The Isolation and Characterization of Murine Macrophages

Ricardo Gonçalves1, David M. Mosser2

1 Department of General Pathology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, 2 Department of Cell Biology and Molecular Genetics, Maryland Pathogen Research Institute, University of Maryland, College Park, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.1
DOI:  10.1002/0471142735.im1401s111
Online Posting Date:  November, 2015
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Abstract

Macrophages are mononuclear phagocytes that are widely distributed throughout the body. These cells can contribute to development and homeostasis and participate in innate and adaptive immune responses. The physiology of macrophages can vary tremendously depending on the environment in which they reside and the local stimuli to which they are exposed. Macrophages are prodigious secretory cells, and in that role can promote and regulate immune responses and contribute to autoimmune pathologies. Macrophages are highly phagocytic, and in this capacity have long been considered to be essential immune effector cells. The important roles of macrophages in maintaining homeostasis and in contributing to tissue remodeling and wound healing is sometimes overlooked because of their vital role in host defense. © 2015 by John Wiley & Sons, Inc.

Keywords: macrophages; macrophage colony‐stimulating factor (M‐CSF); bone marrow; lung; flow cytometry

     
 
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Table of Contents

  • Introduction
  • Isolation and Characterization of Murine Macrophages
  • Basic Protocol 1: The Isolation of Murine Peritoneal Macrophages
  • Basic Protocol 2: The Isolation of Murine Bone Marrow–Derived Macrophages
  • Basic Protocol 3: Isolation of Murine Alveolar Macrophages
  • Basic Protocol 4: Characterization of Murine Macrophages with Directly Conjugated Antibodies
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: The Isolation of Murine Peritoneal Macrophages

  Materials
  • Donor mice: BALB/c or C57Bl/6, specific‐pathogen‐free, ∼8 weeks oldSpecific pathogen‐free mice are purchased from licensed commercial vendors. It is important to use mice that are clean, stress‐free, and uninfected. Mice should be housed in the facility for at least 1 week following delivery to allow them to recover from transit stress. For resident peritoneal macrophages, we suggest mice around 8 weeks of age, since very small animals can deliver fewer resident cells.Both strains of mice yield macrophages that are fully responsive to exogenous stimuli, and despite the fact that these two strains have been associated with dramatically different adaptive T‐cell responses, the macrophages from these mice do not appear to have similar inherent biases.
  • 3% Brewer thioglycollate medium or 3% protease peptone (see recipe)
  • Harvest medium: Phosphate‐buffered saline (PBS) without calcium and magnesium ( appendix 2A or Mediatech, cat. no. 21‐031‐CV)A 10× stock solution is also available for large‐scale isolations (cat. no. 20‐031‐CV). This stock solution must be diluted in sterile water to 1×.
  • Macrophage culture medium (see recipe)
  • Diff‐Quik stain solutions: Differential Staining Set (Cardinal Health, cat. no. B4132‐1A, www.cardinal.com); includes fixative solution, solution I, and solution II.
  • Forceps and small, straight surgical scissors (immerse in 70% ethanol)
  • 23‐G and 20‐G needles, 1.5‐in. (3.75‐cm) length
  • 5‐cc and 10‐cc syringes
  • 50‐ml polypropylene conical centrifuge tubes, on ice
  • Cytocentrifuge (Shandon Cytospin 4; ThermoScientific, cat. no. A7830002)
  • Cytofunnels (Shandon EZ Cytofunnels; ThermoScientific, cat. no. A78710003)
  • Eppendorf 5810R (or equivalent) centrifuge, 4°C (or equivalent)
  • Additional reagents and equipment for mouse euthanasia (unit 1.8; Donovan and Brown, ), intraperitoneal injection (unit 1.6; Donovan and Brown, ), and counting cells with a hemacytometer ( appendix 3A; Strober, ).

Basic Protocol 2: The Isolation of Murine Bone Marrow–Derived Macrophages

  Materials
  • Donor mice: BALB/c or C57Bl/6, specific‐pathogen‐free (see protocol 1)
  • Phosphate‐buffered saline (PBS) without calcium and magnesium ( appendix 2A or Mediatech, cat. no. 21‐031‐CV), sterile and cold
  • DMEM/F12 medium (see recipe)
  • Macrophage complete medium: DMEM/F12‐10 medium (see recipe)
  • Cellstripper non‐enzymatic cell dissociation solution (Corning, cat. no. 25‐056‐CI)
  • Forceps and scissors (keep in sterile beaker containing 70% ethanol)
  • 25‐cc syringes with 26‐G needles
  • 50‐ml conical centrifuge tube, on ice
  • 15‐ml conical centrifuge tubes
  • Falcon 100 × 15–mm petri dishes (Corning, cat. no. 351029), sterile, non‐treated
  • Eppendorf 5810R (or equivalent) centrifuge, 4°C
  • Falcon cell scraper (Corning, cat. no. 353085)
  • Additional reagents and equipment for mouse euthanasia (unit 1.8; Donovan and Brown, ) and counting cells with a hemacytometer ( appendix 3A; Strober, )

Basic Protocol 3: Isolation of Murine Alveolar Macrophages

  Materials
  • Phosphate‐buffered saline (PBS) without calcium and magnesium ( appendix 2A or Mediatech, cat. no. 21‐031‐CV)
  • Ketamine/Xylazine: 80 to 100 mg/kg ketamine (Ketaset, Fort Dodge) and 10 mg/kg xylazine (Rompun, Bayer) in 100 to 200 μl PBS
  • DMEM/F12 medium (see recipe)
  • Diff‐Quik stain solutions: Differential Staining Set (Cardinal Health, cat. no. B4132‐1A, http://www.cardinal.com). The set includes fixative solution, solution I, and solution II.
  • Forceps and small, straight surgical scissors (keep in 70% ethanol)
  • Surgical suture material: 3‐0 Ethilon black 18‐in. PS‐1 cutting (eSutures.com, cat. no. 1663 http://www.eSutures.com)
  • 18‐G needles, and 5 cc syringes
  • 50‐ml polypropylene conical centrifuge tubes, on ice
  • Cytocentrifuge (Shandon Cytospin 4; ThermoScientific, cat. no. A7830002)
  • Cytofunnels (Shandon EZ; ThermoScientific, cat. no. A78710003)
  • Eppendorf 5810R centrifuge, 4°C (or equivalent)
  • Additional reagents and equipment for intraperitoneal injection (unit 1.6; Donovan and Brown, ), euthanasia of the mouse (unit 1.8; Donovan and Brown, ), counting cells (see appendix 3A and protocol 1, step 7; Strober, ), and differential staining of Cytospin cells (see protocol 1, step 8)

Basic Protocol 4: Characterization of Murine Macrophages with Directly Conjugated Antibodies

  Materials
  • 1.2 × 107 cells/ml test cell suspension: macrophages from any anatomical compartment (see Basic Protocols protocol 11 to protocol 33), or macrophages differentiated in culture from bone marrow precursors
  • Phosphate‐buffered saline (PBS) without calcium and magnesium ( appendix 2A or Mediatech, cat. no. 21‐031‐CV), cold
  • Pharm Lyse Lysing Buffer (BD Biosciences, Cat. No. 555899)
  • Blocking solution: 0.2% BSA (bovine serum albumin) in PBSMany other blocking solutions are available, including 5% FBS, milk protein, or commercial blocking solutions.
  • Fc‐receptor blocking antibody: Anti‐CD16/32, clone 93 (eBioscience, cat. no. 14‐0161‐86; http://www.ebioscience.com)
  • Fluorescence‐labeled monoclonal antibodies (MAbs; Tables 14.1.1, 14.1.2, 14.1.3)
  • 2% paraformaldehyde (see recipe)
  • Anti‐rat and Anti‐hamster Igκ/Negative control compensation particles set (Compbeads; BD Biosciences, cat. no. 552845)
  • 14‐ml round‐bottom polypropylene tubes, 17 × 100–mm (BD Biosciences, cat. no. 352059)
  • Clear 96‐well U‐bottom microtest polystyrene assay plate (BD Biosciences, cat. no. 353910)
  • 96‐well polypropylene cluster tubes with rack (Corning Life Sciences, cat. no. 4410)
  • Falcon round‐bottom polystyrene tubes, 12 × 75‐mm (Fisher Scientific, cat. no. 14‐959‐5)
  • Additional reagents and equipment for flow cytometry (units 5.3 & 5.4; Holmes et al., 14.1; Holmes et al., 14.1).
NOTE: All antibodies should be titrated before use.NOTE: All antibodies should be diluted in blocking buffer unless otherwise indicated.
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Figures

Videos

Literature Cited

Literature Cited
  Anderson, C.F. and Mosser, D.M. 2002. Cutting edge: Biasing immune responses by directing antigen to macrophage Fc gamma receptors. J. Immunol. 168:3697‐3701.
  Austin, P.E., McCulloch, E.A., and Till, J.E. 1971. Characterization of the factor in L‐cell conditioned medium capable of stimulating colony formation by mouse marrow cells in culture. J. Cell Physiol. 77:121‐134. doi: 10.1002/jcp.1040770202.
  Cohn, Z. 1978. The activation of mononuclear phagocytes: Fact, fancy, and future. J. Immunol. 121:813‐816.
  Donovan, J. and Brown, P. 2006a. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
  Donovan, J. and Brown, P. 2006b. Parenteral injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10.
  Edwards, J.P., Zhang, X., Frauwirth, K.A., and Mosser, D.M. 2006. Biochemical and functional characterization of three activated macrophage populations. J. Leuk. Biol. 80:1298‐1307.
  Fortier, A.H., Hoover, D.L. and Nacy, C.A. 1982. Intracellular replication of Leishmania tropica in mouse peritoneal macrophages: Amastigote infection of resident cells and inflammatory exudate macrophages. Infect. Immun. 38:130‐1307.
  Gautier, E.L., Shay, T., Miller, J., Greter, M., Jakubzick, C., Ivanov, S., Helft, J., Chow, A., Elpek, K,G., Gordonov, S., Mazloom, A.R., Ma'ayan, A., Chua, W.J., Hansen, T.H., Turley, S.J., Merad, M., and Randolph, G.J. 2012. Gene expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. Nat. Immunol. 13:1118‐1128.
  Ghosn, E.E., Cassado, A.A., Govoni, G.R., Fukuhara, T., Yang, Y., Monack, D.M., Bortoluci, K.R., Almeida, S.R., Herzenberg, L.A., and Herzenberg, L.A. 2010. Two physically, functionally, and developmentally distinct peritoneal macrophage subsets. Proc. Natl. Acad. Sci. U.S.A. 107:2568‐2573. doi: 10.1073/pnas.0915000107.
  Ginhoux, F. and Jung, S. 2014. Monocytes and macrophages: Developmental pathways and tissue homeostasis. Nat. Rev. Immunol. 14:392‐404. doi: 10.1038/nri3671.
  Gordon, S. 2007. The macrophage: Past, present and future. Eur. J. Immunol. 37(Suppl 1):S9‐17.
  Gordon, S. and Taylor, P.R. 2005. Monocyte and macrophage heterogeneity. Nat. Rev. Immunol. 5:953‐964. doi: 10.1038/nri1733.
  Ho, M.K., and Springer, T.A. 1982. Mac‐1 antigen: Quantitative expression in macrophage populations and tissues, and immunofluorescent localization in spleen. J. Immunol. 128:2281‐2286.
  Holmes, K., Lantz, L. M., Fowlkes, B., Schmid, I. and Giorgi, J. V. 2001. Preparation of cells and reagents for flow cytometry. Curr. Protoc. Immunol. 44:5.3.1‐5.3.24.
  Holmes, K. L., Otten, G. and Yokoyama, W. M. 2002. Flow cytometry analysis using the Becton Dickinson FACS Calibur. Curr. Protoc. Immunol. 49:5.4.1‐5.4.22.
  Hoover, D.L. and Nacy, C.A. 1984. Macrophage activation to kill Leishmania tropica: Defective intracellular killing of amastigotes by macrophages elicited with sterile inflammatory agents. J. Immunol. 132:1487‐1491.
  Jenkins, S. and Hume, D. 2014. Homeostasis in the mononuclear phagocyte system. Trends Immunol. 35:358‐367. doi: 10.1016/j.it.2014.06.006.
  Mahoney, J.A., Haworth, R., and Gordon, S. 2000. Monocytes and macrophages. In Haematopoietic and Lymphoid Cell Culture (M.J. Dallman and J.R. Lamb, eds.) pp. 121‐146. Cambridge University Press, Cambridge.
  Maus, U., Herold, S., Muth, H., Maus, R., Ermert, L., Ermert, M., Weissmann, N., Rosseau, S., Seeger, W., Grimminger, F., and Lohmeyer, J. 2001. Monocytes recruited into the alveolar air space of mice show a monocytic phenotype but upregulate CD14. Am. J. Physiol. Lung Cell Mol. Physiol. 280:L58‐68.
  McGarry, M.P. and Stewart, C.C. 1991. Murine eosinophil granulocytes bind the murine macrophage‐monocyte specific monoclonal antibody F4/80. J. Leukoc. Biol. 50:471‐478.
  Morimoto, K., Amano, H., Sonoda, F., Baba, M., Senba, M., Yoshimine, H., Yamamoto, H., Ii, T., Oishi, K., and Nagatake, T. 2001. Alveolar macrophages that phagocytose apoptotic neutrophils produce hepatocyte growth factor during bacterial pneumonia in mice. Am. J. Respir. Cell Mol. Biol. 24:608‐615. doi: 10.1165/ajrcmb.24.5.4292.
  Mosser, D.M. 2003. The many faces of macrophage activation. J. Leukoc. Biol. 73:209‐212. doi: 10.1189/jlb.0602325.
  Mosser, D.M. and Edwards, J.P. 2008. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8:958‐969. doi: 10.1038/nri2448.
  Mosser, D.M. and Gonçalves, R. 2015. Activation of macrophages. Curr. Protoc. Immunol. 111:14.2.1‐14.2.10.
  Nelson, D.S. and Boyden, V. 1963. The loss of macrophages from peritoneal exudates following the injection of antigens into guinea‐pigs with delayed‐type hypersensitivity. Immunology 6:264‐275.
  Politis, A. D. and Vogel, S. N. 2005. Measurement of Fcγ Receptor–Mediated Binding and Phagocytosis. Curr. Protoc. Immunol. 68:14.8.1‐14.8.9.
  Rabinowitz, S.S. and Gordon, S. 1991. Macrosialin, a macrophage‐restricted membrane sialoprotein differentially glycosylated in response to inflammatory stimuli. J. Exp. Med. 174:827‐836.
  Stanley, E.R. 1985. The macrophage colony‐stimulating factor, CSF‐1. Methods Enzymol. 116:564‐587. doi: 10.1016/S0076‐6879(85)16044‐1.
  Stanley, E.R., and Heard, P.M. 1977. Factors regulating macrophage production and growth. J. Biol. Chem. 252:4305‐4312.
  Steele, C., Marrero, L., Swain, S., Harmsen, A.G., Zheng, M., Brown, G.D., Gordon, S, Shellito, J.E., and Kolls, J.K. 2003. Alveolar macrophage‐mediated killing of Pneumocystis carinii f. sp. muris involves molecular recognition by the Dectin‐1 beta‐glucan receptor. J. Exp. Med. 198:1677‐1688. doi: 10.1084/jem.20030932.
  Strauss‐Ayali, D., Conrad, S.M., and Mosser, D.M. 2007. Monocyte subpopulations and their differentiation patterns during infection. J. Leukoc. Biol. 82:244‐252.
  Strober, W. 2001. Monitoring cell growth. Curr. Protoc. Immunol. 21:A.3A.1‐A.3A.2.
  Warren, M.K. and Vogel, S.N. 1985. Bone marrow‐derived macrophages: Development and regulation of differentiation markers by colony stimulating factor and interferon. J. Immunol. 134:982‐989.
  Wynn, T.A., Chawla, A., and Pollard, J.W. 2013. Macrophage biology in development, homeostasis and disease. Nature 496:445‐455. doi: 10.1038/nature12034.
  Yoshikawa, K., Suzuki, Y., Kawai, M., Fukada, M., and Yokochi, T. 1991. Novel cell surface antigens expressed on mouse alveolar macrophages. Microbiol. Immunol. 35:803‐807.
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