Cd105 apc

Rat synovial MSCs at passage 3 were detached by treatment with TrypLE (Thermo Fisher Scientific) for 10 min and used for surface marker analysis. The cell fluorescence and percentage of antigen-positive cells were evaluated with a FACSVerse instrument (BD Biosciences). CD90-PE-Cy7 (eBioscience, San Diego, CA, USA), CD44-PE (eBioscience), CD105-APC (Novus Biologicals, Littleton, CO, USA), CD45-FITC (BD Pharmigen, San Jose, CA, USA) and CD34-PerCP-Cy5.5 (Novus Biologicals) antibodies were used. Cells were incubated with conjugated antibodies at 4 °C for 1 h in the dark. Cells positively stained with Ghost Dye Violet 510 (Tonbo Biosciences, San Diego, CA, USA) were removed as dead cells. Isotype controls were prepared as negative controls.

Colony-forming cells from SF were detached at passage 2 with TrypLE (Thermo Fisher Scientific) and suspended in FACS buffer (2% FBS and 5 mM EDTA in PBS). The cells were stained with the CD34-PerCP (Novus), CD44-PE (R&D Systems, Minneapolis, MN, USA), CD45-FITC (BD Biosciences, San Jose, CA, USA), CD90-PE-Cy7 (BD), CD105-APC (Novus), and Ghost Dye Violet 510 for dead cells (Tonbo Biosciences, CA, USA). For CD73, a mouse anti-CD73 antibody (BD) was used, followed by the secondary antibody conjugated with Alexa Fluor 488 (Abcam).
The synovial tissue was analyzed by mincing the synovium and digesting it for 3.5 h at 37 °C with 0.4 mg/mL Liberase (Roche Diagnostics, Mannheim, Germany) and 0.1 mg/mL DNase (Sigma-Aldrich, St Louis, MO, USA). Residual red blood cells were lysed with ACK Lysing Buffer (Thermo Fisher Scientific), the cells were passed through a 70 μm strainer (Greiner Bio-One GmbH, Frickenhausen, Germany) to yield single-cell suspensions. The cells were then stained with the CD44-PE, CD73-PE-Cy7 (Bioss), and CD90-APC (BD), as well as with Ghost Dye Violet 450 (Tonbo Biosciences) to identify dead cells. The proportion of antigen-positive cells was evaluated using a FACSVerse instrument (BD).