Autophagy is an intracellular lysosomal degradation pathway by which cytoplasmic cargoes are removed to maintain cellular homeostasis. Monitoring autophagy flux is crucial to understand the autophagy process and its biological significance. However, assays to measure autophagy flux are either complex, low throughput or not sensitive enough for reliable quantitative results. Recently, ER-phagy has emerged as a physiologically relevant pathway to maintain ER homeostasis but the process is poorly understood, highlighting the need for tools to monitor ER-phagy flux. In this study, we validate the use of the signal-retaining autophagy indicator (SRAI), a fixable fluorescent probe recently generated and described to detect mitophagy, as a versatile, sensitive and convenient probe for monitoring ER-phagy. This includes the study of either general selective degradation of the endoplasmic reticulum (ER-phagy) or individual forms of ER-phagy involving specific cargo receptors (e.g., FAM134B, FAM134C, TEX264 and CCPG1). Crucially, we present a detailed protocol for the quantification of autophagic flux using automated microscopy and high throughput analysis. Overall, this probe provides a reliable and convenient tool for the measurement of ER-phagy.
Keywords: ER; ER-phagy; SRAI; autophagy; autophagy flux; fluorescent protein; lysosome; reporter.