Bodipy 493 503

Lipid droplet assay was performed according to a previous method using a BSA-conjugated oleic acid system in Huh7 cells as described before (Yen et al., 2018 (link)). Briefly, cells seeded in μClear^® 96-well plates (Greiner Bio-ONE, Frickenhausen, Germany) were treated with oleic acid and the tested samples or DMSO for 18 h. Cells were stained with 2 μg/ml Hoechst 33342 and 1 μg/ml BODIPY^® 493/503 and fixed in paraformaldehyde. A High-content imaging (HCS) instrument was used to take and analyze images of the nuclei and lipid droplets (ImageXpress Micro System, Molecular Devices, Sunnyvale, CA, United States). The diameter settings were 8–25 μm for the nuclei and 0.5–2 μm for the lipid droplets.

Lipid droplet assay was performed according to a previous method using a BSA-conjugated oleic acid system in Huh7 cells as described previously [22 (link)]. Briefly, cells seeded in μClear® 96-wells plates (Greiner Bio-ONE, Frickenhausen, Germany) were treated with oleic acid and the tested sample or DMSO for 18 h. Paraformaldehyde was used to fix the cells, which were stained with 2 μg/mL Hoechst 33342 and 1 μg/mL BODIPY® 493/503. High Content Imaging (HCS) instrument was used to take and analyze images of the nuclei and lipid droplets (ImageXpress Micro System, Molecular Devices, Sunnyvale, CA, USA). The diameter settings were 8–25 μm for the nuclei and 0.5–2 μm for the lipid droplets.

An LD assay was performed as described in previous research [36 (link)]. The accumulation of LDs was detected with the BODIPY^® 493/503 dye (Thermo Fisher Scientific). LD accumulation resulted from treating cells with bovine serum albumin (BSA)-conjugated oleic acid (OA). Cells were seeded in μClear^® 96-well plates (Greiner Bio-ONE, Frickenhausen, Germany) and loaded with 125 μM of OA with testing compounds at a concentration of 40 μM for 16 h. Cells were then fixed with paraformaldehyde and stained with 2 μg/mL of Hoechst 33342 and 1 μg/mL of BODIPY^® 493/503. Nine fields for each well were picked, and images of nuclei and LD were acquired and analyzed automatically with an HCS instrument (ImageXpress Micro System, Molecular Devices, Sunnyvale, CA, USA). A granularity analyzing module was used to identify nuclei and LDs. The diameter settings for defining nuclei and LDs were 8–25 and 0.5–2 μm, respectively. The average LD counts/cell of BSA-conjugated OA + drug vehicle (DMSO)-treated wells (hereinafter referred to as OA) were used as the standard for 100% of fatty loading.