Monitoring Autophagy using MDC Staining

Autophagy was detected by staining autophagic vacuoles with monodansylcadaverine (MDC) as previously described [9 (link)]. HCT116 cells (7 × 10³ cells/well) were plated in 96-well plates and treated for the indicated times. After treatments, cells were stained with 0.05 mM MDC in PBS at 37 °C for 10 min in the dark. Cells were then washed with PBS and analyzed under a Leica DMR fluorescence microscope (Wetzlar, Germany) equipped with a 4′,6-diamidino-2-phenylindole (DAPI) filter system. Pictures were acquired by a computer imaging system (Leica DC300F camera) and three different visual fields were examined for each condition.