Protocol to generate human monocyte-derived macrophages in the absence of exogenous proteases and protease inhibitors to study proteolysis

Investigating proteases and protease inhibitors produced and secreted by primary cells is complicated by the presence of exogenous ones in cell culture medium containing fetal bovine serum. With this protocol, we generate human monocyte-derived macrophages in the absence of exogenous proteases and protease inhibitors by using a serum replacement for the final 24 h of macrophage culture. We describe steps for seeding and differentiating monocytes into macrophages. We then detail procedures for macrophage activation, culturing, and protein collection. For complete details on the use and execution of this protocol, please refer to Bernaerts et al.1.