Sight ds 5m l1 photo camera

Adipogenic differentiation [26 (link),27 (link)] was performed treating rBM-MSCs with three cycles of induction medium (containing: rh-insulin, mesenchymal cell growth supplement, 3-isobuty-lmethylxanthine, dexamethasone, indomethacin, l-glutamine, penicillin/streptomycin), and maintenance medium (basal medium supplemented with rh-insulin, mesenchymal cell growth supplement, penicillin/streptomycin, l-glutamine) (Lonza Walkersville, Inc., Walkersville, MD, USA), and incubated in a humidified incubator at 37 °C and 5% CO2 for 21 days. In all cultures, the medium was changed every 3 days.
Oil red staining. The adipogenic differentiation was assessed by the analysis of the lipid droplets using the Oil Red O (ORO) staining (BioVision Inc., Milpitas, CA, USA). Briefly, after fixing in 4% paraformaldehyde for 15 min at RT, cultures were washed with PBS, rinsed with 60% isopropanol for 10 min at RT, washed in distilled H₂O, and stained with 500 μL of ORO solution (ORO 0.3% in isopropanol mixed with H₂Od (3:2)) for 20 min at RT. Stained cultures were washed twice with distilled H₂O, and the staining was evaluated by the microscopy (Eclipse-TS100, Nikon, Tokyo, Japan) equipped with a digital SIGHT DS-5M-L1 photo camera (Nikon, Tokyo, Japan).

rFFFs and rBM-MSCs were seeded on glass coverslips at the optimal density of 1 × 10³ cells. After fixing with 4% paraformaldehyde for 15 min at room temperature (RT), and some washing with PBS, cells were incubated with 200 μL of Haematoxylin solution (Sigma-Aldrich, St. Louis, MO, USA) for 5 min at RT. After washing with sterile H₂O, cells were added with 1% of Eosin solution (Sigma-Aldrich, St. Louis, MO, USA) for 3 min at RT and further washed with sterile H₂O. All procedures were according to the manufacturer’s instructions. Stained cells on coverslips were mounted with Vectashield Antifade Mounting Medium (Vector Laboratories Inc., Burlingame, CA, USA). Image acquisition was performed by using inverted microscopy (Eclipse-TS100, Nikon, Tokyo, Japan) equipped with a digital SIGHT DS-5M-L1 photo camera (Nikon, Tokyo, Japan).

Osteogenic differentiation [26 (link),27 (link)] was achieved by culturing rBM-MSCs in mesenchymal stem cell differentiation basal medium-osteogenic (OM) (Lonza Walkersville, Inc., Walkersville, MD, USA) supplemented with the SingleQuots (containing: dexamethasone, ascorbate, L-glutamine, pen/strep, β-glycerophosphate, mesenchymal cell growth supplement (Lonza Walkersville, Inc., Walkersville, MD, USA), for 21 days in a humidified incubator at 37 °C and 5% CO₂. In all cultures, the medium was changed every 3 days.
All osteogenic and adipogenic differentiation experiments were performed in triplicate.
Alizarin red staining. The rBM-MSCs osteogenic differentiation was assessed by the analysis of the calcium precipitation through the Alizarin red staining. Briefly, after the osteogenic treatment, the cells were washed with PBS twice, fixed with 4% paraformaldehyde for 15 min at RT, washed in deionized H₂O, and incubated with 500 μL of 2% Alizarin red solution (Sigma-Aldrich, St. Louis, MO, USA). After, washing with distilled water, the staining was evaluated by the microscopy Eclipse-TS100 (Nikon, Tokyo, Japan) equipped with a digital SIGHT DS-5M-L1 photo camera (Nikon, Tokyo, Japan).