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bioRxiv · 10.1101/590513

High-throughput quantitative detection of basal autophagy and autophagic flux using image cytometry

Senthilkumar, G.·Skiba, J. H.·Kimple, R.
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Abstract

Quantitative assessment of changes in macro-autophagy is often performed through manual quantification of the number of LC3B foci in immunofluorescence microscopy images. This method is highly laborious, subject to image-field selection and foci-counting bias, and is not sensitive for analyzing changes in basal autophagy. Alternative methods such as flow cytometry and transmission electron microscopy require highly specialized, expensive instruments and time-consuming sample preparation. Immunoblots are prone to exposure-related variations and noise that prevent accurate quantification. We report a high-throughput, inexpensive, reliable, and objective method for studying basal level and flux changes in late-stage autophagy using image cytometry and acridine orange staining.\n\nMethods summaryA high-throughput, inexpensive, reliable, and objective method for studying both basal autophagy and autophagic flux is reported. This approach uses acridine orange staining of late-stage autophagy and image cytometry to quantify autophagy.

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Senthilkumar, G., Skiba, J. H., Kimple, R.. 2019-03-30. High-throughput quantitative detection of basal autophagy and autophagic flux using image cytometry. https://doi.org/10.1101/590513

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