Isolation and Characterization of Skeletal Stem Cells

Periosteal MSC were harvested as described from C57 BI/6 and CD47-null mice. Post digestion, cells were resuspended in PBS and stained with a panel to mark skeletal stem cells^{24 (link),48 (link)} using conjugated antibodies for CD90, Sca1, CD140a, CD51, CD200, Ly51, CD105, and a lineage negative cocktail. Samples were stained with fixable viability dye for 30 minutes. TrueStain FcX block (Biolegend, San Diego, CA; 156604) was added and cells were pelleted and washed prior to antibody staining at 4 °C for 1 hour. Following staining, samples were pelleted, washed, and fixed using commercially available Fixation/Permeabilization Buffer (Biolegend, 426803). Cells were analyzed on an LSRFortessa Cell Analyzer (BD), and data was analyzed using FlowJo (BD).