Macrophages are tissue-resident professional phagocytes and antigen-presenting cells (APC), which differentiate from circulating peripheral blood monocytes. They perform important active and regulatory functions in innate as well as adaptive immunity.1 Activated macrophages of different phenotypes are routinely classified into M1 macrophages (CAM) or M2 macrophages (AAM). The classically activated M1 macrophages comprise immune effector cells with an acute inflammatory phenotype. These are highly aggressive against bacteria and produce large amounts of lymphokines.2 The alternatively activated, anti-inflammatory M2 macrophages can be separated into at least three subgroups. These subtypes have various different functions, including regulation of immunity, maintenance of tolerance and tissue repair/ wound healing.1,2 Indeed, cells of the macrophage lineage exhibit extraordinary plasticity in response to endogenous as well as exogenous stimuli, which can allow conversion of the initial M1/M2 polarization processes2, for example M2-polarized macrophages can convert to the M1-activated status under certain conditions.
Primary human macrophages are difficult to isolate in sufficient amounts from tissue and do not proliferate in culture. In addition, it is commonly accepted that the obtained cells often exhibit significant phenotypical heterogeneity. Monocyte-derived macrophages provide an excellent alternative, since human blood monocytes are readily available in large numbers and can be differentiated into macrophages in vitro. The PromoCell Macrophage Generation Media were designed for the efficient differentiation of highly pure M1 or M2 macrophages directly from PBMCs as a starting material. The Macrophage Generation Media DXF are defined serum-free/xeno-free and provide a controlled culture environment devoid of all animal component-derived stimuli - a significant benefit in terms of monocytes and macrophages standing for highly reactive immune cells. As a result, these media lack unwanted non-defined and deleterious effects attributable to FBS and therefore enable standardized and controlled macrophage differentiation. In vitro differentiation of monocytes in the presence of the PromoCell M1-Macrophage Generation Medium DXF (C-28055, contains GM-CSF) leads to macrophages exhibiting M1-like polarized CD68+/ CD80+/CD163- macrophages, while the M2-Macrophage Generation Medium DXF (C-28056, contains M-CSF) promotes M2-like polarized CD68+/CD80-/CD163+ macrophages. The Macrophage Base Medium DXF (C-28057) represents the user-customizable version without cytokines and therefore needs appropriate supplementation by the user. If required, customized activation and subtype-specific polarization of the M1/ M2-polarized macrophages may be performed by the user.
Figure 1. Protocol overview using PromoCell M1/M2 Macrophage Generation Medium DXF.
Figure 2. Morphology and phagocytic activity of human PBMC derived M1 macrophages. A) Phase bright images of M1 macrophages 24 hours after plating in PromoCell’s Macrophage Generation Medium DXF. B) PromoCell cryopreserved M1 macrophages ingest large numbers of fluorescently labeled E.coli. bacteria.
Figure 3. Morphology and phagocytic activity of human PBMC derived M2 macrophages. A) Phase bright images of M2 macrophages 24 hours after plating in PromoCell’s Macrophage Generation Medium DXF. B) PromoCell cryopreserved M2 macrophages ingest large numbers of fluorescently labeled E.coli. bacteria.
Figure 4. Flow cytometry analysis of day 10 M1 and M2 macrophages generated in the PromoCell Macrophage Generation Medium DXF. Fresh peripheral blood mononuclear cells (PBMCs) were plated in the Monocyte Attachment Medium. The purified monocytes were differentiated for 10 days without performing the optional activation step. M1 macrophages exhibit the CD68+ (99% positive) and CD80+ (83% positive) marker profile while M2 macrophages exhibit a CD68+ (99% positive) and CD163+ (95% positive) marker profile.