What is a macrophage?

Macrophages are specialized, long-lived, phagocytic cells of the innate immune system. Together with neutrophils, they act as first responders to infections [1]. Macrophages are involved in the recognition, phagocytosis, and degradation of cellular debris and pathogens [2]. Macrophages also function in the presentation of antigens to T cells, as well as in the induction of expression of co-stimulatory molecules on other antigen-presenting cell types, which initiates the adaptive immune response [1]. Furthermore they play an important role in the initiation of inflammation by releasing cytokines and chemokines, which in turn recruit other immune cells to inflammation sites [3].

Macrophages occur in almost all tissues and therefore have different functional abilities. In addition to the immune responses to pathogens and initiation of inflammation responses, macrophages function in the maintenance of tissue homeostasis, as well as in the repair and remodeling of tissues [4] [5]. Unfortunately, this function is associated with many diseases, including metabolic and autoimmune diseases, cancers, infections, obesity, and fibrosis [5] [6]. Hence, macrophages appear to also play a critical role in the tumor microenvironment, specifically in matrix remodeling, angiogenesis, metastasis, and tumor progression [5].

Development of macrophages

Macrophages have many origins. Tissue-resident macrophages can either differentiate from circulating monocytes that develop from hematopoietic stem cells in the bone marrow or arise during embryonic development in the fetal liver, the yolk sac, or an embryonic region near the dorsal aorta and are therefore maintained independently of monocytes during adulthood [3] [5] [6]. The former type of development is achieved by the ability of monocytes to migrate into tissues either in the steady state or upon inflammation, which is followed by differentiation into persistent tissue-specific macrophages, including macrophages of the bone (osteoclasts), central nervous system (microglia), connective tissue (histiocytes), and liver (Kupffer cells), as well as macrophages of the alveoli (dust cells), intestine, spleen, and peritoneum [7]. Microglial cells and Kupffer cells are able to self-renew in the presence of interleukin 34 (IL-34), which is expressed in these tissues and binds to the same receptor as macrophage-colony stimulating factor (M-CSF) [3]. Due to their distribution and function in many different tissues and organs, macrophages show a variety of morphologies and phenotypes [5].

Macrophages as phagocytes

Macrophages are one of three types of phagocytic cell types, in addition to granulocytes (neutrophils, eosinophils, and basophils) and dendritic cells (DCs). As soon as a microorganism enters the host and begins to replicate, it is recognized by one of these phagocyte types and ingested for destruction, a process called phagocytosis. Most pathogens enter the host through the respiratory system, gut mucosa, skin lesions, or the urogenital tract. Since macrophages populate submucosal tissues, they are usually the first defensive cells to encounter pathogens. Phagocytosis is initiated when certain phagocyte receptors—usually carbohydrate or lipid structures specific to pathogens—interact with the pathogen surface [3].

Following this first interaction, the phagocyte plasma membrane surrounds and internalizes the pathogen in a large membrane-enclosed endocytic vesicle called a phagosome. The phagosome is then fused with one or more lysosomes, small vesicles containing antimicrobial peptides and enzymes, to form the phagolysosome. The lysosome contents are released into the phagolysosome, which in turn undergoes acidification and enzymatic processes that produce highly reactive nitric oxide and superoxide radicals to kill the pathogen [3].

Two examples of phagocyte receptors are Dectin-1 and mannose receptor (CD206). Both are members of the C-type lectin-like family. Dectin-1 is expressed by macrophages and neutrophils and binds to glucose polymers that are commonly found in fungal cell walls. CD206 is expressed by macrophages and DCs and binds to a variety of mannosylated ligands shared by fungi, bacteria, and viruses [3].

Two illustrations of macrophage phagocytic receptors and phagocytosis. The left panel shows cell surface macrophage markers that bind pathogens. The right shows the engulfment of bacteria and yeast and the fusion process.
Figure 2. Phagocytosis.First panel: Tissue macrophages carry a variety of surface receptors, some of which are required for phagocytosis. Mannose receptor recognizes mannosylated ligands on bacteria, fungi, and viruses. Dectin-1 binds to certain glucose polymers on fungal cell walls. Complement receptors bind and internalize complement-coated bacteria. Most phagocytosis receptors recognize pathogen-specific carbohydrate or lipid structures. Second panel: Phagocytosis occurs, followed by fusion of the phagosome with one or more lysosomes to create the phagolysosome.

Macrophage classification

The classification of macrophages is still a very controversial topic, as different phenotypes and macrophage activation states can be achieved in response to various factors, including signaling molecules, growth factors, transcription factors, and epigenetic and post-transcriptional mechanisms and changes, as well as niche signals such as cytokines, cell-cell contacts, and metabolites [5] [8] [9]. In addition, macrophages can alter their activation state directly in response to microbes and microbial products such as lipopolysaccharide (LPS) [10]. However, the activation of macrophages plays a crucial role in tissue homeostasis, as well as in inflammation and development of diseases [5]. In general, macrophages can be classified based on their function and activation and are divided into two subtypes: classically activated M1 macrophages and alternatively activated M2 macrophages.

Classically activated M1 macrophages

M1 macrophages mainly function in Th1 cell recruitment, pathogen resistance, and tumor control via innate and adaptive immune responses [13]. The polarization into M1 macrophages can be classically activated by pathogens, LPS, granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-α), and the T helper 1 (Th1) cell cytokine interferon gamma (IFN-γ). To drive macrophages to M1 polarization, many pathways are involved, including the IRF/STAT, LPS/TLR4, and NF-κB/PI-3 kinase pathways [14].

Characteristically, M1 macrophages show high antigen presentation activity and high production of pro-inflammatory cytokines, such as interleukin 1 (IL-1), IL-6, TNF-α, and of nitric oxide (NO) and reactive oxygen species (ROS) [12]. Furthermore, they display an upregulation of IL-12 and IL-23 and a downregulation of IL-10 [13] [5]. Stimulation of M1 macrophages results in high secretion levels of IL-1b, TNF-α, IL-12, IL-18, and IL-23. Moreover, the M1 macrophage phenotype has been shown to express high levels of major histocompatibility complex class II (MHC II), CD68, CD80, and CD86, as well as of the Th1 cell-attracting chemokines, including CXCL9 and CXCL12 [12].

Table 1. Human M1 macrophage markers. These markers can be used to distinguish M1 macrophages from other cell types.

SpeciesMacrophage typeMarkerMarker type
HumanM1CD369 (Dectin-1)Surface
HumanM1Mer (MerTK)Surface
HumanM1MHC IISurface
HumanM1IRF5Intracellular/transcription factor
HumanM1STAT1Intracellular/transcription factor
Abbreviations: CD, cluster of differentiation; IFN, interferon; IL, interleukin; IRF, interferon regulatory factor; MHC, major histocompatibility complex; NOS, nitric oxide synthase; STAT, signal transducer and activator of transcription; TNF, tumor necrosis factor.

Table 2. Mouse M1 macrophage markers. These markers can be used to distinguish M1 macrophages from other cell types.

SpeciesMacrophage typeMarkerMarker type
MouseM1CD369 (Dectin-1)Surface
MouseM1Mer (MerTK)Surface
MouseM1MHC IISurface
MouseM1IRF5Intracellular/transcription factor
Abbreviations: CD, cluster of differentiation; IFN, interferon; IL, interleukin; IRF, interferon regulatory factor; MHC, major histocompatibility complex; TNF, tumor necrosis factor.

Alternatively activated M2 macrophages

M2 macrophages can be alternatively activated by parasitic or fungal infection, immune complexes, apoptotic cells, macrophage colony-stimulating factor (M-CSF), IL-13, TGF-b, and T helper 2 (Th2) cytokine IL-4, as well as by IL-33 and IL-25 via Th2 cells [12] [15]. Underlying signaling that drives macrophages into the M2 state involves STAT6, IRF4, PPARδ, and PPARγ [11].

In contrast to the classically activated subtype, the alternatively activated subtype shows the opposite expression profile: downregulation of IL-12 and IL-23 and upregulation of IL-10 and IL-1RA [13] [5]. In addition, M2 macrophages show low production of the inflammatory cytokines IL-1, IL-6, and TNF-α. M2 macrophages function in pathogen clearance, anti-inflammatory response, and metabolism, as well as in wound healing, tissue remodeling, immunoregulation, tumor progression, and malignancies [12] [15].

The M2 phenotype can be characterized by the expression of CD206, CD163, CD209, FIZZ1 and Ym1/2. In general, this subtype shows high expression of receptors required for the phagocytosis and scavenging of mannose and galactose, as well as high production of ornithine and polyamines via the arginase pathway [12]. Chemokines expressed by this macrophage type are CCL1, CCL17, CCL18, CCL22, and CCL24 [11].

Subtypes of M2 macrophages

Dependent on the respective stimulation of M2 macrophages, four M2 subtypes have been established: M2a, M2b, M2c, and M2d. These subtypes differ from each other based on their cell surface markers, secreted cytokines, and biological functions. However, all M2 macrophage subtypes have IL-10 expression in common [11].

  • M2a macrophages: Activation by IL-4 or IL-13. IL-4 in turn leads to the expression of the mannose receptor (CD206). Further upregulation of IL-10, TGF-b, CCL17, CCL18, and CCL22 have been shown to promote cell growth, tissue repair, and endocytosis [11].
  • M2b macrophages: Activation by immune complexes, Toll-like receptor (TLR) ligands and IL-1b. Upon activation, this subtype releases pro- and anti-inflammatory cytokines TNF-α, IL-1b, IL-6, and IL-10. M2b macrophages function in the regulation of the immune response and inflammation [11].
  • M2c macrophages: Activation by glucocorticoids, IL-10, and TGF-b (and inactivated macrophages). This subtype is characterized by high expression of anti-inflammatory IL-10, pro-fibrotic TGF-b, CCL16, CCL18, as well as the Mer receptor tyrosine kinase (MerTK), which promotes phagocytosis of apoptotic cells [16].
  • M2d macrophages: Activation by TLR antagonists, IL-6, and adenosines. Adenosines lead to the expression of IL-10 and vascular endothelial growth factor (VEGF), which in turn triggers angiogenesis and tumor progression [11] [16].

Table 3. Human M2 macrophage markers. These markers can be used to distinguish M2 macrophages from other cell types.

SpeciesMacrophage typeMarkerMarker type
HumanM2CD206 (MMR)Surface
HumanM2CD209 (DC-SIGN)Surface
HumanM2IRF4Intracellular/transcription factor
HumanM2STAT6Intracellular/transcription factor
Abbreviations: CD, cluster of differentiation; FceR1, Fc fragment of IgE receptor I; IDO, indolamin-2,3-dioxygenase; IL, interleukin; IRF, interferon regulatory factor; STAT, signal transducer and activator of transcription; TGF, transforming growth factor; VSIG4, V-set and immunoglobulin domain containing 4.

Table 4. Mouse M2 macrophage markers. These markers can be used to distinguish M2 macrophages from other cell types.

SpeciesMacrophage typeMarkerMarker type
MouseM2CD206 (MMR)Surface
MouseM2CD209 (DC-SIGN)Surface
MouseM2IRF4Intracellular/transcription factor
MouseM2RELM-aIntracellular/transcription factor
MouseM2STAT6Intracellular/transcription factor
Abbreviations: CD, cluster of differentiation; CSF1R, colony stimulating factor 1 receptor; FceR1, Fc fragment of IgE receptor I; IDO, indolamin-2,3-dioxygenase; IL, interleukin; IRF, interferon regulatory factor; RELMa, resistin-like molecule-alpha; TGF, transforming growth factor.

Macrophages in disease

Next to their regulated roles in fighting diseases as part of the immune system, macrophages can also play a negative role in chronic inflammation, autoimmune diseases, and cancers. In regular immune responses, pro-inflammatory macrophages are regulated such that their pro-inflammatory signals are limited in magnitude and time. During prolonged insults, dysregulated macrophages continuously secrete inflammatory cytokines and recruit other immune cells. These processes sustain chronic inflammation and are believed to play an important role in tumor initiation and promotion. Once a tumor has formed, it can cause macrophages to differentiate from an immunologically active to an immunosuppressive state. Most cancers are highly populated by these so-called tumor-associated macrophages (TAMs), which are associated with poor prognosis in patients. In inadequately controlled responses during wound healing, macrophages may also lead to fibrosis. Other chronic diseases in which macrophages are key players include atherosclerosis, asthma, inflammatory bowel disease, and rheumatoid arthritis [6].

Methods of study

Macrophages can be isolated and analyzed from whole blood (PBMC isolation, macrophage analysis, monocyte stimulation to macrophages) or from tissue or tumors (single-cell suspension preparation from tissue or tumor samples). Additionally, human and mouse monocyte cell lines are commercially available, including THP-1 cells (acute monocytic leukemia), U937 cells (histiocytic lymphoma), as well as RAW264.7 cells (mouse leukemic monocyte macrophage) and J774A.1 cells (mouse BALB/c monocyte macrophage) [17].

There are several methods to isolate macrophages, including magnetic bead–conjugated antibody cell isolation, density-gradient separation, laser-capture microdissection, and fluorescence-activated cell sorting (FACS) [18]. Following isolation, macrophage functions and phenotypes can be analyzed and characterized by gene expression analysis, functional studies, assessment of cytokine and chemokine production, and analysis of protein expression and cell surface markers using immunofluorescent staining, immunoassays, flow cytometry, western blotting, or PCR. Note that not every analysis method requires isolation [18].

Macrophage isolation

When analyzing or processing macrophages from tissues or tumors, typically either a tissue or tumor section or a single-cell suspension is prepared. Tissue or tumor sections can be directly used for immunohistochemistry (IHC) staining and analysis. To prepare a single-cell suspension, the tissue or tumor is dissected and then enzymatically digested. Single-cell suspensions can be used for direct analysis using methods such as flow cytometric analysis of surface markers.

When required, macrophages can be isolated from a single-cell suspension using fluorescence-activated cell sorting (FACS) or magnetic bead–conjugated antibody cell isolation. For isolation by FACS, fluorophore-conjugated antibodies specific for cell surface markers are used to stain a cell subset, and then a cell sorter is used to sort stained cells based on defined sorting criteria. In magnetic bead–conjugated antibody cell isolation, antibodies specific to a cell-specific surface marker are attached to magnetic beads, and the cell suspension is incubated with these beads. Once the cells that feature the selected surface marker are bound to the beads, all unbound cells are washed from the suspension before detaching the cell type of interest from the beads.

Additionally, isolated macrophages can be further cultivated and stimulated. Typically, M1 macrophages can be reprogrammed to M2 macrophages by stimulation and vice versa. Cultivation of macrophages can be used for stimulation or generation of cell lysates or cell culture supernatants that can be used for qPCR assays, western blotting, or various immunoassays (IA), including ELISAs, bead-based IAs such as Invitrogen ProcartaPlex Immunoassays, and PCR based IAs such as Invitrogen ProQuantum High-Sensitivity Immunoassays for quantitation of various cytokines. Macrophage function may also be investigated by performing phagocytosis assays [17].

When analyzing or processing macrophages from whole blood, the usual procedure is to isolate monocytes and differentiate them to macrophages. Therefore, PBMCs are isolated from whole blood via density-gradient separation using Ficoll Paque. This method takes advantage of the density differences between the different cell types in whole blood. Subsequently, CD14+ monocytes are isolated from the PBMC suspension using either FACS or magnetic bead–conjugated antibody cell isolation, e.g., with Invitrogen Dynabeads Untouched Human Monocytes Kit (Cat. No. 11350D), Invitrogen Dynabeads CD14 (Cat. No. 11149D), or Invitrogen Dynabeads FlowComp Human CD14 Kit (Cat. No. 11367D). Another cost-efficient isolation method is the cultivation of the PBMC suspension in tissue culture plasticware, to which the monocytes preferentially adhere; monocyte cell numbers increase with the use of gelatin-coated surfaces.

Macrophage differentiation in vitro

There is a variety of stimulation/differentiation techniques used to obtain naive macrophages or polarized M1 or M2 macrophages in vitro. If further isolation of monocytes is not required, isolated PBMCs can directly produce macrophages by incubation with Gibco RPMI 1640 Medium supplemented with 15% fetal calf serum (FCS).

Isolated monocytes can be differentiated into naive macrophages using special differentiation media. For example, polarized macrophages can be produced from monocytes using the growth factors GM-CSF and M-CSF. Monocytes can be stimulated in RPMI 1640 medium supplemented with 10% FCS and 10ng/ml GM-CSF (Gibco GM-CSF Recombinant Human Protein, Cat. No. PHC2013) or 10ng/ml M-CSF (Gibco M-CSF Recombinant Human Protein, Cat. No. PHC9501) for 7 days, which leads to the differentiation of M1 and M2 macrophages, respectively. After stimulation, the cells should be adherent with an elongated shape.

Alternatively, M1 and M2 polarized macrophages can be obtained by sequential stimulation of monocytes with different cytokines. Cells harvested with RPMI 1640 medium supplemented with 10% FCS can be subsequently treated for 24 hours with 10ng/mL lipopolysaccharide (Invitrogen eBioscience Lipopolysaccharide (LPS) Solution, Cat. No. 00-4976-03) and 50 ng/mL IFN-γ (Gibco IFN-γ Recombinant Human Protein, Cat. No. PHC4031) to obtain M1 macrophages or with 20 ng/mL IL-4 (Gibco IL4 Recombinant Human Protein, Cat. No. PHC0041) to obtain M2/M2a macrophages. Treatment with immune complexes and IL-1b or LPS leads to M2b activation, and addition of IL-10, TGF-b, or glucocorticoids lead to M2c activation [14] [19] [20]. Macrophage types can be identified by the expression of certain markers on their surface using immunohistochemical staining or flow cytometry (Table 5).

Identification and characterization of macrophages

Table 5 lists markers that can be used for the identification of macrophages (general phenotypics) and for their characterization (functional characteristics). The marker location (surface or intracellular) has implications for the methods used to detect these antigens.

Table 5. A nonexhaustive list of cellular markers. The listed markers could be used to characterize human and mouse macrophages.

SpeciesMarker typeMarkerCloneLocation of marker
HumanGeneral phenotypicsCD11bICRF44Surface
Functional characteristicsIDOEyedioIntracellular
CD163eBioGHI/61 or Mac 2-158Surface
CD204 (MSR-1)PSL204Surface
CD369 (Clec7a, Dectin-1)1500Surface
GPNMB (Osteoactivin)HOST5DsSurface
MouseGeneral phenotypicsCD11bM1/70Surface
Functional characteristicsAxlMAXL8DSSurface
GPNMB (Osteoactivin)CTSREVLSurface
VSIG4 (CRIg)NLA14Surface
Nos2 (iNOS)CXNFTSurface
Abbreviations: CD, cluster of differentiation; CXCL13, C-X-C motif chemokine 13; GPNMB, glycoprotein nonmetastatic melanoma protein B; HLA, human leukocyte antigen; IDO, indolamin-2,3-dioxygenase; LYVE-1, lymphatic vessel endothelial hyaluronan receptor 1; NOS, nitric oxide synthase; RELMa, resistin-like molecule-alpha; VSIG4, V-set and immunoglobulin domain containing 4.

Flow cytometry—example for characterizing macrophages

Although the traditional M1/M2 model can be applied to in vitro cultured and stimulated cells to describe different types of macrophage activation, new insights into the intricate network of tissue macrophages clearly reveal that it fails to adequately describe the functional and phenotypic diversity of these cells in vivo [21] [22]. Therefore, we recommend replacing the simplistic M1/M2 model with a more flexible and comprehensive description of macrophages, based on the expression of multiple markers and functional characteristics. Canonic markers used to define the M1 and M2 phenotypes, however, still play an essential role in macrophage characterization. Here, we describe these traditional markers, including inducible nitric oxide synthase (iNOS, NOS2), arginase-1 (Arg1), mannose receptor (CD206), RELM-a (FIZZ1), and CD163, for characterizing macrophages.

iNOS and Arginase 1

Two historically canonic markers of classical and alternative activation of macrophages (at least in mouse cells) are iNOS and Arg1, respectively (Figure 4). iNOS is an enzyme that catalyzes the NADPH-dependent oxidization of L-arginine to L-citrulline and nitric oxide (NO). Nitric oxide is an important mediator of cytotoxicity and other physiological functions (e.g., vasorelaxation). iNOS is not constitutively expressed in macrophages and its presence reliably marks previous stimulation with pro-inflammatory cytokines such as IL-1, TNF-α, or IFN-γ.

The enzyme Arg1 converts L-arginine to urea and L-ornithine. By degrading arginine, Arg1 deprives iNOS of its substrate and downregulates nitric oxide production. Arg1 is strongly induced in macrophages by IL-4 and IL-13 but not by anti-inflammatory cytokines such as IL-10 or TGF-b. Contrary to iNOS expression, baseline expression of Arg1 can be found in multiple populations of tissue macrophages. For instance, most mouse large peritoneal macrophages (F4/80 hi macrophages) are Arg1 positive in the steady state. As shown in Figure 4, Arg1 can be detected even in some macrophages that have been stimulated with IFN-γ. Therefore, Arg1 should not be considered an entirely specific marker of M2 polarization, particularly when analyzing primary uncultured cells.

Two-panel graph showing macrophage marker expression in relation to stimulation

Figure 4. Macrophages stimulated with IFN-γ express mainly iNOS, with a small subpopulation being positive for both iNOS and low levels of Arg1. Macrophages stimulated with IL-4 express Arg1 and no iNOS. C57BL/6 mouse bone marrow-derived macrophages were polarized for 24 hours with either LPS and IFN-γ (M1) or IL-4 (M2a), and subsequently surface stained with Invitrogen F4/80 Monoclonal Antibody (clone BM8), eFluor 450 (Cat. No. 48-4801-82). Cells were then fixed and permeabilized with the Invitrogen eBioscience Intracellular Fixation & Permeabilization Buffer Set (Cat. No. 88-8824-00) followed by intracellular staining with Invitrogen iNOS Monoclonal Antibody (clone CXNFT), APC (Cat. No. 17-5920-82) and Invitrogen Arginase 1 Monoclonal Antibody (clone A1exF5), PE (Cat. No. 12-3697-82). Viable F4/80+ cells were used for analysis.

Mannose receptor (CD206)

The macrophage mannose receptor, known as CD206 or mannose receptor C type 1 (MRC1), mediates phagocytic and endocytic uptake of fungal, bacterial, protozoan, and viral antigens, and plays an important role in the immune defense and its regulation. The mannose receptor was the original identifying marker of alternatively activated macrophages. In contrast to Arg1, CD206 is expressed across a broad spectrum of alternatively activated macrophages, including tolerogenic cells treated with IL-10 or glucocorticoids. Interestingly, TGF-b has been shown to downregulate the expression of CD206. Other factors inhibiting expression of this receptor include LPS, IFN-γ, and TNF-α. Similar to Arg1, CD206 is constitutively expressed by multiple types of tissue macrophages, although the pattern of expression does not entirely overlap with that of Arg1. The right panel in Figure 5 shows the constitutive expression of CD206 in small (F4/80 lo) and large (F4/80 hi) peritoneal macrophages.

Two panel graph showing constitutive expression of CD206 in mouse small and large peritoneal macrophages

Figure 5. Substantial fractions of mouse small peritoneal macrophages (F4/80 lo) and large peritoneal macrophages (F4/80 hi) constitutively express CD206. Mouse resident peritoneal exudate cells were surface stained with Invitrogen F4/80 Monoclonal Antibody (clone BM8), eFluor 450 (Cat. No. 48-4801-82) followed by fixation and permeabilization with the Invitrogen eBioscience Intracellular Fixation & Permeabilization Buffer Set (Cat. No. 88-8824-00). Cells were then intracellularly stained with either Invitrogen Rat IgG2b kappa Isotype Control (clone eB149/10H5), APC (Cat. No. 17-4031-82) (left panel) or Invitrogen CD206 Monoclonal Antibody (clone MR6F3), APC (Cat. No. 17-2061-80) (right panel). Total cells were used for analysis; the majority of double-negative cells are B cells.


RELM-a, also known as resistin-like alpha or FIZZ1, is a small cytokine strongly induced by IL-4 and IL-13 and involved in the response against parasitic infection. Similar to Arg1, RELM-a is not induced by glucocorticoids and IL-10. Despite this, the in vivo expression pattern of these two commonly used markers of M2 activation is very different. For instance, in the absence of stimulation, RELM-a is expressed by a majority of small peritoneal macrophages (F4/80 lo) and few large peritoneal macrophages (F4/80 hi) (Figure 6).

Two panel graph showing RELM-a expression in mouse peritoneal macrophages in the absence of stimulation

Figure 6. Most small peritoneal macrophages (F4/80 lo) and very few large peritoneal macrophages (F4/80 hi) spontaneously express RELM-a. C57BL/6 mouse resident peritoneal exudate cells were surface stained with Invitrogen F4/80 Monoclonal Antibody (clone BM8), eFluor 450 (Cat. No. 48-4801-82). The cells were then fixed and permeabilized using the Invitrogen eBioscience Intracellular Fixation & Permeabilization Buffer Set (Cat. No. 88-8824-00), and intracellularly stained with either Invitrogen Rat IgG1 kappa Isotype Control (clone eBRG1), APC (Cat. No. 17-4301-82) (left panel) or Invitrogen RELM alpha Monoclonal Antibody (clone DS8RELM), APC (Cat. No. 17-5441-82) (right panel). All peritoneal cells were used for analysis; the majority of double-negative cells are B cells.


CD163 is a haptoglobin and hemoglobin receptor. As its main function is to aid the process of iron recycling, the highest expression of CD163 can be observed in splenic red pulp macrophages. CD163 can also detect certain bacterial compounds and is expressed, although to a lower degree, in other subsets of tissue macrophages, including Kupffer cells, intestinal lamina propria macrophages, and a fraction of large peritoneal macrophages (Figure 7). Unlike human CD163, mouse CD163 is not as readily induced by M2 polarizing cytokines, and it is not a good marker of murine M2 macrophages. Instead, its expression seems indicative of tissue-specific macrophage functions (e.g., hemoglobin recycling).

Two panel graph showing expression of CD163 in both mouse small and large peritoneal macrophages

Figure 7. A significant fraction of the large peritoneal macrophages (F4/80 hi) and virtually no small peritoneal macrophages (F4/80 lo) constitutively express CD163. BALB/c mouse splenocytes were stained with Invitrogen F4/80 Monoclonal Antibody (clone BM8), eFluor 450 (Cat. No. 48-4801-82) and either Invitrogen Rat IgG2a kappa Isotype Control (clone eBR2a), PerCP-eFluor 710 (Cat. No. 46-4321-82) (left panel) or Invitrogen CD163 Monoclonal Antibody (clone TNKUPJ), PE (Cat. No. 12-1631-82) (right panel). Total cells were used for analysis.



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