Autophagy is an intracellular homeostasis pathway in which random cytoplasmic contents are recycled. Autophagy commences with the formation of a phagophore originating from either the lipid layer of the endoplasmic reticulum, the Golgi apparatus, the mitochondria, or the plasma membrane. The phagophore expands and engulfs bits of cytoplasm into a double-membraned vesicle known as an autophagosome. The mature double-membrane autophagosome now merges with the lysosome to form a new vesicle called an autolysosome, where lysosomal acid proteases digest the contents. The resulting molecular building blocks are transported back into the cytoplasm and reused by the cellular machinery.

Furthermore, autophagy can selectively remove protein aggregates and damaged organelles. Various studies have reported selective autophagic degradation of pathogens like bacteria and viruses, macromolecules like lipids and protein aggregates, and organelles such as mitochondria, peroxisomes, lysosomes, and endoplasmic reticulum. As with any other cellular pathway, autophagy is a dynamic process with a flux. The term "autophagic flux" denotes all the steps of the autophagy process, including autophagosome formation, its maturation and fusion with lysosomes, the breakdown of cargo in the lysosome, and the further release of the molecular building blocks into the cytoplasm. Understanding and deciphering autophagic flux by monitoring its dynamics is key to assessing autophagic activity in any biological system.

There is currently no single gold standard methodology for determining autophagic flux that is applicable in all experimental contexts or models. This Methods Collection will describe robust and reproducible methods for detecting non-selective and selective autophagy in humans, mice, and less studied farm animal models.