Macrophage-Mediated Myogenic Differentiation

Macrophages were obtained from bone marrow precursor cells extracted from 4 distinct mice that were cultured in DMEM containing 20% FBS and 30% conditioned medium of L929 cell line (enriched in CSF-1) for 7 days. Macrophages were activated with hrANXA1 for 3 days (10 nM) in DMEM containing 10% FBS. After the washing steps, serum-free DMEM was added for 24 hours to obtain macrophage-conditioned medium. Murine myogenic precursor cells (MPCs) were obtained from TA muscle isolated from 4 mice and cultured using standard conditions in DMEM/F12 medium (Gibco Life Technologies) containing 20% heat-inactivated FBS and 2% G/Ultroser (Pall Inc.). MPCs were seeded at 30,000 cells/cm² on Matrigel (diluted 1:10) and incubated for 3 days with conditioned medium containing 2% heat-inactivated horse serum. In the case of direct treatment of MPCs, cells were directly incubated with 10 nM hrANXA1 for 3 days in the presence of 2% heat-inactivated horse serum. Cells were then incubated with an anti-desmin antibody (ab32362, Abcam), followed by a Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc.) (13 (link), 29 (link)). Cells were stained with Hoechst (Sigma-Aldrich) and mounted in Fluoromount (Interchim), and pictures were taken on Axio Observer.Z1 (Zeiss) at ×20 magnification connected to a CoolSNAP HQ2 CCD Camera (Photometrics).