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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Rybstein MD, Bravo-San Pedro JM, Kroemer G, Galluzzi L (2018) The autophagic network and cancer. Nat Cell Biol 20(3):243–251. https://doi.org/10.1038/s41556-018-0042-2
Levine B, Kroemer G (2019) Biological functions of autophagy genes: a disease perspective. Cell 176(1–2):11–42. https://doi.org/10.1016/j.cell.2018.09.048
Kimmelman AC, White E (2017) Autophagy and tumor metabolism. Cell Metab 25(5):1037–1043. https://doi.org/10.1016/j.cmet.2017.04.004
Zhang J, Tripathi DN, Jing J, Alexander A, Kim J, Powell RT, Dere R, Tait-Mulder J, Lee JH, Paull TT, Pandita RK, Charaka VK, Pandita TK, Kastan MB, Walker CL (2015) ATM functions at the peroxisome to induce pexophagy in response to ROS. Nat Cell Biol 17(10):1259–1269. https://doi.org/10.1038/ncb3230
Sumpter R Jr, Sirasanagandla S, Fernandez AF, Wei Y, Dong X, Franco L, Zou Z, Marchal C, Lee MY, Clapp DW, Hanenberg H, Levine B (2016) Fanconi anemia proteins function in mitophagy and immunity. Cell 165(4):867–881. https://doi.org/10.1016/j.cell.2016.04.006
Wang Y, Zhang N, Zhang L, Li R, Fu W, Ma K, Li X, Wang L, Wang J, Zhang H, Gu W, Zhu WG, Zhao Y (2016) Autophagy regulates chromatin ubiquitination in DNA damage response through elimination of SQSTM1/p62. Mol Cell 63(1):34–48. https://doi.org/10.1016/j.molcel.2016.05.027
Klionsky DJ et al (2021) Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)(1). Autophagy 17(1):1–382. https://doi.org/10.1080/15548627.2020.1797280
Tsuboyama K, Koyama-Honda I, Sakamaki Y, Koike M, Morishita H, Mizushima N (2016) The ATG conjugation systems are important for degradation of the inner autophagosomal membrane. Science 354(6315):1036–1041. https://doi.org/10.1126/science.aaf6136
Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19(21):5720–5728. https://doi.org/10.1093/emboj/19.21.5720
Tanida I, Minematsu-Ikeguchi N, Ueno T, Kominami E (2005) Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy. Autophagy 1(2):84–91. https://doi.org/10.4161/auto.1.2.1697
Shvets E, Fass E, Elazar Z (2008) Utilizing flow cytometry to monitor autophagy in living mammalian cells. Autophagy 4(5):621–628. https://doi.org/10.4161/auto.5939
Kaminskyy V, Abdi A, Zhivotovsky B (2011) A quantitative assay for the monitoring of autophagosome accumulation in different phases of the cell cycle. Autophagy 7(1):83–90. https://doi.org/10.4161/auto.7.1.13893
Eng KE, Panas MD, Karlsson Hedestam GB, McInerney GM (2010) A novel quantitative flow cytometry-based assay for autophagy. Autophagy 6(5):634–641. https://doi.org/10.4161/auto.6.5.12112
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2071-7_5
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2070-0
Online ISBN: 978-1-0716-2071-7
eBook Packages: Springer Protocols