Differentiation and Polarization of Monocyte-Derived Cells into Macrophage-Like Cells

To initiate macrophage differentiation, dilute the cells to a 5 x 10⁶ cells per milliliter of serum-free medium concentration, and seed 2 milliliters of cells into individual wells of a 6-well plate. Place the plate in the cell culture incubator for two to three hours to allow the cells to adhere to the well bottoms.

At the end of the incubation, wash the wells three times with 1 milliliter of serum-free medium per wash, and add 2 milliliters of complete RPMI medium supplemented with 50 nanograms per milliliter of GM-CSF, or 50 nanograms per milliliter of M-CSF, for M1 or M2 differentiation respectively, to each well of adherent cells.

Three days after polarization initiation, carefully replace 1 milliliter of supernatant from each well, with 1 milliliter of fresh complete medium supplemented with the appropriate growth factors.

On day 6 of culture, add 50 nanograms per milliliter of interferon-gamma and 10 nanograms per milliliter of LPS to the GM-CSF-treated wells, and 20 nanograms per milliliter of IL4 to the M-CSF-treated wells.

Check the morphology of the monocyte-derived cell cultures regularly by light microscopy to ensure that the smaller monocytes are differentiating into larger macrophage-like cells.