Differentiating M1- and M2-Macrophages

Macrophages—tissue-resident phagocytes and antigen-presenting cells (APC)—typically differentiate from circulating peripheral blood monocytes. They carry out crucial active and regulatory innate functions as well as adaptive immunity [1].

Activated macrophages of various phenotypes are regularly categorized into M1-macrophages (CAM) and M2-macrophages (AAM). The classically activated M1-macrophages consist of immune effector cells with an acute inflammatory phenotype. These are extremely aggressive against bacteria and create large quantities of lymphokines [2].

The alternatively activated, anti-inflammatory M2-macrophages can be divided into a minimum of three subgroups. These sub-types have many different functions, such as maintenance of tolerance, regulation of immunity, and tissue repair/wound healing [1, 2]. Cells of the monocyte/macrophage lineage display unexpected plasticity in reaction to endogenous as well as exogenous stimuli, which can override the preliminary M1/M2-polarization processes [2]. For instance, M2-polarized macrophages can change to the M1-activated status under specific conditions.

Primary human macrophages are hard to isolate from tissues in adequate amounts and do not multiply in culture. Furthermore, it is generally accepted that the acquired cells often display substantial phenotypical heterogeneity. An outstanding alternative is provided by monocyte-derived Macrophages (MDM), as human blood monocytes are easily available in large amounts and can be differentiated into macrophages in vitro.

The PromoCell Macrophage Generation Media was engineered for simple and efficient differentiation of extremely pure M1- or M2-macrophages straight from PBMC as a starting material (refer to Figure 2). Earlier monocyte purification is not required. Just like all PromoCell’s DXF media series, the Macrophage Generation Media DXF are defined and xeno-free, and thus, offer a regulated culture environment devoid of all animal component-derived stimuli. Consequently, these media lack undesirable non-defined and deleterious effects that are attributed to FCS and thus allow standardized and regulated differentiation of macrophages.

For M1-macrophages, a customarily accepted marker profile is CD68+/ CD80+, while M2-macrophages are defined as CD68+/CD163+ [4]. Moreover,  in vitro differentiation of monocytes exposed to the PromoCell M1- Macrophage Generation Medium DXF (C-28055, contains GM-CSF) results in macrophages displaying a CD68+/ CD80+/CD163-/low M1-like polarized phenotype, while the M2-Macrophage Generation Medium DXF (C-28056, contains M-CSF) promotes M2-like polarized CD68+/CD80-/low/CD163+ macrophages (refer Figure 4 for exemplary flow cytometry plots). The Macrophage Base Medium DXF (C-28057) signifies the user-customizable model of this new product series. It is available without cytokines and thus needs suitable supplementation by the user.

If necessary, tailored activation and subtype-specific polarization of the M1/M2-polarized macrophages may be carried out by the user in an optional step of the protocol mentioned below (also refer to Figure 2/3 and Step 8 of the protocol). Macrophage activation may actually prompt a modified expression pattern of some markers, as compared to non-activated cells [5].

Day 10 culture of activated M2-Macrophages differentiated in the PromoCell M2-Macrophage Generation Medium DXF. M2a-activation was achieved by performing the optional activation step described in the protocol using 20 ng/mL IL-4. Note the typical “fried egg” morphology.

Figure 1. Day 10 culture of activated M2-Macrophages differentiated in the PromoCell M2-Macrophage Generation Medium DXF. M2-activation was achieved by performing the optional activation step described in the protocol using 20ng/mL IL-4. Note the typical “fried egg” morphology.

Schematic overview of the user-customizable M1-/M2-Macrophage differentiation process using the PromoCell Macrophage Generation Media.

Figure 2. Schematic overview of the user-customizable M1-/M2-Macrophage differentiation process using the PromoCell Macrophage Generation Media.

Protocol overview using PromoCell M1-/M2-Macrophage Generation Medium DXF (10 days).

Figure 3. Protocol overview using PromoCell M1-/M2-Macrophage Generation Medium DXF (10 days).

Use Aseptic Methods and a Laminar Flow Bench

Macrophage Differentiation

A) Media and Solutions

  • M1- or M2-Macrophage Generation Medium DXF (C-28055 or C-28056)
  • Monocyte Attachment Medium (C-28051)
  • endotoxin-free PBS w/o Ca2+/Mg2+/2 mM EDTA/0.1% HSA
  • endotoxin-free PBS w/o Ca2+/Mg2+
  • optional: Macrophage Detachment Solution DXF (C-41330, refer to protocol step 11)
  • optional: additional activation/polarization factors (refer to protocol Step 8)

B) Differentiation Protocol

1. Isolate MononUClear Cells (Day 0)

Fresh PBMC has to be isolated from buffy coats using the standard protocol. Tuesday is a good day to start so as to avoid working through the weekend.

Note: It is recommended not to use buffy coats older than 20 hours since this will considerably impair the experimental results. Buffy coats less than 8 hours old are ideal to use.

2. Examine MononUClear Cells (Day 0)

The isolated PBMC for monocyte content has to be counted and examined; for example, using the FSC/SSC plot of a flow cytometer. Afterward, re-suspend the cells at 100 million PBMC per ml in Monocyte Attachment Medium.

Note: The protocol may be carried out without the determination of the monocyte content of the PBMC (refer to Step 3). However, this might cause a lower yield because of suboptimal initial plating density.

3. Allow the Monocytes to Attach (Day 0)

Freshly isolated PBMC needs to be plated in a suitable amount of Monocyte Attachment Medium - e.g. 15mL Medium per T-75 flask. A seeding density of 1 million/cm2 for Mononuclear Cells with a monocyte content of ≥25% and 1.5 million/cm2 for a monocyte content of <25% should be used. Incubation should be done for 1–1.5 hours at 5% CO2 and 37 °C in the incubator without any additional manipulation.

Note: Nunc plasticware with NunclonTM surface has to be used for ideal results. A plating density of 1.5 million PBMC per cm2 needs to be used when step 2 was excluded.

4. Prepare the Complete Macrophage Generation MEDIUM DXF (Day 0)

The Macrophage Generation Medium DXF needs to be prepared by incorporating the thawed Supplement Mix aseptically to the Basal Medium. To obtain a homogeneous mixture, it should be swirled gently. Then, Cytokine Mix M1 or M2 has to be added, respectively.

5. Wash the Adherent Cell Fraction (Day 0)

By swirling the tissue culture vessel vigorously, non-adherent cells will loosen and aspirate. The adherent cells, monocytes, need to be washed three times with warm Monocyte Attachment Medium, by swirling the vessel and aspirating the supernatant.

6. Start the Macrophage Differentiation (Day 0)

A right amount of complete M1- or M2-Macrophage Generation Medium DXF has to be added to the cells (for instance, 20mL per T-75 flask) and incubated for six days at 37 °C and 5% CO2 without a medium change.

Note: The monocytes separate into M1-like or M2-like polarized macrophages under these settings. If necessary, activation and subtype-specific polarization can be realized by carrying out the optional activation step (refer to Step 8).

7. Continue the Differentiation Process (Day 6)

Another 50% to 75% has to be added by the volume of fresh whole M1- or M2-Macrophage Generation Medium DXF to the cells. The immature macrophages have to be incubated for an additional three days at 37 °C and 5% CO2.

Note: Both adherent and suspension cells may be present. Do not take off any of the used medium from the cells, simply add the fresh medium.

8. Optional Step: Macrophage Activation (Day 7)

To activate specific macrophage, the total volume of the culture is supplemented with sufficient stimuli of the customers´ choice. Do not carry out a medium change; simply incorporate the activation factors.

Classically activated M1-macrophages can be made by adding IFN-γ (50ng/mL) and LPS (10ng/mL) to M1-macrophages. M2a-activation of M2-macrophages is realized by 20ng/mL IL-4. Supplementation with immune complexes and IL-1β or LPS will trigger M2b-activation, while IL-10, TGFβ or glucocorticoids result in M2c-activation of M2-macrophages. A substitute type of M1-activated macrophage can be attained by activating M2-macrophages with LPS and IFN-γ [2].

9. Medium Change (Day 9)

The medium including suspension cells needs to be aspirated and collected in a centrifugation tube. Pipet fresh complete PromoCell Macrophage Generation Medium DXF, supplemented with suitable cytokines/activation factors, to the cells. The cells in the tube have to be centrifuged for 15 minutes at 350 x g at room temperature. The supernatant has to be discarded and the cells should be re-suspended carefully in a small amount of fresh medium. The re-suspended cells in the tube have to be combined with the adherent cells in the fresh medium and held in the tissue culture vessel. It should be incubated until the next day at 37 °C and 5% CO2.

Note: Both adherent and non-adherent cells may be perceived at this stage.

10. The Macrophages are Ready (Day 10)

The macrophages can be used immediately in the plates where they reside, for example, when carrying out phagocytosis assays. Otherwise, they can be harvested (view instructions in optional Step 11). The culture needs to be maintained for up to three weeks by carrying out weekly medium changes with the new complete Macrophage Generation Medium DXF.

Note: Macrophages appear as adherent cells with distinctive morphology: a noticeable nucleus with a flatly outspread cytoplasm and numerous pseudopodia.

Exemplary flow cytometry analysis of day 10 M1-Macrophages generated in the PromoCell M1-Macrophage Generation Medium DXF. Fresh peripheral blood mononuclear cells were plated in the Monocyte Attachment Medium. The purified Monocytes were differentiated for 10 days without performing the optional activation step. Note that the cells exhibit the CD68+ (99% positive) and CD80+ (89% positive) marker profile, typical for M1-Macrophages.

Figure 4a. Exemplary flow cytometry analysis of day 10 - M1-Macrophages generated in the PromoCell M1-Macrophage Generation Medium DXF. Fresh peripheral blood mononuclear cells were plated in the Monocyte Attachment Medium. The purified Monocytes were differentiated for 10 days without performing the optional activation step. Note that the cells exhibit the CD68+ (99% positive) and CD80+ (89% positive) marker profile, typical for M1-Macrophages.

Exemplary flow cytometry analysis of day 10 M2-Macrophages generated in the PromoCell M2-Macrophage Generation Medium DXF. Fresh peripheral blood mononuclear cells were plated in the Monocyte Attachment Medium. The purified Monocytes were differentiated for 10 days without performing the optional activation step. Note that the cells exhibit the CD68+ (99% positive) and CD163+ (95% positive) marker profile, typical for M2-Macrophages.

Figure 4b. Exemplary flow cytometry analysis of day 10 M2-Macrophages generated in the PromoCell M2-Macrophage Generation Medium DXF. Fresh peripheral blood mononuclear cells were plated in the Monocyte Attachment Medium. The purified Monocytes were differentiated for 10 days without performing the optional activation step. Note that the cells exhibit the CD68+ (99% positive) and CD163+ (95% positive) marker profile, typical for M2-Macrophages.

11. Optional Step: Harvesting/Sub-Cultivation of Macrophages (Day 10+)

The medium has to be aspirated and then discarded. The adherent macrophages should be washed twice with endotoxin-free PBS w/o Ca2+/Mg2+. Add a suitable amount of cold (2-8 °C) Macrophage Detachment Solution DXF directly to the cells (for example, 25mL per T-75 flask). The tissue culture vessel has to be sealed and the cells have to be incubated for a period of 40 minutes at 2-8 °C. If needed, incubate for another 20 minutes at room temperature to enforce the release of cells from the culture surface.

The tissue culture vessel has to be strongly tapped to facilitate cell detachment. Ensure that most of the cells have already detached, or are only roughly adherent to the surface of the tissue culture vessel. After that, a cell scraper can be used to dislodge the remaining macrophages.

The harvested macrophages in centrifugation tubes have to be collected and diluted in a ration of 1:1 with PBS/2 mM EDTA/0.1% HSA. The cells have to be centrifuged for 15 minutes at 350 x g at room temperature. Two washes need to be carried out with PBS/2 mM EDTA/0.1% HSA on the cells, which should be subsequently counted. The macrophages are then ready to be used in the researcher's experiments.

Note: The percentage of attaching cells after re-seeding is based on the general health status of the macrophages prior to detachment and the effective performance of the detachment process itself. Therefore, a certain degree of variation is inevitable.

Demonstration of the phagocytosis capabilities of Macrophages generated in the PromoCell Macrophage Generation Media. Day 10 M2-Macrophages were exposed to florescently labeled E.coli for 24 hours before removing non-ingested bacteria. Left: Phase contrast image of M2-Macrophages after phagocytosis of fluorescently labeled bacteria. Note that a portion of the cells already starts to detach from the culture surface, indicative for the approaching death of the cells. It is a commonly observed phenomenon, that a macrophage dies from its own toxic metabolic products, for example, reactive oxygen species, after destroying approximately 50-100 bacteria. Right: Fluorescence image of the phagocytizing Macrophages. Green fluorescence shows labeled bacteria ingested by the Macrophages. Note that the roundish cells (Macrophages starting to detach/dying) have phagocytized most bacteria, whereas the still attached Macrophages exhibit reduced fluorescence because they have taken up less bacteria and are therefore still healthy.

Figure 5. Demonstration of the phagocytosis capabilities of Macrophages generated in the PromoCell Macrophage Generation Media. Day 10 - M2-Macrophages were exposed to fluorescently labeled E.coli for 24 hours before removing non-ingested bacteria.
Left: Phase contrast image of M2-Macrophages after phagocytosis of fluorescently labeled bacteria. Note that a portion of the cells already starts to detach from the culture surface, indicative for the approaching death of the cells. It is a commonly observed phenomenon, that a macrophage dies from its own toxic metabolic products, for example, reactive oxygen species, after destroying approximately 50-100 bacteria.
Right: Fluorescence image of the phagocytizing Macrophages. Green fluorescence shows labeled bacteria ingested by the Macrophages. Note that the roundish cells (Macrophages starting to detach/dying) have phagocytized most bacteria, whereas the still attached Macrophages exhibit reduced fluorescence because they have taken up fewer bacteria and are therefore still healthy.

 

References

[1] Murray PJ, Wynn TA (2011). Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol, 11(11):723-37. DOI: 10.1038/nri3073.

[2] Murray PJ, Wynn TA (2011). Obstacles and opportunities for understanding macrophage polarization. J Leukoc Biol, 89(4):557-63.

[3] Gordon S (2003). Alternative activation of macrophages. Nat Rev Immunol, 3(1):23-35.

[4] Khramtsova G, Liao C, Khramtsov A, Li S, Gong C, Huo D, Nanda R (2009). The M2/Alternatively Activated Macrophage Phenotype Correlates with Aggressive Histopathologic Features and Poor Clinical Outcome in Early Stage Breast Cancer. Cancer Res, 69(24): Supplement 3, DOI:10.1158/0008-5472.

[5] Menzies FM, Henriquez FL, Alexander J, Roberts CW (2010). Sequential expression of macrophage anti-microbial/inflammatory and wound healing markers following innate, alternative and classical activation. Clin Exp Immunol, 160(3):369-79.

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Last updated: May 21, 2019 at 9:39 AM

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