Make each cell count: How to assess autophagy using flow cytometry

Kristy R. Howell, PhD

The cellular recycling process known as autophagy may be induced by a variety of conditions including reduced nutrient availability, serum starvation and pharmacological agents (e.g., Rapamycin (mTOR)). Upon autophagy induction, a process of increased sequestration of dysfunctional cellular components into autophagosomes ensues. The autophagosome fuses with lysosomes, forming an autolysosome, whose components are subsequently degraded via a regulated signaling pathway. To fully understand the mechanisms behind autophagy, an assessment of cell cycle phase and organelle function using advanced technological methods such as flow cytometry is warranted.

LC3B and some of its caveats in monitoring autophagy

Although light chain 3 (LC3) proteins are in both the cytoplasm and nucleus, LC3 is known as an autophagosome marker that is often measured to determine autophagic activity. Particularly, the key biological marker of autophagy is the microtubule associated protein LC3-II (LC3B). LC3B is recruited to the autophagosome’s inner membrane as a cleaved, lipidated form of LC3-I ^1,2. The autolysosome’s content, including LC3B, is then degraded and recycled for cell repair and regeneration.

Various detection methods provide means to track LC3 protein levels, including use of western blot to monitor autophagy signaling. The sole biochemical use of LC3B expression as a marker of autophagy, however, becomes difficult to interpret given this protein correlates only with the number of autophagosomes, and not how the cells are undergoing this process. Another critical point is that LC3B expression increases through the cell cycle in a normal state² in addition to being an autophagy substrate, preventing interpretation of whether autophagy is being induced or blocked. It is important, therefore, to consider other methods for measuring autophagy that may provide better strategies for quantifiable and functional data.

Measuring autophagic flux via flow cytometry

Flow cytometry allows for the assessment of a variety of dynamic cellular properties including cell morphology, cell cycle distribution, organelle function and specific organelle changes (e.g., size, organelle specific autophagy), which are critical for the analysis of autophagic activity².

A commonly used approach to monitor and analyze autophagy flux is via blockade of autophagic activity. For example, chloroquine can be used for its unique properties of inhibiting lysosome acidification, thereby preventing lysosome-autophagosome fusion and the subsequent degradation of metabolic stress products². Chloroquine thus creates a higher autophagic substrate-gradient for quantification by flow cytometry. One way to maximize outcomes of flow cytometry is through the use of tandem-fluorescently tagged LC3; LC3B-GFP/mCherry³ to determine autophagic flux as introduced in a previous blog: Best way to quantitatively measure Autophagic Flux. The benefit of this approach is the ability to monitor changes in the fluorescence of tandem-fluorescently tagged LC3 as it transitions from autophagosome to an autolysosome destined for degradation.

Physiological functions require lysosomes to have a lower pH to perform degradation. Thus, LC3B within the autolysosome would be cleared of the pH sensitive activated fluorophore (GFP), only showing the signal of the pH stable fluorophore (mCherry), while intact LC3B would be positive for both fluorophores. An autophagosome, therefore, would be positive for both the GFP and mCherry, while an autolysosome will only show red fluorescence due to the elimination of the GFP in the acidic environment. Autophagic flux, or LC3 recycling, is determined by a ratio of the stable fluorophore-mCherry signal and the GFP signal.

Another method to monitor autophagy flux is multispectral imaging flow cytometry (MIFC), which provides high throughput capabilities, multiplexing potential and can acquire images of every cell for precise quantitative analyses⁴. MIFC allows the visualization of fluorescently labeled LC3 puncta and/or the co-localization of fluorescently labeled LC3 and lysosomal markers. There are four experimental designs that have used MIFC to examine various readouts for autophagy: 1. Bright Detail Intensity, 2. Spot Count-measure LC3 puncta accumulation, 3. Bright Detail Similarity-visualizes autolysosome formation, and 4. Bright Detail Similarity/Spot Count combination method-assesses autophagosome and lysosome co-localization in addition to LC3 puncta⁴. While all methods are appropriate, solely using LC3 puncta counts or autolysosome formation as a readout for autophagy, particularly after stressful conditions or pharmacological agents that can induce the process, may lead to misrepresentation of cellular functions that could be better depicted by assessing autophagic product turnover.

The benefit of using combined methods such as these would reveal a multitude of phenotypic changes that can provide researchers with functional data in their models. The Bright Detail Similarity/Spot Count combination method, therefore, may be best for measuring autophagic flux due to its ability to quantify lysosome-autophagosome fusion in addition to puncta, which can then help one visualize the intracellular morphological changes occurring in real time. Given that changes in autophagy are observed with both aging and various diseases⁷, the effective measurement and determination of the cellular consequences of autophagy would provide knowledge for an array of disease models and age-related disorders. Previous work has validated use of LC3 quantification by flow cytometry^5,6 using NBP2-46892 and NB100-2220. Further validation of these antibodies using MIFC, however, would significantly advance the autophagy field.