Sorting cells for basal and induced autophagic flux by quantitative ratiometric flow cytometry

Abstract

We detail here a protocol using tandem-tagged mCherry-EGFP-LC3 (C-G-LC3) to quantify autophagic flux in single cells by ratiometric flow cytometry and to isolate subpopulations of cells based on their relative levels of autophagic flux. This robust and sensitive method measures autophagic flux rather than autophagosome number and is an important addition to the autophagy researcher’s array of tools for measuring autophagy. Two crucial steps in this protocol are i) generate cells constitutively expressing C-G-LC3 with low to medium fluorescence and low fluorescence variability, and ii) correctly set up gates and voltage/gain on a properly equipped flow cytometer. We have used this method to measure autophagic flux in a variety of cell types and experimental systems using many different autophagy stimuli. On a sorting flow cytometer, this technique can be used to isolate cells with different levels of basal autophagic flux, or cells with variable induction of flux in response to a given stimulus for further analysis or experimentation. We have also combined quantification of autophagic flux with methods to measure apoptosis and cell surface proteins, demonstrating the usefulness of this protocol in combination with other flow cytometry labels and markers.

Keywords: :

• autophagy
• flow cytometry
• autophagic flux
• GFP-LC3
• cell sorting
• quantification

Disclosure of Potential Conflicts of Interest

The authors report no potential conflicts of interest.

Acknowledgments

We are grateful to K Helm, L Acosta and C Childs of the University of Colorado Cancer Center Flow Cytometry Core for their invaluable guidance and assistance. This work was supported by National Institutes of Health grants R01 CA111421 and CA150925 (AT) and Shared Resources supported by P30 CA46934. JMG was previously supported by 5T32 CA82086-10 (UCAMC Department of Pediatrics) and the American Cancer Society.