Pe cy7 cd105

Cultured Sk-MSC-3d cells were suspended by trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA; Life Technologies, Tokyo, Japan) and labeled with rat monoclonal anti-mouse CD31 (FITC conjugated, BD or eFluor450 conjugated; eBioscience, San Diego, CA), CD44 (PE-Cy7; BioLegend, San Diego, CA), CD73 (PE, BD or eFluor450; eBioscience), CD105 (PE-Cy7; BioLegend) and CD271 (p75; rabbit polyclonal anti-p75, CST, Boston, MA; and anti-rabbit IgG Dylight 649 or PE, BioLegend, were used as secondary antibodies). Live cells were counted after cells positive for propidium iodide (PI) were excluded as dead cells. All cell analyses and sorting procedure in this study were carried out using FACSAria (Becton Dickinson Japan, Tokyo, Japan). Experiments were repeated three times using 3 mice/experiment in each cell type.

Upon reaching confluence, cells were collected and characterized for the expression of MSC-associated markers (CD105, CD90, and CD105) with a FortessaTM cell analyzer (Becton Dickinson), under the assistance of the HMRI Flow Cytometry Core. Antibodies used (APC/Cy7-CD73, PE/Cy7-CD105, and Alexa Fluor 700-CD90) were purchased from BioLegend. Briefly, MSC were recovered by trypsin method and by spinning down at 500 g for 5 min. They were then washed with FACS buffer labeled with directly conjugated antibodies according to the manufacturer’s indications.
At passage 3, cells were also assessed for their capability to undergo osteogenic and chondrogenic differentiation following a previously reported procedure (Corradetti et al., 2015 (link)). Briefly, to induce osteogenesis, MSC were seeded at the density of 5,000 cells/cm² in 12-well plates. Osteogenic induction was performed over 14-day period using a StemPro Osteogenesis Differentiation Kit (Gibco). To confirm differentiation, conventional von Kossa was performed. For chondrogenesis, cells were seeded at the density of 5,000 cells/cm². Induction was performed using the StemPro Chondrogenesis Differentiation Kit (Gibco) for a 14-day period. ECM constituted by proteoglycans was visualized by conventional Alcian Blue staining.