Macrophages.

Format:

Book

Author/Creator:

Paulnock, Donna M.

Series:

Practical Approach

Practical Approach ; v.239

Language:

English

Subjects (All):

Methodology.

Physical Description:

1 online resource (228 pages)

Edition:

1st ed.

Other Title:

Macrophages

Place of Publication:

Oxford : Oxford University Press, 2000.

Summary:

Macrophages are an important part of the immune response and are characterized by their ability to phagocytose foreign matter. However the difficulties involved in macrophage isolation mean they are some of the body's least explored cells. Macrophage Methodology describes how to isolate moderate to high yields of viable cells from a variety of specific tissue sites under both normal and pathological conditions and then goes on to give protocols for macrophage purification. The third chapter covers techniques used to identify and measure endocytic and phagocytic capabilities using immunochemistry and fluorescent analysis. Chapter four identifies the key issues relating to the study of macrophages as antigen presenting cells and has protocols for the major assays used to measure antigen processing and presentation. Also covered are the theoretical and practical issues related to the processing and presentation of intracellular pathogens for which macrophages are the major host cell. The methods described for measuring macrophage secretory products concentrate on bioassays for molecules where no ELISA is available.; The next two chapters cover measuring macrophage activity in vitro and in vivo. Finally methods are described for the analysis of gene expression in macrophages. A variety of broad techniques have been brought together in one affordable volume to make Macrophage Methodology an essential buy for anyone studying macrophages.

Contents:

Intro

Contents

Preface

List of protocols

Abbreviations

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

1 Introduction

2 The heterogeneity of macrophages

3 Isolation of free mononuclear phagocytes

Murine peritoneal cells

Blood monocytes

Alveolar macrophages

4 Macrophages in haematopoietic tissues

5 Fixed tissue macrophages

Mechanical and enzymatic digestion of tissue

6 Macrophages in immune response sites

Macrophages in infection

Tumour-associated macrophages

References

2 Purification of macrophages

2 Purification of macrophages by adherence-based methods

Adhesion properties of macrophages

Macrophage adhesion molecules

Effect of adherence on macrophage gene expression

Effect of adherence on macrophage functional activities

3 Technical approaches for macrophage purification by adherence-based methods

Adherence to uncoated plastic or glass surfaces

Adherence to gelatin-coated surfaces

Adherence to microexudate-coated surfaces

Adherence to collagen matrices

4 Methods for detachment of adherent macrophages

Mechanical detachment

Recovery of adherent cells by EDTA treatment

Recovery of adherent macrophages by lignocaine treatment

5 General considerations on the adherence-based methods

Effects of different surfaces on macrophage morphology and functions

Effects of different detachment procedures on macrophage physiology

6 Physical methods of macrophage purification

Purification of macrophages by isopycnic gradient centrifugation

Isolation of whole mononuclear cells from peripheral blood by the Ficoll-Hypaque gradient

Purification of monocytes by Percoll gradient

Importance of the control of experimental conditions in the preparation of a gradient

Counterflow centrifugal elutriation.

7 Additional issues to be considered in monocytes/macrophages purification

Macrophage heterogeneity

Comparison of the efficacy of different purification techniques with respect to the source of macrophages

Non-adherent versus adherent culture of macrophages

Problems caused by LPS contamination during the course of macrophage purification and culture

8 Conclusions

Acknowledgements

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

2 Immunochemical labelling of monocytes and macrophages

Introduction

Practical considerations for immunofluorescent staining of macrophage populations in vitro

Preparation of in vitro macrophage cultures for immunochemical staining

3 Detection techniques for fluorescent analysis of macrophages

Flow cytometry

Fluorescent microscopy

4 Immunohistochemical staining of macrophages in mouse tissues

Preparation of tissue

Immunochemical staining of tissue

5 Fluorescent analysis of macrophage endocytic function

Introduction to the endocytic pathway

Practical considerations for testing macrophage endocytic function

Practical considerations for testing macrophage phagocytic function

6 Conclusion

4 Analysis of antigen processing and presentation

2 Preliminary considerations in study design

A definition of antigen processing and presentation

T cell choice restricts functional interpretation

'In vivo veritas'

The dendritic cell issue

Pathogens are not simple antigens

3 Analysis of class I and II antigen processing

Pathways of processing

Modified protocols for use with pathogens

Cell biology of antigen processing

4 Analysing antigen presenting function of macrophages.

Correlative studies of cell phenotype

Functional assays of co-stimulation

5 Macrophage secretory products

2 Cytokines and chemokines: chemotaxis

3 Leukocyte transmigration

4 Reverse transmigration

5 Soluble cytokine receptors

6 Cross-linking of soluble receptors

6 Analysis of macrophage lytic functions

Biological perspective

Technical notes

2 Macrophage-mediated anti-tumour activity

Morphological tumour cell counting assay

Macrophage-mediated cytolysis: release of radioisotopes

Macrophage-mediated cytostasis of tumour cells: incorporation of radioactive labels

3 Target cell sensitivity

4 Microbicidal activity

Anti-Leishmania activity of monocytes/macrophages

Anti-fungal activity

7 Analysis of macrophage activity In vivo

2 Previous methods for blocking of phagocytosis

Silica, carrageenan, and dextran sulfate

Gadolinium chloride

Anti-macrophage antibodies and receptor antagonists

Competition

3 The liposome-mediated macrophage suicide technique

Principles

Liposomes

Liposome-encapsulated clodronate

Injection of liposomes and access to tissue macrophages

Selectivity of the approach with respect to macrophages

Duration of macrophage depletion

4 Practical applications of the technique

Improved efficacy of carrier-mediated gene transfer

Improved survival of human cells in immunodeficient (SCID) mice

Suppression of inflammatory reactions

Improved graft survival and functioning

8 Analysis of gene expression In mononuclear phagocytes

2 Detection and quantification of specific RNA levels

Basic principles

Preparation of total cellular RNA

Nuclear run-on analysis

Quantification of specific mRNAs

3 Gene transfer.

Basic principles

Experimental strategies

Transient transfection

Stable transfection

4 Measurement of protein-nucleic acid interactions

Sources and characteristics of binding factors

Measurements of DNA and RNA binding proteins

A1 List of suppliers

Index.

Notes:

Description based on publisher supplied metadata and other sources.

ISBN:

1-280-37557-4

9786610375578

0-585-48401-5

OCLC:

1027194088