78 Steps Health Journal

Protocol list

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Isolation of free mononuclear phagocytes

Harvesting of resident peritoneal macrophages 3 Elicitation of peritoneal macrophages using thioglycollate 5 Peritoneal macrophage elicitation using Bio-Gel polyaciylamide beads 5 Isolation of human blood monocytes 6

Isolation of human alveolar macrophages from tumour lung biopsies 8 Isolation of murine alveolar macrophages by lung lavage 8 Isolation of alveolar macrophages from whole murine lung 9

Macrophages In haematopoietic tissues

Isolation of resident bone marrow macrophages 11 Isolation of murine bone marrow-derived macrophages 12

Fixed tissue macrophages

Isolation of murine splenic macrophages 14 Isolation of human splenic macrophages 16 Isolation of murine Kupffer cells 18

Isolation of human Kupffer cells from liver wedge biopsies 19 Isolation of osteoclasts from murine bone 20 Isolation of rat microglial cells 21 Isolation of human microglial cells 22

Isolation and purification of lamina propria macrophages from human small intestine 23 Isolation of murine Langerhans cells 24

Macrophages in immune response sites

Isolation of granuloma macrophages from the livers of infected mice 25 Isolation of macrophages from solid tumours (mechanical dissociation) 27 Isolation of tumour-associated macrophages (enzymatic method) 27

Technical approaches for macrophage purification by adherence-based methods

Purification of macrophages by adherence to uncoated plastic or glass surfaces 36 Purification of macrophages by adherence to gelatin-coated surfaces 37 Purification of macrophages by adherence to microexudate-coated surfaces 38 Purification of macrophages by adherence to collagen-coated surfaces 39

Methods for detachment of adherent macrophages

Mechanical detachment of macrophages 40

Detachment of macrophages by EDTA treatment 41 Detachment of macrophages by treatment with lignocaine 42

Physical methods of macrophage purification

Isolation of mononuclear cells by Ficoll-Hypaque gradient separation 47 One-step continuous Percoll gradient separation of monocytes 48 Purification of macrophages by counterflow centrifugal elutriation (CCE) 52

Immunochemical labelling of monocytes and macrophages

Preparation of isolated macrophages for immunochemical staining 66 Detaching cultured macrophages from plastic and glass surfaces using

EDTA/Lidocaine 67 Preparation of 4% paraformaldehyde 68

Indirect immunofluorescent staining of cultured macrophages 68

Immunohistochemical staining of macrophages in mouse tissues

Preparation of fresh tissue for immunohistochemistry 72

Preparation of periodate-lysine paraformaldehyde for perfusion of mouse tissue

Perfusion of mouse tissue 74

Immunochemical staining of mouse tissue 77

Fluorescent analysis of macrophage endocytic function

Fluorescent labelling of proteins or particles 83 Quantitation of macrophage endocytic function 84 Phagocytic uptake of bacteria by macrophages 89

Analysis of class I and II antigen processing

Preparation of bone marrow macrophages 97 Induction of MHC class II antigens on bone marrow macrophages 99 Pulsing macrophages with soluble antigens for analysing class II-restricted processing 101

Assessment of the cellular characteristics of class II-restricted antigen processing 103

Loading soluble antigens into macrophages by osmotic lysis of pinosomes 104 Stimulation of class I-restricted responses by BMM0 105 Pulsing macrophages with particulate antigens in suspension 106 Enumeration of microbe uptake by macrophages using direct staining 108 Evaluating antigen transfer between APC populations 109

Analysing antigen presenting function of macrophages

Assay of co-stimulatory function of macrophages 111

Cytokines and chemokines: chemotaxis

Assessment of macrophage migration using a Boyden chamber 117

Leukocyte transmigration

Assessment of transendothelial migration by radioisotopic detection 119

Reverse transmigration

Assessment of leukocyte reverse transmigration in vitro 121

Cross-linking of soluble receptors

Identification of soluble cytokine receptors using radiolabelled ligands 124

Macrophage-mediated anti-tumour activity

Measurement of macrophage anti-tumour activity by the tumour cell counting assay 131

Measurement of macrophage-mediated cytolysis by the 51Cr release assay 133 Assessment of macrophage-mediated cytotoxicity using [3H]TdR and [125I]dUrd release assays 135

Measurement of macrophage cytotoxicity using the mindium release assay 138 Assessment of macrophage-mediated cytostasis of tumour cells based on target cell incorporation of radioactive label 140

Microbicidal activity

Assessment of macrophage anti-Leishmania cytolytic activity 145

Detection of phagocytosis of C. albicans by macrophages 148

Assessment of macrophage-mediated intracellular Candida killing using a colony counting technique 150 Assessment of macrophage-mediated extracellular Candida killing using a colorimetric assay 151

The llposome-medlated macrophage suicide technique

Preparation of multilamellar clodronate-liposomes 162 Spectrophotometry: determination of the amount liposome-encapsulated clodronate 164

Detection and quantification of specific RNA levels

Preparation of total cellular RNA from cultured macrophages 175 Detection of nuclear RNA by nuclear run-on assay 178 Analysis of specific gene expression by RT-PCR 182

Gene transfer

Purification of supercoiled plasmid DNA 187 DEAE dextran-mediated transfection of macrophages 189 Transfection of macrophages using lipophilic reagents 190 Transfection of macrophages by electroporation 191

Measurement of protein-nucleic acid interactions

Preparation of nuclear extracts from macrophages 196 Preparation of oligonucleotide probes for EMSA 198 Electrophoretic mobility shift assay (EMSA) 199

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