Cells were cultured in SDCA medium to a density of about 5×107 cells/ml, and autophagy was induced by addition of rapamycin. Cells were harvested, spun at RT with a microcentrifuge, and subjected to fluorescence microscopy using an IX83 inverted system microscope (Olympus) equipped with a UPlanSApo100×/1.40 Oil (Olympus) and a CoolSNAP HQ CCD camera (Nippon Roper). A U-FGFP and U-FMCHE filter sets (Olympus) were used for GFP/mNeonGreen and octadecyl rhodamine B (R18, Invitrogen)/FM4-64 staining visualization, respectively. Images were acquired using the MetaVue imaging software (Molecular Devices). For determination of IM length, fluorescence intensities of mNeonGreen-labeled Atg proteins were measured using the ‘linescan’ function of the MetaView software, and the full width at half maximum was calculated manually.
For R18 staining, cells were stained with 10 μg/ml of R18 (1 mg/ml stock dissolved in dimethyl sulfoxide) for 10 min in nutrient-rich medium at 30°C. After cells were washed three times with fresh medium, rapamycin was added to induce autophagy. FM 4–64 staining was performed as previously described [16 (link)].
For R18 staining, cells were stained with 10 μg/ml of R18 (1 mg/ml stock dissolved in dimethyl sulfoxide) for 10 min in nutrient-rich medium at 30°C. After cells were washed three times with fresh medium, rapamycin was added to induce autophagy. FM 4–64 staining was performed as previously described [16 (link)].