Jc 10

HCC1143, HCC1806, and HCC1395 cells were treated by free GA or NP-GA at its IC₅₀ for 3h (30.27µm for HCC1143; 14.51µm for HCC1806, 15.76µm for HCC1395). Cells cultured in media served as controls. Changes in mitochondrial membrane potential were measured by cationic lipophilic dye JC-10 (Abcam) and the data were presented as the ratio of green fluorescent signal (520nm) to red fluorescent signal (590nm).

The dissociated HUES8 SC-β cells were seeded onto 3.5 cm Matrigel-coated glass-bottom petri dishes for 24 h and loaded with JC-10 (Abcam, #ab112134, 10 mg ml⁻¹) for 1 h. During the period of JC-10 staining, several dishes of HUES8 SC-β cells were treated with FFAs in parallel for 0 min or 30 min or 45 min, or 60 min, respectively. A confocal microscope was used to take images with the excitation = 540 nm, emission = 590 nm for aggregated imaging, and excitation = 490 nm, emission = 525 nm for monomeric imaging. The mitochondrial membrane potential was measured by the ratio of green monomeric to red aggregated fluorescence.

Mitochondrial membrane potential was assessed by flow cytometry using a fluorogenic dye, JC-10 (Abcam; Cambridge, MA). Treated cells were loaded with JC-10 dye according to the manufacturer's instructions with modifications: spent medium was aspirated and complete medium added to scrape cells. JC-10 solution was added at equal volume and incubated in the dark at 37°C for 15 minutes prior to analysis. For the positive control, cells were incubated with FCCP (carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone), preceding the JC-10 solution. Monomeric (green) and J-aggregate (red) fluorescence were measured using the FL1 and FL2 channels, respectively, and analyzed following compensation for spectral overlap.