Experimental design and applied gating strategy for flow cytometry.
a Skeletal muscles were harvested from Pax7-zsGreen animals and mononuclear and myofiber-associated cells were purified using a two-step enzymatic and mechanical digestion procedure. All cells were stained with the viability markers PI and calcein, as well as negatively selecting markers. Due to fluorophore overlap, positive selecting antibodies were split between two staining conditions including all hematopoietic and stromal factors (Sca1, CD31, CD45, Mac1, and Ter119) and either β1-integrin, CXCR4, and VCam1 or β1-integrin, CXCR4, α7-integrin, and CD34. Cells were analyzed and sorted by flow cytometry. b Gating conditions applied to all downstream analysis included initial physical parameter gate to exclude cells larger than our target population, viability selection of cells that were PI− and calcein+, and exclusion of hematopoietic and stromal cell types. All comparative flow cytometric analyses (see Figs. 2, 3, and 4) began with this Sca1, CD31, CD45, Mac1, and Ter119 negative cell population (red)