Murine Macrophage Isolation and Stimulation

Tissues were collected from WT, HEM, and KO mice and primary bone-marrow-derived macrophages (BMDMs) were prepared from bone marrow cells of the hind limbs of mice, as described^{31 (link)}. Macrophages were obtained growing cells in Dulbecco’s Modified Eagle’s Medium supplemented with 20% FBS and 30% L-cell (medium obtained from the supernatant of L929 cells used as source of M-CSF, Macrophage-Colony Stimulating Factor) for up to 7 days. Then, cells were scrapped and seeded in plates for experiments. Cells were treated with 100 ng/ml LPS (Lipopolysaccharide) [Sigma] and 1 ng/ml IFNγ [ImmunoTools] for the indicated times. Cells were exposed to ultraviolet B (UVB) irradiation once medium was removed using a UV lamp [UVP Inc.] at the distance of 50 cm, and then fresh medium was added. A Radiometer with UVX-31 sensors was used [UVP Inc] to monitor UVB doses. The doses were 50 and 100 J/m² for UVB.