cells: incorporation of radioactive labels 140

3 Target cell sensitivity 141

4 Microbicidal activity 142

Anti-Leishmania activity of monocytes/macrophages 144 Anti-fungal activity 147

References 153

7 Analysis of macrophage activity In vivo 157

Nico van Rooijen and Esther van Kesteren-Hendrikx

1 Introduction 157

2 Previous methods for blocking of phagocytosis 157

Silica, carrageenan, and dextran sulfate 157 Gadolinium chloride 158

Anti-macrophage antibodies and receptor antagonists 159 Competition 159

3 The liposome-mediated macrophage suicide technique 160

Principles 160 Liposomes 161

Liposome-encapsulated clodronate 162 Injection of liposomes and access to tissue macrophages 165 Selectivity of the approach with respect to macrophages 166 Duration of macrophage depletion 16 7

4 Practical applications of the technique 368

Improved efficacy of carrier-mediated gene transfer 168 Improved survival of human cells in immunodeficient (SCID) mice 169 Suppression of inflammatory reactions 170 Improved graft survival and functioning 170 References 171

8 Analysis of gene expression in mononuclear phagocytes 173

Joyce E. S. Doan, Thomas A. Hamilton, and Donna M. Paulnock

1 Introduction 173

2 Detection and quantification of specific RNA levels 174

Basic principles 174 Preparation of total cellular RNA 174 Nuclear run-on analysis 177 Quantification of specific mRNAs 181

3 Gene transfer 185

Basic principles 185 Experimental strategies 185 Transient transfection 186 Stable transfection 192

4 Measurement of protein-nucleic acid interactions 193

Basic principles 193

Sources and characteristics of binding factors 194 Measurements of DNA and RNA binding proteins 195 References 201

A1 List of suppliers 203 Index 209

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