Traffic light assay

Technical illustration of a method used for monitoring autophagy in live cells, using fluorescence microscopy. In the assay, the molecule of interest is labelled with two fluorescent probes, a green and a red (GFP and mCherry, respectively).

This type of labelling approach is a general method that enables monitoring the intracellular dynamics of the tagged molecule, but what is particularly useful with respect to autophagy, is that when the molecule of interest is located within an autolysosome, the GFP signal gets quenched due to the low pH inside lysosomes. Therefore, when the molecule of interest is accumulating in autophagosomes, puncta with both a red and green signal is observed (“yellow” signal), whereas accumulation in autolysosomes result in red puncta. Counting the number of yellow and red puncta enables monitoring of the autophagic flux.