Cd140b percp cy5

Cultured synovial MSCs from three donors at passage 1 were harvested using a cell-dissociation buffer. Cells were suspended in HBSS at a density of 5 × 10⁵ cells/mL and stained for 30 minutes on ice with the antibodies CD31-PE-Cy7 (Becton, Dickinson and Company; BD, Franklin Lakes, NJ, USA), CD45-APC-H7 (Biolegend, San Diego, CA, USA), CD44-APC-H7 (BD), CD73-BV421 (BD), CD90-PE (BD), CD105-PerCP-Cy5.5 (BD), CD140a-BV421 (BD), CD140b-PerCP-Cy5.5 (BD), CD146-FITC (BD) and CD271-APC (Miltenyi Biotec) for cell surface analysis. Flow cytometric analysis of the cell surface was performed by a triple-laser FACS Verse™ system (BD).

Synovium was digested in a solution of 3 mg/mL collagenase (Sigma-Aldrich Japan, Tokyo, Japan) at 37 °C. After 3 h, the digested cells were filtered through a 70-μm cell strainer (Greiner Bio-One GmbH, Kremsmunster, Austria). The cells from six donors were harvested using a cell-dissociation buffer. Cells were suspended in HBSS at a density of 5 × 10⁵ cells/mL and stained for 30 min on ice with the antibodies. For cell isolation, cells were stained with CD31-PE-Cy7 (BD), CD45-PE-Cy7 (BD), CD235a-PE-Cy7 (BD), CD55-FITC (Miltenyi Biotec), CD90-PE (BD) and CD271-APC (Miltenyi Biotec) were used at day 0. Flow cytometric isolation of cell surface antigens were performed by a double-laser Aria 2 system (BD). For cell surface analysis, cells were stained with CD31-FITC (BD), CD45-FITC (BD), CD44-APC-H7 (BD), CD73-BV421 (BD), CD90-PE (BD), CD105-PerCP-Cy5.5 (BD), CD55-PE (BD), CD271-APC (Miltenyi Biotec), CD140b-PerCP-Cy5.5 (BD) and CD146-FITC (BD) at passage 3. Flow cytometric analysis of cell surface antigens was performed by a triple-laser FACS Verse system (BD). These data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA). Flow cytometric analyses were also performed for expanded cells at passage 3.