Facsdiva data acquisition software

The isolated cells were cultured on 100 mm cell culture dishes until 60% confluence. We analyzed the cell surface antigen expression according to previously described methods [22 (link)]. Cells were resuspended in flow cytometry staining buffer (phosphate-buffered saline (PBS) + 2% FBS) at a concentration of 1 × 10⁶ cells/tube, followed by staining with primary specific antibodies, including phycoerythrin-conjugated CD146, APC-conjugated CD90, APC-conjugated CD73, APC-conjugated CD105, APC-conjugated CD34, APC-conjugated CD45, APC-conjugated CD14, and APC-conjugated HLA-DR (eBioscience, San Diego, CA, USA), and incubating for 30 min at 4 °C. Subclass-matched antibodies were used as negative control. The cells were then washed twice and resuspended in 400 µL of 2% FBS/PBS. Flow cytometry was performed using SH800 cell sorter (Sony, Tokyo, Japan); all the data were analyzed using FACSDiVa data acquisition software (BD Biosciences, San Jose, CA, USA). We confirmed that the isolated cells expressed mesenchymal stem cell markers, and not hematopoietic stem cell markers, and defined the isolated cells as DPSCs.