|Autophagic vacuoles were detected by incubating cells with MDC solution (1:1,000 in Cell-Based Assay Buffer, 50 μM) using an Autophagy/Cytotoxicity Dual Staining Kit (Cayman Chemical Co.). Cells were incubated with MDC for 10 min at 37˚C, washed three times with PBS and immediately analyzed under a fluorescence Nikon ECLIPSE E600 microscope (Nikon, Tokyo, Japan) equipped with a filter system (excitation wavelength of 365 nm, emission wavelength of 400 nm). For quantification of MDC-positive staining of cells, bright-field and fluorescence images were merged, 100 cells were counted in three separate fields by using a fluorescence microscope and the proportion of MDC-positive cells was determined. Images were captured with a Nikon DIGITAL SIGHT DS-Ri1 microscope camera (Nikon) and imported into Photoshop.|
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