Mdc powder

Autophagy inhibitor 3-MA (Sigma-Aldrich Chemical Company, St Louis, MO, USA) was diluted using sterile PBS. The final concentration of stock solution was adjusted to 40 mM, and the stock solution was sub-packed at 20°C for further usage. MDC powder (Sigma-Aldrich Chemical Company, St Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO) to obtain a final concentration of 0.1 M and then stored at -20°C. The macrophages were seeded into six-well plates, treated with 3-MA and cultured in brand-new culture medium containing 50 μm of MDC at 37°C in 5% CO 2 for 10 min. The solutions were centrifuged at 1000 rpm and resuspended for 10 min. The cells were applied to slide glass with a cover glass, observed under an inverted fluorescence microscope OLYMPUS IX71 (Olympus Corp., Tokyo, Japan), activated by ultraviolet light, and imaged.

To evaluate autophagy, MDC powder was purchased from Sigma Aldrich (Merck KGaA) and dissolved in dimethyl sulfoxide at 0.1 mol/l of the stock concentration; the working concentration was 50 µm/l. The CT26.WT cells (1x10 6 cells/ml) were incubated with the MDC dye for 45 min in the dark at 4˚C and then washed with PBS thrice. Subsequently, they were visualized under a fluorescence microscope (Olympus Corporation). The autophagosomes emitted blue fluorescence and appeared in a punctate pattern in CT26.WT cells.