Ideas software

Protein expression of ALP, OPN, OC, and COL1a1 was evaluated by flow cytometry analysis, following a previous protocol [35 (link)]. Samples were processed using a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA), and data were analyzed using FACSDiva v6.1.3, IDEAS software (BD Biosciences, San Jose, CA, USA), and FlowJo v8.3.3 software (Tree Star Inc., Ashland, OR, USA). Data are indicated as a mean fluorescence intensity (MFI) ratio calculated by dividing the MFI of positive events by the MFI of negative events (MFI of secondary antibody).

ROS released by hGDFs cells (20,000 cells/mL) were measured following the treatment with the culture medium (CTRL) or the CR extracts (100%, 50%, and 25%) obtained at soaking times of 24 h and 14 days. The hydrogen peroxide (H₂O₂, 300 µM for 30 min) was used as positive control. After 48 h of exposure, cells were collected and stained using the cell fluorescent reagent “CellROXTM Green Reagent” (C10444, Molecular Probes, ThermoFisher Scientific, MA, USA) at 2.5 µM in PBS for 30 min at 37 °C. Each sample was processed using a FACS Canto II flow cytometer (BD Bioscences, San Jose, CA, USA). All data were analyzed using FACSDiva v 6.1.3, IDEAS software (BD Biosciences) and FlowJo 8.3.3 software (Tree Star Inc., Ashland, OR, USA). The results were obtained as an MFI (Mean Fluorescence Intensity) Ratio calculated by dividing the MFI of positive events by the MFI of negative events (MFI of secondary antibody).

hOBs (5 × 10⁵) were analyzed for the expression of the following bone markers: ALP (rabbit monoclonal anti-ALP; 1:1000; cat.# EPR4477, Abcam, Cambridge, MA, USA), Runx2 (rabbit monoclonal anti-Runx2; 1:800; cat.# 12556, Cell Signalling, Danvers, MA, USA), COL1a1 (rabbit monoclonal anti-COL1a1; 1:1000; Abcam, cat.# P02452), OPN (rabbit polyclonal anti-OPN; 1:1000; Abcam, cat.# P10451), and OC (rabbit polyclonal anti-OC; 1:100; Abcam, cat.# O60422) for 30 min at 4 °C. Anti-rabbit Alexa 488 (1:100, Invitrogen, Thermo Fisher Scientific) was used as secondary antibody. Each sample was processed using an FACS Canto II flow cytometer (BD Bioscences) and data were analyzed using FACSDiva v6.1.3, IDEAS software (BD Biosciences) and FlowJo v8.3.3 software (Tree Star Inc., Ashland, OR, USA). Results were expressed as the MFI (mean fluorescence intensity) ratio calculated by dividing the MFI of positive events by the MFI of negative events (MFI of secondary antibody).