Hoechst 33342 nuclear blue fluorescent probes

The intracellular ROS levels were marked using a DCFH-DA fluorescent probe (Beyotime, Shanghai, China). In brief, HepD cells at a density of 2 × 10⁵ per well were grown in a laser confocal glass-bottom culture dish and incubated for 24 h. HepD cells were treated with the Im and La mixture (0, 20, 50, 75, and 100 μg/mL, respectively) for 24 h. Then, the cells were stained with 5 μL DCFH-DA green fluorescent probes and 0.2 μL Hoechst 33342 nuclear blue fluorescent probes (Beyotime, Shanghai, China) at 37 °C for 30 min in the dark. Finally, the cells were washed with PBS twice, and the intracellular ROS levels were imaged using a fluorescence microscope (Olympus Corporation, Tokyo, Japan). The fluorescence intensity was analyzed with ImageJ software.

The cytoplasmic Ca²⁺ concentration was related to ER stress and mitochondria apoptosis, and the increase of Ca²⁺ occurred at the early and late stages of the apoptotic pathway [36 (link)] To determine the Ca²⁺ levels in cells, HepD cells were seeded in a laser confocal glass-bottom culture dish at a density of 2 × 10⁵ and incubated for 24 h up to 80% confluence. Then, the cells were treated with the Im and La mixture (0, 20, 50, 75, and 100 μg/mL, respectively) for 24 h. Next, the cells were incubated with 5 μM Fluo-4 AM fluorescent probes (Beyotime, Shanghai, China), 0.2 μL Hoechst 33342 nuclear blue fluorescent probes, and 50 nM Mito-Tracker Red CMXRos probes at 37 °C for 30 min. The intracellular Ca²⁺ levels and mitochondrial apoptosis were imaged using the fluorescence microscope (Olympus Corporation, Tokyo, Japan). The fluorescence intensity was analyzed with ImageJ software.