Isolation of Murine Macrophages

Macrophages isolated from murine peritoneal cavity, bone marrow, and spleen are suitable samples for studying the activation properties of this immunologically important cell type. The peritoneal cavity provides an accessible site for harvest of fair numbers of resident macrophages. However, the number of macrophages present in the noninflamed peritoneum is insufficient for large-scale studies. To increase macrophage yield, sterile inflammatory agents, such as proteose peptone or thioglycollate, can be injected into the peritoneal cavity prior to cell harvest. Although these inflammatory agents increase the number of peritoneal macrophages present, concerns have been raised as to how inflammatory agents affect the activation state and contribute to heterogeneity of macrophages (see also UNIT 14.2). In some cases, therefore, it is desirable to isolate macrophages from noninflamed peritoneum. The first basic protocol describes the isolation of murine macrophages from the peritoneal cavity under inflammatory and noninflammatory conditions. To investigate macrophage activation using a more homogeneous population of cells, macrophages derived from immature progenitor cells in bone marrow can be studied. These cells can be propagated by incubation with colony stimulating factors (CSFs) or interleukin-3 (IL-3). Use of this culture technique has helped elucidate the mechanism(s) by which macrophage heterogeneity may develop. The second basic protocol describes the isolation of bone marrowderived progenitor cells and propagation of these immature macrophages by CSFs or IL-3. NOTE: All solutions and equipment coming in contact with cells must be sterile, and proper sterile techniques must be employed. Additionally, solutions must be free of endotoxin, as macrophages are acutely sensitive to its biological effects. Polypropylene plasticware must be used for adequate cell recovery, because macrophages tend to stick to most surfaces. All reagents and harvested cells should be kept at 4C prior to use. ISOLATION OF MURINE PERITONEAL MACROPHAGES Murine peritoneal macrophages suitable for use in protocols outlined elsewhere in this chapter are recruited and isolated as described below. Although the peritoneal cavity provides an accessible site for harvest of fair numbers of resident macrophages, in vivo manipulations prior to cell harvest can enhance macrophage yield. However, such inflammatory induction will also alter the physiologic characteristics of the cells collected. Generally, the normal mouse peritoneal cavity will yield mature, resident macrophages and the inflamed peritoneal cavity will yield immature, inflammatory macrophages recruited from the circulating and marginal pool. Materials
For recipes, see Reagents and Solutions in this unit; for common stock solutions, see APPENDIX 2; for suppliers, see APPENDIX 5.

UNIT 14.1

BASIC PROTOCOL 1

Donor mice 3% proteose peptone or 3% Brewer thioglycollate medium (see recipes; for collecting inflammatory macrophages) 70% ethanol Harvest medium: Dulbeccos minimum essential medium (DMEM; e.g., GIBCO/BRL) with 5% sterile, endotoxin-free FBS (HyClone) Diff-Quik stain solutions I, II, and III (Baxter) 25-G needles 6- and 30-cc syringes
Copyright 1994 by John Wiley & Sons, Inc.

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Forceps and small, straight surgical scissors (keep in 70% ethanol) 19-G needles, 1.5-in. (3.75-cm) 50-ml polypropylene conical centrifuge tubes, on ice Cytocentrifuge and cytocentrifuge containers (Shandon/Lipshaw) Cytospin filter cards (Shandon/Lipshaw) Sorvall RT6000 centrifuge with H1000B rotor (or equivalent) Additional reagents and equipment for mouse euthanasia (UNIT 1.8), intraperitoneal injection (UNIT 1.6), and counting cells with a hemacytometer (APPENDIX 3) Harvest peritoneal cells 1a. To collect resident peritoneal cells: Euthanize untreated mice by decapitation or CO2 asphyxiation. 1b. To collect inflammatory macrophages: Fill 6-ml syringe with 1.0 ml of 3% proteose peptone. Attach 25-G needle and inject solution into peritoneal cavity of each mouse. Allow inflammatory response to proceed for 3 days and euthanize by decapitation or CO2 asphyxiation. Alternatively, inject 1.0 ml of 3% Brewer thioglycollate medium into peritoneum 5 to 7 days prior to cell harvest (this will yield a larger number of inflammatory macrophages).
Use only mice bred and housed in clean environments (preferably barrier facilities, UNIT 1.2). Chronic, endemic infectious diseases, such as those caused by Sendai virus and mouse hepatitis virus, have a profound effect on macrophage physiology and will affect cell responsiveness and capacity for function. Decapitation or CO2 asphyxiation are the preferred methods of euthanasia for macrophage isolation because they limit the chance of contaminating the peritoneal cavity with blood.

2. Wet the abdomen of each mouse with 70% alcohol to sterilize the area. 3. Make a midline incision with sterile scissors. Retract abdominal skin with forceps to expose the intact peritoneal wall. 4. Attach 30-cc syringe to 19-G needle and fill with harvest medium. Push on syringe plunger to allow a small amount of medium to pass through the needle as the needle penetrates the peritoneum to avoid hitting the intestines. With bevelled end of needle facing up, insert needle through peritoneal wall at the midline. Inject 10 ml harvest medium into each mouse.
Inject and collect fluid from three mice using a single 30-cc syringe. Passing a small amount of medium through the needle serves to eliminate any air bubbles.

5. Using the same syringe and needle, insert needle bevelled end down into peritoneum. Raise needle slightly to cause tenting of peritoneal wall. Withdraw peritoneal fluid slowly.
Expect fluid recovery of 8 ml/mouse.

6. Remove needle from syringe and dispense pooled peritoneal fluid to 50-ml polypropylene centrifuge tubes on ice. Count cells and adjust cell concentration 7. Remove 20-l sample and count cells using a hemacytometer. 8. Transfer 0.2 ml peritoneal fluid to a cytocentrifuge container to prepare a cell smear for differential stain. Follow manufacturers instructions for collecting cells using cytocentrifuge. Cytospin 6 min at 600 rpm. Remove slide and allow to air dry.

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9. Stain cytocentrifuged sample with Diff-Quik staining solutions and perform a differential cell count.
Follow manufacturers instructions for Diff-Quik staining. For additional information about cell counting see Critical Parameters.

10. Centrifuge peritoneal lavage fluid in 50-ml tubes 10 min at 400 g (1000 rpm in H1000B), 4C. 11. Discard supernatant and resuspend cell pellet by gently tapping bottom of tube. Adjust to appropriate cell concentration in harvest medium.
Expect 23 106 total peritoneal cells from an untreated mouse, containing 50% to 70% macrophages. Proteose peptonetreated mice should yield 34 106 macrophages per mouse. Thioglycollate-treated mice will yield 107 macrophages per mouse. Both inflammatory agents recruit young, immature macrophages into peritoneum. Thioglycollate-elicited cells may be better than proteose peptoneelicited cells for generation of an oxidative burst in response to PMA but not as responsive to cytokines for increased NO generation (Hoover and Nacy, 1984). Different experiments will require different irritants (see Critical Parameters). Cells are now ready for phenotypic analysis (UNIT 14.2) or functional assays (e.g., UNIT 14.4).

ISOLATION OF MURINE BONE MARROWDERIVED MACROPHAGES In this protocol, bone marrow cells are harvested and placed in a liquid culture containing colony-stimulating factor (CSF) or interleukin-3 (IL-3). The initial 24-hr adherence step removes bone marrow stromal cells and any mature resident bone marrow macrophages. Removal of nonadherent cells at this point allows progenitor cells to expand and differentiate under the influence of specific CSFs. After 7 days in culture, only adherent cells are harvested for assaying; any contaminating nonadherent, nonresponsive progenitors are eliminated. Bone marrowderived macrophages can be plated at a known cell density for later functional or molecular assays to examine activation and the generation of macrophage heterogeneity. This protocol has been used to examine the functional capabilities of subpopulations of macrophages in the absence of uncontrolled environmental stimulation. In addition, bone marrowderived macrophages have also been used to examine the differential response of subpopulations of macrophages to environmental stimuli such as LPS or IFN as an alternate method for analyzing the heterogeneity observed in tissue macrophages. Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see APPENDIX 2; for suppliers, see APPENDIX 5.

BASIC PROTOCOL 2

Donor mice Phosphate-buffered saline (PBS; APPENDIX 2) Lymphocyte separation medium (LSM; Organon Teknika Cappel) Supplemented EMEM and EMEM-10: supplemented EMEM (see recipe) with and without 10% heat-inactivated FBS EMEM with 10% FCS, heat-inactivated (EMEM-10) Species-reactive CSF or IL-3 (e.g., Genzyme) 1.0 mg/ml dispase grade II (Boehringer Mannheim) Forceps and scissors (keep in sterile beaker containing 70% ethanol) 25-cc syringes 26-G needles 50-ml conical centrifuge tube, on ice

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15-ml conical centrifuge tubes Sorvall RT6000 centrifuge and HB1000B rotor (or equivalent) 25- and 75-cm2 tissue culture flasks (Corning) Sterile rubber scraper Additional reagents and equipment for mouse euthanasia (UNIT 1.8) and counting cells with a hemacytometer (APPENDIX 3) Harvest bone marrow cells 1. Euthanize mice. Using aseptic technique, peel skin from the top of each hind leg and down over the foot. Cut off foot with the skin and discard. Cut off the hind legs and place in dish containing sterile PBS.
Use only mice bred and housed in clean environments (preferably barrier facilities, UNIT 1.2). Chronic, endemic infections diseases, such as those caused by Sendai virus and mouse hepatitis virus, have a profound effect on macrophage physiology and will affect cell responsiveness and capacity for function.

2. Remove excess muscle from legs by holding end of bone with forceps and using scissors to push muscle downward away from forceps. Sever leg bones between joints. 3. Attach 25-cc syringe to 26-G needle and fill with sterile serum-free supplemented EMEM or PBS. 4. Insert needle into bone marrow cavity of femur or tibia. Flush bone cavity with 2 to 5 ml medium or PBS, or until bone cavity appears white. Allow wash medium to collect in sterile 50-ml conical centrifuge tube on ice. 5. Transfer medium containing bone marrow cells into 50-ml conical centrifuge tubes on ice. Centrifuge cells 10 min at 500 g (1600 rpm in H1000B), room temperature. 6. Discard supernatant. Resuspend cell pellet in 3 to 5 ml serum-free supplemented EMEM.
The volume of EMEM will depend on the number of mice harvested.

Separate mononuclear cells 7. Place 5 ml lymphocyte separation medium (LSM) in a 15-ml conical tube. Overlay with 5 ml bone marrow suspension.
The use of high-density solutions such as LSM for the separation of lymphocytes is described in detail in the second support protocol of UNIT 3.1 (using an alternative to the overlaying technique described here in which the separation medium is underlaid beneath the cell suspension). Generally each gradient can be loaded with the bone marrow cells derived from five mice. Do not overload, as this will reduce the effective yield at the interface and cause retention of contaminating cells trapped during centrifugation.

8. Centrifuge gradient 20 min at 500 g, room temperature, with brake off.

For optimal separation, the separation medium used to suspend the cells must be serum-free and the centrifuge must be at room temperature.

9. Gently remove cells at interface with a Pasteur pipet or 5-ml pipet by placing pipet tip at interface and slowly moving it over interface while slowly drawing cells into pipet.
Isolation of Murine Macrophages

Withdrawing cells slowly helps to avoid removing substantial amounts of separation medium.

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10. Pool cells from gradients and centrifuge 10 min at 500 g, 4C. 11. Resuspend cells in 10 ml supplemented EMEM-10. Centrifuge 10 min at 500 g, 4C. 12. Resuspend cells in supplemented EMEM-10 to a final concentration of 5 106 cells/ml.
The yield of bone marrow cells obtained per mouse varies with the strain; therefore, the volume of medium needed to resuspend the cells must be empirically determined. A yield of 310 106 cells per mouse can be expected.

Stimulate immature progenitor cells 13. Aliquot 13 107 cells into 25-cm2 tissue culture flasks containing 10 ml supplemented EMEM-10 and an appropriate concentration of species-reactive growth factor (CSF or IL-3).
Because the number of CSF-responsive progenitors varies depending on which CSF is used, the initial number of bone marrow cells must be adjusted accordingly. For instance, progenitors responsive to CSF-1 or M-CSF require initial plating at 1 107 cells/ml, and those responsive to GM-CSF or IL-3 require plating at 3 107 cells/ml. In general, a concentration of CSF that gives optimal colony formation in soft agar provides an adequate initial concentration for growth using liquid culture techniques: e.g., 10 ng/ml GM-CSF or IL-3, or 500 to 1000 U/ml CSF-1 is sufficient to promote optimal growth.

14. Incubate cells 24 hr in humidified 37C, 5% CO2 incubator. 15. Transfer nonadherent cells to 75-cm2 tissue culture flask. Add 10 ml supplemented EMEM-10 containing growth factor. Incubate cells 4 days in humidified 37C 5% CO2 incubator.
In the authors experience (L.F.), Corning brand 75-cm2 flasks are superior to other brands, because they permit easier removal of adherent cells without significant trauma to the cells.

16. Add a further 10 ml supplemented EMEM-10 containing growth factor. Continue incubation another 3 days. Harvest mature macrophages 17. Remove and discard culture supernatant. Wash remaining adherent cells with 15 ml PBS. 18. Prepare 1.0 mg/ml dispase (5 ml per flask of cells) in PBS. Filter sterilize and warm to 37C. 19. Discard PBS wash. Add 5 ml dispase to flask and incubate 5 min at 37C.
To minimize amount of time that cells are incubated with enzyme, treat only five flasks at any given time.

20. Bang side of flask to loosen adherent cells. Gently remove cells by unidirectionally scraping flask with sterile rubber scraper.
Use of a rubber scraper limits the destruction of cells often seen if a plastic scraper is used. To further limit damage, scrape in one direction onlydo not use a back and forth motion.

21. Add 10 ml supplemented EMEM-10 to flask and harvest detached cells. 22. Centrifuge cells 10 min at 500 g, 4C.
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23. Resuspend cells in 3 to 5 ml medium and count cells using a hemacytometer.

The volume of EMEM-10 will vary with the number of flasks harvested. The resuspension volume should be determined based on volume necessary for subsequent assays. Cells can now be replated for further assaying.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2; for suppliers, see APPENDIX 5.

Brewer thioglycollate medium, 3% Add 3% (w/v) Brewer thioglycollate medium (Difco) to distilled H2O. Bring solution to boil and autoclave to sterilize. Store 6 months at room temperature. Discard if turbidity develops (indicating bacterial contamination). Eagle minimal essential medium (EMEM), supplemented EMEM (e.g., GIBCO/BRL) supplemented with: 2 mM glutamine 15 mM HEPES buffer 0.02% (w/v) sodium bicarbonate 100 IU/ml penicillin 100 g/ml streptomycin Store up to 2 months at 4C Proteose peptone, 3% Add 3% (w/v) proteose peptone (Difco) to distilled H2O. Bring solution to boil and autoclave to sterilize. Store up to 3 months at 4C and warm to room temperature before use. Discard if turbidity develops (indicating bacterial contamination). COMMENTARY Background Information
Resident, inflammatory, and bone marrow derivedmurine macrophages have been the biological tools used to dissect the complex process of cytokine activation that leads to cellular cytotoxicity (Ruco and Meltzer, 1978; Belosevic et al., 1988; Green et al., 1990). The murine peritoneal cavity provides a site to obtain relatively large numbers of either resident or inflammatory macrophages. Inflammatory cells can be recruited by inoculating mice with sterile irritants, such as proteose peptone or thioglycollate. Inflammatory macrophages not only differ in number but are also qualitatively different from resident cells. They have increased plasma membrane and rate of membrane turnover, increased phagocytic and respiratory burst capacity, and they respond better to cytokines for tumor killing but more poorly to signals for intracellular parasite killing (Fortier et al., 1982; Hoover and Nacy, 1984; Nacy et al., 1984). Cells isolated from the peritoneum, whether resident or inflammatory, are a heterogeneous population. They therefore exhibit a spectrum of morphological and functional phenotypes characteristically associated with successive levels of macrophage activation, such as differences in complement receptor expression, Ia antigen expression, ability to secrete TNF, or ability to be activated for tumor cell killing (Cohn, 1978; Rutherford et al., 1993). A more homogeneous cell population can by obtained by culturing bone marrow macrophages. These culture techniques allow examination of the functional heterogeneity previously reported in the literature and the study of macrophage development under defined conditions. The differentiation of the monocyte/macrophage lineage from bone marrow cells to mature effector cell populations in peripheral tissues is a complex developmental process. Subpopulations of progenitor cells, which differ in their capacities for replication and differentiation, respond to a variety of distinct growth factors by dividing and differentiating. Once released into the circulation, monocytes can enter peripheral tissues and mature into mac-

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rophages under the influence of environmental signals. These mature macrophages also exhibit a spectrum of morphological and functional phenotypes characteristically associated with successive levels of macrophage differentiation (Cohn, 1978; Rutherford et al., 1993; UNIT 14.2). Early studies demonstrated that bone marrow progenitors could be induced to differentiate in liquid culture by CSF-1 (M-CSF). The resulting macrophages were shown to respond to macrophage activating agents (Moore et al., 1984; Warren and Vogel, 1985). The use of bone marrowderived macrophages has expanded the understanding of the apparent redundancy of cytokines, as well as the mechanisms by which functionally distinct macrophage populations are derived. In general, CSF-1-derived macrophages are larger, express higher levels of TGF- receptors, produce higher levels of IFN-/, and express lower levels of Ia antigen compared to GM-CSF- or IL-3-derived macrophages. Establishment of CSF-derived macrophage populations makes it possible to analyze the contribution of distinct CSFs to macrophage activation and provides a more homogeneous population of macrophages for the further study of macrophage biology (Falk et al., 1988).

Critical Parameters
Macrophages are acutely sensitive to the biological effects of endotoxin, thus all reagents must be of high quality and guaranteed endotoxin-free. Use of pathogen-free mice is equally critical, because macrophages responding to an ongoing infectious process in vivo may be refractory to signals initiated in vitro. If peritoneal harvests from untreated mice seem unusually high, cell morphology can be checked for evidence of inflammation (presence of polymorphonuclear leukocytes). If inflammation is apparent, these cells may not be useful. Serious consideration should be given to the advantages of discarding peritoneal cells that do not appear to be resident cells rather than using them in functional assays that will yield contrary results. When macrophages are harvested from bone marrow, the number of cells obtained per mouse will vary, depending on the efficiency of flushing of the bone cavity. Lymphocyte separation medium (LSM) gradients should always be layered at room temperature, using cells that have been resuspended in serum-free medium. For best results, the gradient must be

centrifuged at room temperature with the centrifuge brake off. The differential cell count is performed to determine what percentage of the peritoneal fluid cell population is macrophages. Fluids from the uninflamed peritoneal cavity will contain 50% lymphocytes and 50% macrophages. Fluids from inflamed peritoneal cavities will vary depending on the inflammatory agent used to induce inflammation, and can range from 30% to 70% macrophages. Using a standard hematological criteria for differentiating lymphocytes from macrophages, 100 random cells are counted in a Diff-Quikstained cytospin preparation and scored as lymphocytes, macrophages, polymorphonuclear leukocytes, or basophils (mast cells). To quantitate the number of macrophages in peritoneal fluids, the total cell count from the hemacytometer is multiplied by the percent of macrophages (determined from the differential cell count). The number of macrophages harvested at the end of the culture period will vary greatly depending on the CSF used to stimulate the cultures. GM-CSF and IL-3 generally result in the production of fewer murine macrophages than CSF-1. The choice of which cytokine to use depends on the experimental design and requires familiarity with the biological and biochemical characteristics of different cytokines. The functional properties of specific hematopoietic growth factors should be taken into account when designing macrophage experiments. Growth factors should be recombinant and species reactive. Human CSF-1 does act on murine cells, but GM-CSF and IL-3 are species-specificthe human cytokines will not stimulate murine cells. If it is necessary to use conditioned medium, murine L cell fibroblast conditioned medium has been used as a source of CSF-1, WEHI-3B-conditioned medium as a source of IL-3, and con Astimulated spleen cells as a source of GM-CSF and IL-3.

Anticipated Results
Peritoneal harvest In general, one can expect to harvest 23 106 total peritoneal cells from an unmanipulated, clean mouse. This harvest will contain 50% to 70% macrophages, and the remainder of the cells will be lymphocytes. Cell yields much greater than this suggest an ongoing inflammatory response. The peritoneal cell population harvested from mice previously inoculated with sterile

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irritants (proteose peptone or thioglycollate) will contain inflammatory macrophagesrelatively young cells recruited from the periphery. Total cell yields and subpopulations of cells will differ depending on the irritant used. Easily digestible inflammatory agents will induce a short-lived inflammatory response and the animal will be able to return to homeostasis within days. On the other hand, agents that are more difficult to digest, such as thioglycollate or colloidal starch, can induce an inflammatory response in vivo that lasts for 5 to 7 days. Proteose peptoneinduced inflammation will yield 34 106 macrophages per mouse when harvest occurs 3 days after inoculation. Macrophages will represent 50% to 70% of the total cell yield. Thioglycollate-induced inflammation will yield 107 macrophages per mouse 5 to 7 days after inoculation. Macrophages will constitute 80% of the total cell yield. Bone marrow harvest The yield of LSM-purified bone marrow cells should be 310 106 cells per mouse. This number may vary somewhat depending on strain of mouse used. The yield of mature macrophages following 7 days of liquid culture in CSF-1 should be 23 106 cells per 107 input murine bone marrow cells. The yield of mature macrophages following 7 days of liquid culture in GM-CSF or IL-3 should be 0.71 106 macrophages per 107 input murine bone marrow cells.

Literature Cited
Belosevic, M., Davis, C.E., Meltzer, M.S., and Nacy, C.A. 1988. Characterization of the lymphokines that cooperate with interferon to induce activated macrophage resistance to infection with amastigotes of Leishmania. J. Immunol. 141:890-895. Cohn, Z. 1978. The activation of mononuclear phagocytes: Fact, fancy, and future. J. Immunol. 121:813-816. Falk, L.A., Wahl, L.M., and Vogel, S.N. 1988. Analysis of Ia antigen expression in macrophages derived from bone marrow cells cultured in granulocyte-macrophage colony-stimulating factor or macrophage colony-stimulating factor. J. Immunol. 140:2652-2660. 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. 38:13041307. Green, S.J., Crawford, R.M., Hockmeyer, J.T., Meltzer, M.S., and Nacy, C.A. 1990. Leishmania major amastigotes initiate the L-arginine-dependent killing mechanism in IFN--stimulated macrophages by induction of TNF-. J. Immunol. 145:4290-4296. 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. Moore, R.N., Larson, H.S., Horohov, D.W., and Rouse, B.T. 1984. Endogenous regulation of macrophage proliferative expansion by colony stimulating factor-induced interferon. Science 223:178-180. Nacy, C.A., Oster, C.N., James, S.L., and Meltzer, M.S. 1984. Activation of macrophages to kill Rickettsia and Leishmania: Dissociation of intracellular microbicidal activities and extracellular destruction of neoplastic and helminth targets. Contemp. Top. Immunobiol. 13:147-153. Ruco, L.P. and Meltzer, M.S. 1978. Macrophage activation for tumor cytotoxicity: Development of macrophage cytotoxic activity requires the completion of a series of short-lived intermediary interactions. J. Immunol. 121:2035-2041. Rutherford, M.S., Witsell, A., and Shook, L.B. 1993. Mechanisms generating functionally heterogeneous macrophages: Chaos revisited. J. Leukocyte Biol. 53:602-618. Warren, M.K. and Vogel, S.N. 1985. Bone marrowderived macrophages: Development and regulation of differentiation markers by colony stimulating factor and interferon. J. Immunol. 134:982-989.

Time Considerations
Isolation of peritoneal macrophages. Harvesting peritoneal cells and performing cell counts and differential counts on 10 mice will take 1 hr. Isolation of bone marrow macrophages. The time required to harvest the cells and set up the liquid cultures depends on how quickly femurs and tibias are obtained and the bone marrow extracted. Harvesting bone marrow from 10 to 15 mice should take 1 hr. Once bone marrow is harvested, gradient preparation, cell separation, and counting should take 112 hr. On day 1, the transfer and feeding of flasks should take 1 hr. This will increase if the experimental design calls for setting up large numbers of flasks. Feeding cultures on day 4 should take 12 hr. On day 7, the time needed to harvest cells will increase, depending on the number of flasks involved, but should not take more than 1 hr.

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Key References
Falk, L.A., Wahl, L.M., and Vogel, S.N. 1988. See above. Demonstrates the functional heterogeneity that can be observed and dissected when examining homogeneous populations of macrophages derived from stimulation of bone marrow progenitors with specific CSFs. Rutherford, M.S., Witsell, A., and Shook, L.B. 1993. See above. Describes the concept of functional heterogeneity and provides a complete review of tissue-derived and bone marrowderived macrophage phenotypes.

Warren, M.K. and Vogel, S.N. 1985. See above. Describes in detail the method of generating bone marrowderived macrophages and discusses the effects of CSF-1 on macrophage differentiation as a function of culture time.

Contributed by Anne H. Fortier (peritoneum) EntreMed, Inc. Rockville, Maryland Lydia A. Falk (bone marrow) Food and Drug Administration Bethesda, Maryland

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