Eclipse ts200

Acidic intracellular compartments were visualized by AO staining. After 24 h of incubation with various concentrations of CP and CP/MH, HepG2^DR cells were washed with PBS and stained with AO (10 μg/mL) for 15 min at 37 °C. Subsequently, the cells were washed with PBS and visualized for red fluorescence under a fluorescence microscope (Eclipse TS200; Nikon Corp., Tokyo, Japan) at 400× magnification.
To determine the specific location of autophagic vacuoles, HepG2^DR cells were seeded on glass coverslips (SPL, Korea) in 12-well cell-culture plates. Cells were then treated with various concentrations of the corresponding drugs for 24 h at 37 °C. After treatment, cells were fixed with 4% paraformaldehyde in PBS for 10 min. Fluorescence microscopy of cells stained with Cyto-ID Green Detection Reagent (Enzo Life Sciences, Plymouth Meeting, PA, USA) was performed according to the manufacturer’s protocol. In brief, cells were washed with PBS, stained with Cyto-ID in an indicator-free medium supplemented with 5% FBS for 30 min at 37 °C. Cells were counter stained with Hoechst-33342 to stain the nucleus. The coverslips were washed with assay buffer and visualized under a fluorescence microscope (Nikon Eclipse TS200, Nikon Corp., Tokyo, Japan) at 400× magnification.

HepG2^DR cells were treated with the Hoechst 33342 stain according to Singh et al. Briefly, cells were cultured and treated with different concentrations of CP and MH for 24 h at 37 °C, then washed twice with PBS and fixed with cold 4% formaldehyde. The cells were then washed again with PBS and incubated with Hoechst 33342 (1 µg/mL) for 10 min at 37 °C. After washing cells with PBS, fluorescence was detected under a fluorescence microscope (Nikon Eclipse TS200) at 400× magnification.