Acridine orange assay for autophagy

Autophagy is characterized by formation of acidic vesicular organelles (AVO). To detect AVOs, acridine orange (AO) was used. Acidic compartment causes accumulation of AO, which gives bright red fluorescence upon excitation by 488-nm laser. Measurement of red fluorescence is proportional to increase in AVOs (20).

Cells (2 × 10⁵) were subjected to suspension culture for 48 hours, after which they were trypsinized, counted, and 1 × 10⁵ cells were stained with acridine orange (1 µg/mL) for 15 minutes at 37°C in DMEM + 10% FBS. Post staining, cells were washed thrice in PBS. The cells were analyzed in BD FACS CantoII (Becton & Dickinson) equipped with a blue (488 nm) Coherent Sapphire Solid State laser. Red (564–606 nm) fluorescence emission from 10⁴ cells was measured. Data were analyzed using Summit V5.2.1.12465 software.