Isolation and Polarization of Mouse Macrophages

Bone marrow was flushed from mouse femurs and tibias with Ca²⁺ and Mg²⁺-free Hepes-buffered saline solution (HBSS). Pellets were stored at −80°C for protein analysis by western blot, or were frozen in liquid nitrogen in 10% DMSO/90% FCS for cell culture experiments. For in vitro differentiation of bone marrow-derived macrophages, the method was adapted from (39 (link)). Cells were plated at 2 × 10⁶ cells per 10 cm dish in 10 ml macrophage media consisting of αMEM (Gibco) supplemented with 10% FCS, 1% penicillin/streptomycin, 2 mM L-glutamine and 10 ng/ml M-CSF (Peprotech). Functional assays were performed starting on day 7 of culture. For polarization, macrophages were removed from plates using Cell Dissociation Buffer (Gibco), washed, and plated in either M0 conditions (macrophage media), M1-polarizing conditions (macrophage media supplemented with 100 ng/ml LPS and 20 ng/ml recombinant IFNγ) or M2-polarizing conditions (macrophage media supplemented with 20 ng/ml recombinant murine IL-4) for 24 h before analysis. To measure CD206 expression on M2 polarized mouse macrophages, cells were removed from plates using Cell Dissociation Buffer (Gibco) and stained for subsequent analysis by flow cytometry as outlined below. To measure cytokine production by M1 polarized macrophages, supernatants were harvested and analyzed by multiplexed ELISA.