Evolution of tools and methods for monitoring autophagic flux in mammalian cells

Autophagy is an evolutionarily conserved lysosome-mediated degradation and recycling process, which functions in cellular homeostasis and stress adaptation. The process is highly dynamic and involves autophagosome synthesis, cargo recognition and transport, autophagosome–lysosome fusion, and cargo degradation. The multistep nature of autophagy makes it challenging to quantify, and it is important to consider not only the number of autophagosomes within a cell but also the autophagic degradative activity. The rate at which cargos are recognized, segregated, and degraded through the autophagy pathway is defined as autophagic flux. In practice, methods to measure autophagic flux typically evaluate the lysosome-mediated cargo degradation step by leveraging known autophagy markers such as MAP1LC3B (microtubule-associated proteins 1A/1B light chain 3 beta) or lysosome-dependent fluorescent agents. In this review, we summarize the tools and methods used in mammalian cultured cells pertaining to these two approaches, and highlight innovations that have led to their evolution in recent years. We also discuss the potential limitations of these approaches and recommend using a combination of strategies and multiple different autophagy markers to reliably evaluate autophagic flux in mammalian cells.