Measuring autophagic flux by assessing LC3, p62 and LAMP1 co-localization using imaging flow cytometry (TECH2P.908)

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome; during starvation it is a survival mechanism that reallocates nutrients from unnecessary processes to more vital processes in the cell. In autophagy, cytoplasmic LC3 protein is processed and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to cause the breakdown of the autophagosome vesicle and its contents. The ubiquitin-associated protein p62 which binds to LC3 can also be used to measure autophagy. p62 is an important marker for the induction of autophagy, clearance of protein aggregates and the inhibition of autophagy. In addition, p62 positive cytoplasmic inclusions have been found in several neurodegenerative diseases. Immunofluorescence microscopy has been used to visually identify LC3 puntca, p62 and/or lysosomes on a per-cell basis; however, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, we employed the ImageStreamX Mark II imaging cytometry platform. We collected large numbers of cell images to assess autophagy and co-localization of LC3, p62 and the lysosomal marker LAMP1 in an objective, quantitative, and statistically robust manner. Defects in autophagic flux have been linked to immune-mediated diseases; we believe this technique may bring insight to these diseases.