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. 2014 Jul;10(7):1327-34.
doi: 10.4161/auto.29394. Epub 2014 Jun 5.

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

Jacob M Gump  1 , Andrew Thorburn  1
Affiliations

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

Jacob M Gump et al. Autophagy. 2014 Jul.

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: GFP-LC3; autophagic flux; autophagy; cell sorting; flow cytometry; quantification.

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Figures

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Figure 1. mCherry-GFP-LC3 reporter cells enable flow cytometric quantification and sorting of cells based on autophagic flux. (A) Quantification of autophagic flux by flow cytometry using reporter cells stably expressing mCherry-GFP-LC3. Using the ratio of mCherry to GFP, autophagic flux can be easily and consistently measured in individual cells. (B) Overview of protocol for sorting cells by autophagic flux. First, cells must be generated which stably express mCherry-GFP-LC3 (C-G-LC3). Second, individual clones (or flow sorted pools) must be characterized based on their expression of mCherry and GFP and ability to induce flux in response to an autophagic stimulus. Third, cells treated with a given stimulus can be flow sorted based on their relative ratios of mCherry and GFP fluorescence into populations of high and low autophagic flux.
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Figure 2. Quantification of autophagic flux by flow cytometry. (A) Example of flow-cytometry data illustrating flow cytometer setup for measuring autophagic flux. Scatter and singlet gates should be used to eliminate debris, dead cells, and mitotic cells. Voltages and gain on GFP and mCherry detectors are set empirically to allow both negative and positive control (starvation) plots to fit the mCherry/GFP ratio histogram. (B) Comparison of this protocol to other previously published methods for flow cytometric quantification of autophagy. C-G-LC3 reporter cells were starved for 4 h in EBSS followed by flow cytometry with or without extraction of cytosolic (nonlipidated) LC3 by saponin. (C) Concurrent measurement of autophagic flux and apoptosis using the established flow cytometry markers ANXA5/annexinV and DAPI. C-G-LC3 reporter cells were treated with an apoptotic stimulus (Fas ligand) for 4 h and stained with ANXA5/annexinV-APC and DAPI followed by flow cytometry.
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