Co-culture of bone marrow-derived macrophages with aged primary myoblasts.

Skeletal muscle regeneration depends on the coordinated activity of multiple cell populations within the muscle stem cell niche, most prominently macrophages, which undergo dynamic phenotypic transitions essential to tissue repair. Aging disrupts this process, impairing macrophage signaling and muscle stem cell function in ways that are not yet fully understood. Existing approaches (e.g. conditioned media transfer and indirect transwell co-culture) fail to recapitulate the direct cell-to-cell contact required for continuous, reciprocal regulation throughout the regenerative cascade. Here, we present a protocol for the isolation of bone marrow-derived and tissue-resident macrophages from skeletal muscle, together with a direct co-culture methodology designed to interrogate macrophage-myoblast interactions in an age-relevant context.