In Vitro Differentiation of Monocyte-Derived Macrophages from Human PBMC

Primary human macrophages are hard to separate from tissue in adequate amounts and they do not grow in culture. It is generally accepted that the cells that are obtained usually show important phenotypical heterogeneity. Monocyte-derived macrophages (MDM) offer a superior alternative, as human blood monocytes are readily available in large amounts and can be generated into macrophages in vitro.

A complete system of the PromoCell Macrophage Base Medium DXF, combined with the Monocyte Attachment Medium, was developed for easy and cost-effective differentiation of pure monocyte-derived macrophages; directly from peripheral blood mononuclear cells, or PBMC (see Figures 1 and 3). Specific equipment and former monocyte purification (for example, with magnetic beads) is not required, thereby saving both costs and time.

Differentiation of monocyte-derived macrophages in 8 days directly from PBMC using the Monocyte Attachment Medium and the Macrophage Base Medium DXF. Activation of the culture can optionally be performed on day 7.

Figure 1. The differentiation of monocyte-derived macrophages in 8 days, directly from PBMC using the Monocyte Attachment Medium and the Macrophage Base Medium DXF. Activation of the culture can be performed on day 7 as an optional procedure.

Protocol overview using Macrophage Base Medium DXF (8 days).

Figure 2. Protocol overview using Macrophage Base Medium DXF (8 days).

A  culture of non-activated human monocyte-derived macrophages (MDM) differentiated directly from PBMC as a starting material. Monocytes were purified using the Monocyte Attachment Medium and differentiated in Macrophage Base Medium DXF containing 100 ng/ml GM-CSF for 10 days.

Figure 3. A  culture of non-activated human monocyte-derived macrophages (MDM), differentiated directly from PBMC as the starting material. Monocytes were purified using the Monocyte Attachment Medium and differentiated in the Macrophage Base Medium DXF containing 100ng/ml GM-CSF for 10 days.

The Macrophage Base Medium DXF is the user-adaptive version of the macrophage media product line. It lacks cytokines and is a widely applicable MDM culture system; featuring a completely user-adaptive macrophage differentiation and activation process (see Table 1 for suggestions).

Table 1. The human macrophage activation reference table according to the common framework consensus nomenclature [7] (see also Background, below). The published differentiation factor/activator combinations are listed to serve as a basic guide. Specific effects of activation on macrophages should be tested in comparison to the most appropriate non-activated M(-)-baseline variant as a control. DEX = dexamethasone, IC = immune complexes, IFN = interferon, IgG = immunoglobulin G, GC = glucocorticoids, (G)M-CSF = (granulocyte/)macrophage colony stimulating factor, IL = interleukin, LPS = lipopolysaccharide, TAM = tumor-associated macrophages, TGF = transforming growth factor

Activation state Former designation Differentiation factor (day 0+)* Activator (day 7+) Activation process reference
M1 M(IFN-γ) M1 GM-CSF or M-CSF IFN-γ (50ng/ml) [1]
M(LPS+IFN-γ) M1 GM-CSF or M-CSF IFN-γ (50ng/ml) + LPS (10ng/ml) [1]
M(LPS) M1 GM-CSF or M-CSF LPS (100ng/ml) [1]
M(-) M1, non-activated GM-CSF - [2]
M(-) M0 / Mϕ 2% human AB serum - [1, 3]
M(-) M2, non-activated M-CSF - [2]
M(GC) M2c M-CSF DEX (100nM) [2]
M(TGFb) M2c M-CSF TGF-b1 (20ng/ml) [2]
M(IL-10) M2c M-CSF IL-10 (10ng/ml) [4]
M(IC+LPS) M2b M-CSF IgG (immobilized) + LPS (100ng/ml) [5]
M2 M(IL-4) M2a M-CSF IL-4 (20ng/ml) [4, 5]

TAM M2-like tumor microenvironment tumor microenvironment [6]

* Use the differentiation factors M-CSF or GM-CSF at 50-100ng/ml final concentration with the Macrophage Base Medium DXF (= complete medium: see section II., protocol step 4).

As with all Promocell’s DXF media series, the Macrophage Base Media DXF shows a xeno-free/defined formula. Hence, these media offer a controlled culture environment lacking all animal component-derived stimuli—an important advantage with respect to macrophages and monocytes, representing highly reactive immune cells. Thus, without the impact of animal-derived or undefined components, these media properties allow customized and standardized macrophage differentiation and activation.

Macrophage Generation

  1. Materials
  • Differentiation and activation factors (if necessary, refer to Table 1)
  • Monocyte Attachment Medium (C-28051)
  • PBS w/o Ca2+/Mg2+/2 mM EDTA/0.1% HSA
  • PBS w/o Ca2+/Mg2+ (C-40232)
  • Macrophage Base Medium DXF (C-28057)
  • Optional: Macrophage Detachment Solution DXF (C-41330, refer to protocol step 11)
  1. Monocyte-derived macrophage (MDM) Differentiation Protocol
  1. PBMC isolation (Day 0)

Fresh PBMC from buffy coats is separated using a standard protocol.

Note: Buffy coats should be as fresh as possible before use. Buffy coats older than 24 hours should not be used as this will considerably damage the experimental result.

  1. Count the PBMC (Day 0)

The isolated PBMC is counted and the cells are resuspended at 100 million PBMC per mL in Monocyte Attachment Medium.

  1. Let the monocytes attach (Day 0)

Freshly isolated PBMC is plated in an adequate amount of Monocyte Attachment Medium (e.g.15mL medium per T-75 flask). A seeding density of 1.5 million/cm2 is used. Without any further treatment, the resultant is incubated for 1.5 hours at 5% CO2 and at 37 °C in the incubator.

  1. Prepare the complete Macrophage Base Medium DXF (Day 0)

The Macrophage Base Medium DXF is prepared by adding the thawed SupplementMix aseptically to the Basal Medium. To obtain a homogeneous mixture, the mixture is swirled lightly.

Note: To promote survival and efficient differentiation of the monocytes in the specified defined culture conditions, it is strongly recommended to add a cytokine that acts as a survival factor to the medium, for example, GM-CSF or M-CSF at 100 ng/ml. On the other hand, 2% human AB serum can be added to the medium instead of GM-CSF or M-CSF to generate non-activated/unpolarized M0 macrophages. Macrophage Base Medium DXF with additional survival factors (GM-CSF, human AB serum, or M-CSF) is mentioned as the“complete medium” in the following sections.

Note that GM-CSF, despite being a stronger survival factor, will produce a more heterogeneous population comprising of some moDC-like cells (myeloid dendritic cell-like cells) [7], while M-CSF yields a homogeneous population of differentiated MDM.

  1. Wash the adherent cell fraction (Day 0)

The tissue culture vessel is vigorously swirled, followed by aspirating the non-adherent cells. The adherent cells, i.e. monocytes, are washed three times with warm Monocyte Attachment Medium by completely swirling the vessel and aspirating the supernatant.

  1. Start the macrophage differentiation (Day 0)

A quantity of complete medium is added to the cells, for example, 20 mL per T-75 flask, and incubated for 6 days at 37 °C and 5% CO2 without any change in the medium.

Note: Under these conditions, the monocytes differentiate into macrophages. If necessary, activation can be achieved by carrying out the optional activation step (refer to step 9).

  1. Add fresh complete medium (Day 4)

Another 50% to 75% fresh complete medium of the current volume is added to the cells. The immature macrophages are incubated for another 3 days at 37 °C and 5% CO2.

Note: The fresh medium is simply added to the cells without removing any of the used medium.

  1. Medium change (Day 7)

The medium including suspension cells is aspirated and then collected in a centrifugation tube. Fresh complete medium is instantly pipetted into the adherent cells. The cells are centrifuged in the tube for 15 minutes at 350 x g at room temperature.

The supernatant is discarded and the cells are carefully resuspended in a small amount of fresh complete medium. The resuspended cells in the tube are mixed with the adherent cells in the fresh complete medium contained in the tissue culture vessel. The resultant mixture is incubated for at least one day at 37 °C and 5% CO2.

Note: At this stage, suspension, as well as adherent cells, may be present.

  1. Optional activation step (Day 7)

Soon after the previous media change, the entire volume of the culture is supplemented with sufficient stimuli (refer to Table 1 for suggestions) to activate specific macrophages.

  1. The macrophages are ready (Day 8+)

The macrophages may now be utilized directly in their plates, for example, when conducting phagocytosis assays. Alternatively, they can also be harvested (see instructions in optional step 11). The culture can be maintained for several weeks by changing the medium weekly with fresh complete medium.

Note: Usually, macrophages look like adherent cells with typical morphology: a prominent nucleus that has flatly outspread cytoplasm and multiple pseudopodia.

  1. Optional step: Harvesting/subcultivation of macrophages (Day 8+)

The medium is aspirated and disposed of. The adherent macrophages are washed two times with endotoxin-free PBS w/o Ca2+/Mg2+. A right amount of cold (2-8 °C) Macrophage Detachment Solution DXF is instantly added to the cells, for example, 25 mL per T-75 flask. The tissue culture vessel is sealed and the cells are incubated for 40 minutes at 2–8 °C. If required, the cells are incubated for another 20 minutes at room temperature to enforce the release of cells from the surface of the culture. Next, the tissue culture vessel is firmly tapped to facilitate the detachment of cells. It must be ensured that the majority of the cells have already detached or are at least loosely adherent to the surface of the tissue culture vessel. Only then should a cell scraper be used to remove the remaining macrophages.

The harvested macrophages are collected in centrifugation tubes and then diluted in a ratio of 1:1 with PBS/2 mM EDTA/0.1% HSA. The cells are centrifuged for 15 minutes at 350 x g at room temperature. They are washed twice with PBS/2 mM EDTA/0.1% HSA and subsequently counted. The macrophages are now set to be used for the experiments.

Note: The percentage of attaching cells after re-seeding relies on the overall health status of the macrophages prior to detachment and the effective performance of the detachment process itself. Therefore, some changes cannot be avoided.


Macrophages are antigen-presenting cells (APC) and tissue-resident professional phagocytes, which differentiate from circulating peripheral blood monocytes. They execute significant active and regulatory functions in both innate and adaptive immunity [8]. Macrophages are involved in the manifestation of many diseases, for example, diabetes, autoimmune and allergic disorders, atherosclerosis, metabolic syndrome, cancer, and rheumatoid arthritis [9].

Conventionally, activated macrophages of different phenotypes have commonly been divided into M2- and M1-macrophages. The “classically activated” M1- macrophages contain immune effector cells that have an acute inflammatory phenotype. These produce large amounts of lymphokines and are extremely aggressive against bacteria [10]. On the contrary, the “alternatively activated” anti-inflammatory M2-macrophages adhere to several regulatory functions of many kinds including the maintenance of tolerance, wound healing/tissue repair, and regulation of immunity [8, 10].

This functional heterogeneity of M2 macrophage functions results in their division into three subgroups, that is, M2a, M2b, and M2c. Undeniably, cells of the monocyte/macrophage lineage show excellent plasticity in response to both exogenous and endogenous stimuli, perhaps even leading to a reversal of the initial M1/M2-polarization processes [2]. For instance, M2 polarized macrophages can change to the M1-activated status under specific conditions.

It has now become clear that the conventional M1/M2 model of macrophage polarization/activation is not adequate enough to reflect the entire complexity of activation states of this highly plastic cell lineage [11]. As a result, a common framework proposal for macrophage-activation nomenclature [7] was published by a group of international macrophage specialists. This novel system stipulates the designation of in vitro macrophage activation states in accordance with the stimulus used (for example, 20ng/mL recombinant human (rhu) IL-4) along with clear disclosure of differentiation factors used for MDM generation (for example, 100ng/mL rhu M-CSF). See also Table 1.

Xeno-free and defined macrophage culture systems in association with the published instructions for combined experimental standards for in vitro macrophage activation represent important corner points for effective and purposeful developments in macrophage-related research.


  1. Fujiwara, Y., et al., Guanylate-binding protein 5 is a marker of interferon-gamma-induced classically activated macrophages. Clin Transl Immunology, 2016. 5(11): p. e111.
  2. Zizzo, G., et al., Efficient clearance of early apoptotic cells by human macrophages requires M2c polarization and MerTK induction. J Immunol, 2012. 189(7): p. 3508-20.
  3. Vogel, D.Y., et al., Macrophages migrate in an activation-dependent manner to chemokines involved in neuroinflammation. J Neuroinflammation, 2014. 11: p. 23.
  4. Iqbal, S. and A. Kumar, Characterization of In vitro Generated Human Polarized Macrophages. Journal of Clinical & Cellular Immunology, 2015. 06(06).
  5. Graff, J.W., et al., Identifying functional microRNAs in macrophages with polarized phenotypes. J Biol Chem, 2012. 287(26): p. 21816-25.
  6. Sousa, S., et al., Human breast cancer cells educate macrophages toward the M2 activation status. Breast Cancer Res, 2015. 17: p. 101.
  7. Murray, P.J., et al., Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity, 2014. 41(1): p. 14-20.
  8. Murray, P.J. and T.A. Wynn, Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol, 2011. 11(11): p. 723-37.
  9. Wynn, T.A., A. Chawla, and J.W. Pollard, Macrophage biology in development, homeostasis, and disease. Nature, 2013. 496(7446): p. 445-55.
  10. Murray, P.J. and T.A. Wynn, Obstacles and opportunities for understanding macrophage polarization. J Leukoc Biol, 2011. 89(4): p. 557-63.
  11. Martinez, F.O. and S. Gordon, The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep, 2014. 6: p. 13.

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Last updated: May 21, 2019 at 9:44 AM


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