Differentiation and Immortalization of Primary Mouse Macrophages

Primary mouse macrophages (BMDMs) were differentiated for 6 d and cultured for up to 9 d in DMEM (Gibco) supplemented with 10% FCS (Bioconcept), 20% 3T3 supernatant (MCSF), 10% Hepes (Gibco), and 10% nonessential amino acids (Gibco). Immortalization of macrophages was performed as previously described (Blasi et al, 1985 (link); Broz et al, 2010 (link)). Immortalized macrophages (iBMDMs) were cultured in DMEM complemented with 10% FCS (Bioconcept), 10% MCSF (3T3 supernatant), 10% Hepes (Amimed), and 10% nonessential amino acids (Life Technologies). To passage the BMDMs and iBMDMs, the cells were washed with PBS and left to detach at 4°C for 15 min and scarped using cell scrapers (Sarstedt), spun down at 300g for 5 min at 4°C, and resuspended in the appropriate amount of medium.