Fractured Femur Callus Tissue Isolation

Callus tissue was pooled from 2 fractured femurs 5 DPF and digested.^{24 (link)} In brief, muscle tissue was dissected from the bone, bone marrow flushed and the thickened periosteum around the fracture site (“periosteal callus”) was scraped and digested in PBS containing 0.05% Collagenase P (Roche, IN, USA) and 0.2% hyaluronidase (Sigma Aldrich, St Louis, MO, USA) for 1 hour at 37°C. Cells were washed and stained with Zombie aqua (1:500, BioLegend, San Diego, CA, USA) to exclude dead cells. To exclude hematopoietic and endothelial cells staining for CD45 violet Fluor 450 (Clone: 30-F11, Tonbo biosciences), Ter119 eFluor 450 (Clone: TER-119, Invitrogen, Waltham, MA, USA) and CD31 eFluor 450 (Clone: 390, eBioscience) was performed. LSR II flow cytometry system was used (BD Bioscience, San Jose, CA, USA). Voltages and gates were set based on unstained samples and single stained controls. Diva8 software (BD Bioscience, San Jose, CA, USA) was used for analyzing the data.