LysoTracker Staining to Aid in Monitoring Autophagy in <i>Drosophila</i>

LysoTracker is an acidotropic dye that stains cellular acidic compartments, including lysosomes and autolysosomes. LysoTracker has been used to detect autophagy-associated lysosomal activity in Drosophila tissues including the fat body, midgut, salivary gland and ovary, as well as in Drosophila cell culture. A low level of LysoTracker staining can be observed under resting or well-fed conditions, and is increased following autophagic stimuli such as starvation. Here we provide a protocol for examining LysoTracker levels in Drosophila cultured cells in vitro using standard cell culture methods and flow cytometry. We also describe how to examine LysoTracker in fixed and nonfixed Drosophila tissues using fluorescence microscopy. Ovary tissue is used as an example. Dissections of ovaries are relatively easy to perform, given their large size.