Hi,
I am looking for an established protocol to differentiate M1 and M2 macrophages from human PBCMs? I want to use the M1 and M2 macrophages to expand them and perform killing assays with T-cells. Any protocol and support will be appreciated. I'm happy to provide my email.
Thank you1
Hi there Hitesh, you can use the protocol we used in here: https://www.frontiersin.org/articles/10.3389/fimmu.2019.01789/full
M1/M2 Macrophage Polarization Negatively selected monocytes, as described before, were cultured in complete RPMI medium (Merck) supplemented with either 20 ng/mL human GM-CSF (PeproTech), for M1 differentiation, or 20 ng/mL human M-CSF (PeproTech), for M2 differentiation, and incubated for 6 days adding fresh media with the respective factors on day 3 (I strongly recommend adding, half the amount of initial media in here, rather than replacing all the media) . On day 6 M0 macrophages were activated with 100 ng/mL human IFN-γ (PeproTech) and 50 ng/mL LPS, for M1 macrophage polarization, or 20 ng/mL IL-13 (PeproTech) and 20 ng/mL IL-4 (PeproTech), for M2 macrophage polarization, and incubated for 24 h. M1 macrophages were stimulated with 100 ng/ml LPS for 4 h (Figure 3A). For M1/M2 macrophage characterization using flow cytometry, monocytes seeded at a density of 1 ×106 cell/ml were cultured in tissue culture-treated 6 well plates, stimulated and activated with their respective factors. On day 7 cells were washed twice with DBPS without Ca2+ and Mg2+, and detached using DPBS with EDTA 10 mM. Cells were washed twice with DPBS (2% FBS), pelleted and resuspended in BSB with human and mouse serum for 30 min on ice. Cells were washed, pelleted, and stained with the surface markers for M1 (CD14+, HLA-DR+, CD274+ and CD86+) and M2-type (CD14+, HLA-DR-, and CD206+) for 30 min on ice. Then, cells were washed, pelleted, fixed, and permeabilized for 20 min on ice. The intracellular markers IL-10 and TNF were then added to fully characterize the M1/M2 macrophages. After a final wash cells were resuspended in BSB in FACS collection tubes. All antibodies used are listed in detail in the table of regents. All the gating strategies are shown in Supplementary Figure 2.
You can grow the macrophages in 6 well plates at a density of 1 ×10^6 cell/ml in 1 ml of media, or in 12 well plates or even 24 well plates (decreasing the media volume and macrophages number respectively. As you mentioned to harvest the cells Acutase work fine for M1 and PBS+EDTA for M2, you still have to give them a hard wash to detach them. I hope your experiments work.
The method described by Reyna is great, you just need to be careful that you have to differentiate M1 and M2 in the number you require as they won't expand in number after differentiation.
I recommend Samuel J Lara Reyna answer, with using of heat inactivated FBS in RPMI culture media, to avoid the blocking of IL-4 stimulation.
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