Quantifying autophagy: Measuring LC3 puncta and autolysosome formation in cells using multispectral imaging flow cytometry

•A review of the predominant methods to measure autophagy via MIFC.•Bright Detail Intensity and Spot Count work for measuring LC3 puncta accumulation.•Bright Detail Similarity (BDS) can measure the formation of the autolysosome.•Spot Count vs BDS provides the most comprehensive information on autophagy.•There is not a single correct method for measuring autophagy using MIFC. The use of multispectral imaging flow cytometry has been gaining popularity due to its quantitative power, high throughput capabilities, multiplexing potential and its ability to acquire images of every cell. Autophagy is a process in which dysfunctional organelles and cellular components that accumulate during growth and differentiation are degraded via the lysosome and recycled. During autophagy, cytoplasmic LC3 is processed and recruited to the autophagosomal membranes; the autophagosome then fuses with the lysosome to form the autolysosome. Therefore, cells undergoing autophagy can be identified by visualizing fluorescently labeled LC3 puncta and/or the co-localization of fluorescently labeled LC3 and lysosomal markers. Multispectral imaging flow cytometry is able to collect imagery of large numbers of cells and assess autophagy in an objective, quantitative, and statistically robust manner. This review will examine the four predominant methods that have been used to measure autophagy via multispectral imaging flow cytometry.