Protocol for in vivo lineage tracing of regeneration-associated macrophages from injured skeletal muscle of adult mice

Macrophages undergo phenotypic transitions that are essential for successful skeletal muscle (SkM) regeneration. Here, we present a protocol for in vivo lineage tracing of regeneration-associated macrophages, combining genetic labeling with transplantation of fluorescence-activated cell sorting (FACS)-isolated cells. Macrophages isolated from congenic CD45.1⁺ donor mice are transplanted into pre-injured SkMs of CD45.2⁺ mice and phenotyped by flow cytometry at designated time points of the regenerative process. We describe steps for muscle injury, SkM tissue processing, macrophage isolation, transplantation, and flow cytometry phenotyping. For complete details on the use and execution of this protocol, please refer to Sousa et al.¹.