SpringerProtocols: Abstract: 7. Isolation and Culture of Murine Macrophages

7. Isolation and Culture of Murine Macrophages

By: John Q. Davies³, Siamon Gordon³

Abstract

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The two most convenient sources of primary murine macrophages are the bone marrow and the peritoneal cavity. Resident peritoneal macrophages can readily be harvested from mice and purified by adherence to tissue culture plastic. The injection of Bio-Gel polyacrylamide beads or thioglycollate broth into the peritoneal cavity produces an inflammatory response allowing the purification of large numbers of elicited macrophages. The production of an activated macrophage population can be achieved by using Bacillus-Calmette-Guerin as the inflammatory stimulus. Resident bone marrow macrophages can be isolated following enzymatic separation of cells from bone marrow plugs and enrichment on 30% fetal calf serum containing medium or Ficoll-Hypaque gradients. Bone marrow-derived macrophages can be produced by differentiating nonadherent macrophage precursors with medium containing macrophage colony-stimulating factor.

Affiliation(s): (3) Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

Book Title: Basic Cell Culture Protocols

Series: Methods in Molecular Biology | Volume: 290 | Pub. Date: Oct-15-2004 | Page Range: 91-103 | DOI: 10.1385/1-59259-838-2:091

Subject: Cell Biology

Key Words: Macrophage - murine - culture - peritoneum - bone marrow - resident - Bio-Gel - thioglycollate - BCG

References

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1.	Leijh, P. C. J., Van Furth, R., and Van Swet, T. L. (1986) In vitro determination of phagocytosis and intracellular killing by polymorphonuclear and mononuclear phagocytes, in Handbook of Experimental Immunology (Weir, D. M., ed.), Blackwell Scientific, Oxford, pp. 46.1–46.21.

2.	Stein, M. and Gordon, S. (1991) Regulation of tumor necrosis factor (TNF) release by murine peritoneal macrophages: role of cell stimulation and specific phagocytic plasma membrane receptors. Eur. J. Immunol 21, 431–437.

3.	Adams, D. H. and Lloyd, A. R. (1997) Chemokines: leucocyte recruitment and activation cytokines. Lancet 349, 490–495.

4.	Peiser, L., Gough P. J., Darley, E., and Gordon, S. (2000) Characterisation of macrophage antigens and receptors by immunohistochemistry and fluorescent analysis: expression, endocytosis and phagocytosis, in Macrophages (Paulnock, D. M., ed.), Oxford University Press, Oxford, pp. 61–91.

4a.	Martinez-Pomares, L., Platt, N., McKnight, A. J., da Silva, R. P., and Gordon, S. (1996) Macrophage membrane molecules: markers of tissue differentiation and heterogeneity. Immunobiology 195, 407–416.

5.	Smith, M. J. and Koch, G. L. (1987) Differential expression of murine macrophage surface glycoprotein antigens in intracellular membranes. J. Cell Sci. 87(Pt. 1), 113–119.

6.	Martinez-Pomares, L., Reid, D. M., Brown, G. D., et al. (2003) Analysis of mannose receptor regulation by IL-4, IL-10, and proteolytic processing using novel monoclonal antibodies. J. Leukocyte Biol. 73, 604–613.

7.	Haworth, R. and Gordon, S. (1998) Isolation and measuring the function of professional phagocytes: murine macrophages, in Methods in Microbiology (Kaufmann, S. and Kabelitz, D., eds.), Academic, London, Vol. 25, pp. 287–311.

8.	Fortier, A. H. (1994) Isolation of Murine Macrophages. In Current Protocols in Immunology (Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M., and Strober, W., eds.), Green Publishing/Wiley, New York, unit 14.1.1.

9.	Eraser, I., Hughes, D., and Gordon, S. (1993) Divalent cation-independent macrophage adhesion inhibited by monoclonal antibody to murine scavenger receptor. Nature 364, 343–346.

10.	Rosen, H. and Gordon, S. (1987) Monoclonal antibody to the murine type 3 complement receptor inhibits adhesion of myelomonocytic cells in vitro and inflammatory cell recruitment in vivo. J. Exp. Med. 166, 1685–1701.

11.	Brunt, L. M., Portnoy, D. A., and Unanue, E. R. (1990) Presentation of Listeria monocytogenes to CD8+ T cells requires secretion of hemolysin and intracellular bacterial growth. J. Immunol. 145, 3540–3546.

12.	Stanley, E. R., Cifone, M., Heard, P. M., and Defendi, V. (1976) Factors regulating macrophage production and growth: identity of colony-stimulating factor and macrophage growth factor. J. Exp. Med. 143, 631–647.

13.	Li, Y. M., Baviello, G., Vlassara, H., and Mitsuhashi, T. (1997) Glycation products in aged thioglycollate medium enhance the elicitation of peritoneal macrophages. J. Immunol. Methods 201, 183–188.

14.	Hume, D. A. and Gordon, S. (1983) Optimal conditions for proliferation of bone marrow-derived mouse macrophages in culture: the roles of CSF-1, serum, Ca²⁺, and adherence. J. Cell Physiol 111, 189–194.

15.	Fauve, R. M., Jusforgues, H., and Hevin, B. (1983) Maintenance of granuloma macrophages in serum-free medium. J. Immunol. Methods 64, 345–351.

16.	Gordon, S. (1995) The macrophage. Bioessays 17, 977–986.

17.	Mahoney, J. A., Haworth, R., and Gordon, S. (2000) Monocytes and macrophages, in Haematopoietic and Lymphoid Cell Culture (Dallman, M. J. and Lamb, J. R., eds.), Cambridge University Press, Cambridge, pp. 121–146.

18.	Ezekowitz, R. A., Austyn, J., Stahl, P. D., and Gordon, S. (1981) Surface properties of bacillus Calmette-Guerin-activated mouse macrophages. Reduced expression of mannose-specific endocytosis, Fc receptors, and antigen F4/80 accompanies induction of Ia. J. Exp. Med. 154, 60–76.

19.	Haworth, R., Platt, N., Keshav, S., et al. (1997) The macrophage scavenger receptor type A is expressed by activated macrophages and protects the host against lethal endotoxic shock. J. Exp. Med. 186, 1431–1439.

20.	Crocker, P. R. and Gordon, S. (1985) Isolation and characterization of resident stromal macrophages and hematopoietic cell clusters from mouse bone marrow. J. Exp. Med. 162, 993–1014.

21.	Handel-Fernandez, M. E. and Lopez D. M. (2000) Macrophages in tissues, fluids and immune response sites, in Macrophages (Paulnock, D. M., ed.), Oxford University Press, Oxford, pp. 1–30.

22.	Peiser, L., Gough, P. J., Kodama, T., and Gordon, S. (2000) Macrophage class A scavenger receptor-mediated phagocytosis of Escherichia coli: role of cell heterogeneity, microbial strain, and culture conditions in vitro. Infect. Immun. 68, 1953–1963.

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