Measuring autophagy: a study of tools and methods for application and suitability in any cell type

Authors

• AW Gee
• SS Kurapaty
• RR Calvo
• J Marugan
• M Ferrer

Abstract

Autophagy is a process by which cells can regulate their metabolism in times of starvation. It is also used to recycle damaged proteins and other cellular macromolecules. A growing number of human illnesses, including cancers and neurodegenerative diseases, have autophagy misregulation as either a component or a cause. We were therefore interested in assessing methods of analyzing autophagy in any cell, including in primary patient samples. Multiple publicly available dyes have been tested for ability to selectively stain autophagosomes, as marked by LC3, or lysosomes, as marked by LAMP-1. Additionally, a tandem-tagged LC3-GFP-RFP reporter construct was assessed. The tandem tag takes advantage of GFP degradation in the lysosome. Early autophagosomes, the LC3-GFP-RFP protein fluoresces both green and red, whereas the GFP signal is degraded in later autophagosomes as the pH decreases, so the vesicles become red-fluorescent only. This construct can therefore be used to track autophagic flux. Autophagy was induced with several methods and in multiple cell lines to map differences in mechanism of action. Both high-content imaging and flow cytometry were used to capture readouts. We here report our findings on the tools most flexible for use in any system.

Scientific Focus Area: Cell Biology

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