Abstract
Autophagy is deregulated in cancer cells and often activated as a cellular stress response to anticancer therapies. Flow cytometry–based assays enable detection and quantification of various cellular markers in live or fixed cells. Here, a flow cytometry–based assay to characterize autophagy across the cell cycle is described. This method is based on selective plasma membrane permeabilization with digitonin and extraction of membrane-unbound LC3 protein followed by staining of the autophagosome-bound LC3 protein with antibody and labeling of DNA with propidium iodide. Staining with the LC3 antibody described here can be also combined with the staining of other cellular markers, allowing to quantitatively assess autophagy in relation to different cellular processes by flow cytometry.
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Acknowledgments
I gratefully thank my mentor Professor Boris Zhivotovsky (Karolinska Institutet, Sweden) in whose group we developed the method described here. I also want to thank my current PIs Assoc. Professors Erik Norberg and Helin Norberg (both at the Dept. of Physiology and Pharmacology, Karolinska Institutet, Sweden) for their support in continuation of my research on metabolism and autophagy in their groups. The labs are supported by grants from The Swedish Cancer Society (Cancerfonden), The Swedish Research Council (VR) and The Ragnar Söderberg Foundation.
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Kaminskyy, V.O. (2022). A Quantitative Flow Cytometry–Based Method for Autophagy Detection Across the Cell Cycle. In: Norberg, H., Norberg, E. (eds) Autophagy and Cancer. Methods in Molecular Biology, vol 2445. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2071-7_5
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DOI: https://doi.org/10.1007/978-1-0716-2071-7_5
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