Mdc fluorescent staining kit

The autophagosomes were detected using MDC fluorescent staining kit (C3018S, Beyotime, Shanghai, China), following the manufacturer’s instructions. Cells were cultured in 24-well plates and treated with a 1:1000 dilution of MDC for 30 min at 37 °C in the absence of light. Then, the cells underwent three consecutive washes with assay buffer. The degree of autophagy was assessed utilizing a fluorescence microscope (Leica, MHG, Germany).

MDC fluorescent staining kit (Beyotime, Shanghai, China) was used to detect the level of autophagy occurring in cells. After different treatments, cells were harvested and incubated with 1 ml MDC staining solution for 30 min at 37°C in the dark. After washing three times with washing solution, cells were resuspended in the collection buffer, and assayed under the flow cytometry (Beckman coulter, Brea, CA, United States).

The bladder cancer cells were cultured on the glass coverslips for 24 h in 6-well plate, followed by the experimental treatment for 4 or 8 h. The autophagosomes were visualized using MDC fluorescent staining kit (Beyotime, Shanghai, China). In brief, when the treatment was completed, the adherent cells were washed with PBS for twice, incubated with 1 ml MDC staining solution in 37°C for 30 mines. Then, the MDC staining solution was removed, and the cells were washed with assay buffer for 3 times, and the slides were mounted with mounting solution containing DAPI. The images were captured under a fluorescence microscope, which were displayed on the computer and analyzed using ImageJ software package.