Fitc conjugated anti cd90

Flow cytometry analyses were performed on hDPSCs cultured in medium supplemented with C-FBS or NZ-FBS at first passage of culture. Cells were incubated with FITC-conjugated anti-CD90, PerCP-Cy5.5-conjugated anti-CD105, APC-Cy7-conjugated anti-CD45 (all purchased from BD Pharmingen, San Diego, CA), and PE-conjugated anti-CD34 (Miltenyi Biotech, Calderara di Reno, Bologna, Italy) for phenotypic characterization; and anti-Bone sialoprotein (BSP) (Abcam, Cambridge, UK), anti-CFS-conjugated anti-Osteopontin (OPN), PE-conjugated anti-Osteocalcin (OC) (both from R&D Systems, Minneapolis, MN) and PerCP-Cy 5.5-conjugated anti-Nanog (BD Pharmingen, Milan, Italy) to evaluate osteogenic differentiation and mesenchymal stemness. hDPSCs were sorted by CD34 expression. The purity of sorting was 90%. As negative controls, cells were stained with an isotype control antibody. After incubation with the antibody, cells were resuspended in PBS and analyzed with a FACS ARIA III (BD Biosciences, San Jose, CA). For intracellular staining of Osteocalcin, Osteopontin and Nanog, cells were processed using Fix & Perm Kit (Invitrogen, Milan, Italy) following the manufacturer's guidelines. All data were analyzed using FCS express version 3 (De Novo Software, Glendale, CA).

Cytofluorimetric analysis was conducted to analyze the expression of mesenchymal markers on both c-MSC isolates. 2 × 10⁵ cells were incubated with FITC-conjugated anti-CD-90 (BD Biosciences, San Jose, CA, USA, cat n. 555595), PE-conjugated anti-CD-44 (Miltenyi Biotec, Bergisch Gladbach, Germany, cat n. 130-102-606), PE-conjugated anti-CD-105 (Miltenyi Biotec, Bergisch Gladbach, Germany, cat n. 130-118-174), and APC-H7-conjugated anti-CD-45 (BD Biosciences, San Jose, CA, USA, cat n. 641399) as well as dye/isotype-matched antibodies in the dark environment for 30 min at 4°C. All samples were processed by a BD LSR Fortessa (BD Biosciences, San Jose, CA, USA) and analyzed with BD FACS Diva Software. For each sample, 10⁴ events were acquired in all analyses.

The cells diluted in HBSS were stained with the following human antibodies for cell sorting and cell surface analysis: phycoerythrin (PE)-conjugated anti-CD73 (BioLegend, San Diego, CA), allophycocyanin (APC)-conjugated anti-CD29, anti-CD44, APC-conjugated anti-CD73, fluorescein isothiocyanate (FITC)-conjugated anti-CD90, PE-conjugated anti-CD271, PE-Cy7-conjugated anti-CD31, anti-CD45, and anti-CD235a (BD, Franklin Lakes, NJ). Propidium iodide fluorescence was used to gate dead cells. Flow cytometry and sorting were performed on a FACS Aria II instrument (BD).