Murine Macrophage Differentiation Protocol

Murine macrophages were differentiated in vitro as previously described [19 (link)]. In brief, 8–12 week old iRhom2 knockout (KO) or wild-type (WT) mice were sacrificed by carbon dioxide (CO₂) inhalation, and femurs and tibia collected. After removing joints from the bones, femurs and tibia were flushed with 2 ml RPMI (Gibco, part of Thermo Fischer Scientific, Billings, Montana, United States). Cells were strained through a nylon mesh to remove debris and clumps, into a 15 ml centrifuge tube. Then, cells were resuspended in 15 ml complete RPMI, and centrifuged at 230 × g for 5 min. Cell pellet was resuspended in 1 ml of red blood cell lysis buffer (Sigma-Aldrich, St. Louis, Missouri, United States) for 3 min to remove erythrocytes, then diluted in 15 ml RPMI and centrifuged again at 230 × g for 5 min. Pellet was resuspended in RPMI, then 10⁶ cells were plated in a bacteriological Petri Dish with 10 ml complete RPMI supplemented with 100 ng/ml recombinant murine M-CSF (Peprotech, London, UK). After 1 week, cells were differentiated into macrophages (98% of cells were F4/80 positive–not shown) and then used for further analysis (secretome analysis and FACS).