Ratiometric analysis of Acridine Orange staining in the study of acidic organelles and autophagy

Acridine orange (AO) is a cell permeable green fluorophore that can be protonated and trapped in Acidic Vesicular Organelles (AVOs). Its metachromatic shift to red fluorescence is concentration-dependent and, therefore, AO fluoresces red in AVOs, such as autolysosomes. This makes AO staining a quick, accessible and reliable method to assess the volume of AVOs, which increases upon autophagy induction. Here we describe a ratiometric analysis of autophagy using AO, considering the red-to-green fluorescence intensity ratio (R/GFIR) to quantify flow cytometry and fluorescence microscopy data of AO-stained cells. This method measured with accuracy the increase in autophagy induced by starvation or rapamycin and the blockage by bafilomycin A1 or the knockdown of Beclin1 or ATG7. Results obtained with AO, considering R/GFIR, correlated with LC3-I to LC3-II conversion, SQSTM1 degradation and GFP-LC3 puncta formation, thus validating this assay to be used as an initial and quantitative method for evaluating the late step of autophagy in individual cells, complementing other methods.