Oil red powder

For Oil Red O staining, linoleic acid (Wako) was added into the medium at 10 mg/ml and incubated for 30 minutes. After washed with PBS, cells were fixed with 4% PFA at room temperature for 30 minutes and washed with 60% isopropanol, followed by incubation for 30 minutes with Oil Red solution which Oil Red powder (Sigma Aldrich) was dissolved into isopropanol and diluted to 60% with distilled water. Then cells were washed with distilled water three times. For PAS staining, cultured cells were stained by periodic acid-Schiff (MUTO PURE CHEMICALS) following the manufacturer’s instructions. For paraffin-embedded spheroids, the same procedure was carried out after deparaffinization. For ICG uptake assay, indocyanine green (Wako) was added in the medium at 0.1 mg/ml and incubated for 1 hour, followed by washing with PBS three times.

Oil red powder, 0.15 g (Sigma, United States), was dissolved in 30 ml isopropanol and stored in a dark place. The cells were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 30 min in the culture plates. Oil red stock solution was diluted with distilled water (ratio = 3:2) and added to the plate for 15 min. The plates were rinsed with 60% isopropanol and counterstained with hematoxylin for 10 s. After washing with distilled water, the cells in the plates were investigated under a Leica DMI3000B microscope (Leica, Germany).
The intracellular TG levels in HepG2 cells were determined by using a TG quantification kit (Applygen, Beijing, China) according to the manufacturer's instructions. Cellular TG levels were normalized to their protein concentrations.