Quantitative determination of autophagy flux by probes.

Abstract

Macroautophagy is an intracellular degradation system in which autophagosomes and autolysosomes degrade the contents they contain in order to realize cell homeostasis and organelle renewal. Measuring autophagy activity and autophagic flux is very important for studying the role of autophagy, but accurate measurement of autophagic flux is quite complicated. Here, we use the GFP-mRFP-LC3 tandem probe to evaluate the cell autophagic flux. GFP is more sensitive to acidic environment and can be degraded in autolysosome due to the acidic environment. On the contrary, mRFP can be stably present in autolysosome due to its better tolerance to PH reduction. Hence, autophagic flux can be evaluated by calculating the ratio of GFP/RFP signal values. In addition, using this probe, we can more accurately measure the basal autophagic flux and induced autophagic flux in cells or animals. Summarily, the GFP-mRFP-LC3 tandem probe is a simple quantitative method to evaluate autophagic flux of cells and even whole organism.