Bodipy

Osteogenic cells were labelled with Alizarin Red staining according to the following protocol: cells were washed in PBS (Gibco, 10010023) and fixed in 95% methanol for 10 min. Alizarin Red S (Sigma, A5533) solution 2% in deionized water was added for 5 min, then the cells were rinsed multiple times with water until no eluting stain could be seen. Ca²⁺ deposits were observed under an epifluorescence microscope (Zeiss, Rueil-Malmaison, France). Osteogenic differentiation was also assessed using the alkaline phosphatase (ALP) assay (Supplementary information contains the method description and the results—Fig. S1B).
Adipogenic cells were stained with Bodipy: after the cell medium was removed, the cells were incubated for 30 min with 10 µM Bodipy (Sigma, 790,389) prepared in fresh medium. The cells were rinsed with fresh medium. The lipid droplets were visualized using the FITC filter of the epifluorescence microscope. Adipogenic differentiation was also assessed using the Oil Red O staining method (Supplementary information contains the method description and the results—Fig. S1A).
All assessments of cell differentiation were conducted in three biological replicates. To evaluate the differences among the groups, one-way ANOVA followed by Dunnett's multiple comparison test was employed.

HCS LipidTOX™ Phospholipidosis and Steatosis Detection Kit (#H34158, ThermoFisher Scientific) or BODIPY (#790389, Sigma-Aldrich) was used to stain the lipid. LipidTOX Red phospholipid stain was added to the cell culture 18 h before harvesting. Harvested DCs were washed with PBS and stained with LipidTOX Green neutral lipid stain at room temperature for 30 min. DCs were spun down and washed with PBS once before acquisition on flow cytometer. If BODIPY was used, harvested DCs were stained in the same way as LipidTOX Green neutral lipid stain.

Fluorinated boron-dipyrromethene (BODIPY) and 4’,6-diamidino-2-phenylindole (DAPI) staining techniques were used to analyze the size of lipid droplets and the number of nuclei, respectively. Briefly, cells were fixed with 4% paraformaldehyde for 5 min and permeabilized with 0.1% Triton X-100 for 10 min. Cells were then stained with 10 µg/mL of BODIPY (Sigma, USA; Cat#: 121207) for 15 min, followed by incubation with 1 µg/mL of DAPI (Sigma, USA; Cat#: 28718) for 5 min. Images were captured using an inverted fluorescence microscope (Olympus, CK30, Tokyo, Japan) from five different areas per replicate. The number of nuclei and the size distribution of lipid droplets were analyzed using CellProfiler (Broad Institute, Cambridge, MA, USA). Lipid droplet size was determined by calculating the average of the minimum and maximum Feret diameter [28 (link)].