Abstract
Autophagy activation is characterized by the accumulation of double-membrane autophagic vesicles (autophagosomes) in the cytoplasm. The mere presence of autophagosomes in the cytoplasm does not necessarily indicate an increased level of autophagy, since the blockade of any step downstream of autophagosome formation increases the number of autophagosomes. Therefore, quantitative methods for the detection of cytoplasmic protein turnover should be employed in addition to autophagosome monitoring, to verify increased levels of autophagy. At the present, multiple methods are available for the quantification of autophagy and the identification of autophagosomes. Here, we detail the in vitro methods currently available to detect autophagic cell death by flow cytometry analysis.
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
Similar content being viewed by others
References
Klionsky DJ, Emr SD (2000) Autophagy as a regulated pathway of cellular degradation. Science 290:1717–1721
Mizushima N, Ohsumi Y, Yoshimori T (2002) Autophagosome formation in mammalian cells. Cell Struct Funct 27:421–429
Wang CW, Klionsky DJ (2003) The molecular mechanism of autophagy. Mol Med 9:65–76
Kihara A, Kabeya Y, Ohsumi Y, Yoshimori T (2001) Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network. EMBORep 2:330–335
Pattingre S, Tassa A, Qu X (2005) Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 122:927–939
Mizushima N (2004) Methods for monitoring autophagy. Int J Biochem Cell Biol 36:2491–2502
Kuma A, Matsui M, Mizushima N (2007) LC3, an autophagosome marker, can be incorporated into protein aggregates independent of autophagy: caution in the interpretation of LC3 localization. Autophagy 3:323–328
Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140(3):313–326
Tasdemir E, Galluzzi L, Maiuri MC, Criollo A, Vitale I, Hangen E, Modjtahedi N, Kroemer G (2008) Methods for assessing autophagy and autophagic cell death. Methods Mol Biol 445:29–76
Shvets E, Elazar Z (2009) Flow cytometric analysis of autophagy in living mammaliancells. Methods Enzymol 452:131–141
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 19:5720–5728
Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo AA, Adachi H, Adams CM et al (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12(1):1–222
Grimaldi A, Santini D, Zappavigna S, Lombardi A, Misso G, Boccellino M, Desiderio V, Vitiello PP, Di Lorenzo G, Zoccoli A, Pantano F, Caraglia M (2015) Antagonistic effects of chloroquine on autophagy occurrence potentiate the anticancer effects of everolimus on renal cancer cells. Cancer Biol Ther 16(4):567–579
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Zappavigna, S., Lombardi, A., Misso, G., Grimaldi, A., Caraglia, M. (2017). Measurement of Autophagy by Flow Cytometry. In: Di Nardo, P., Dhingra, S., Singla, D. (eds) Adult Stem Cells. Methods in Molecular Biology, vol 1553. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6756-8_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6756-8_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6754-4
Online ISBN: 978-1-4939-6756-8
eBook Packages: Springer Protocols