To isolate periosteal cells,40 (link) femurs and tibiae were placed in ice-cold PBS after the overlying skin and muscle were carefully removed. The periosteum was gently scratched using a scalpel and forceps and then incubated with prewarmed digestion buffer at 37 °C for 30 min on a shaker. The dissociated periosteal cells were washed twice by centrifugation at 600 × g for 5 min with ice-cold staining buffer.
To isolate growth plate cells,28 (link) dissected growth plates were minced using a scalpel and incubated with 0.15% collagenase (Sigma, C6885) at 37 °C for 90 min on a shaking incubator. The cells were pelleted and resuspended in ice-cold staining buffer.
The cells were stained with PerCP/Cy5.5 anti-CD31 (BioLegend, 102420), PerCP/Cy5.5 anti-CD45 (BioLegend, 103132), PerCP/Cy5.5 anti-mouse Ter-119 (BioLegend, 116228), BV421 anti-mouse CD73 (BioLegend, 127217) or Pacific blue anti-Sca1 (BioLegend, 108120) for 30 min and sorted on an MA900 flow cytometer (Sony) with a 100 μm nozzle.
To isolate growth plate cells,28 (link) dissected growth plates were minced using a scalpel and incubated with 0.15% collagenase (Sigma, C6885) at 37 °C for 90 min on a shaking incubator. The cells were pelleted and resuspended in ice-cold staining buffer.
The cells were stained with PerCP/Cy5.5 anti-CD31 (BioLegend, 102420), PerCP/Cy5.5 anti-CD45 (BioLegend, 103132), PerCP/Cy5.5 anti-mouse Ter-119 (BioLegend, 116228), BV421 anti-mouse CD73 (BioLegend, 127217) or Pacific blue anti-Sca1 (BioLegend, 108120) for 30 min and sorted on an MA900 flow cytometer (Sony) with a 100 μm nozzle.
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