Quantification of Autophagy During Senescence

  • Joon Tae Park
  • Young-Sam Lee
  • Sang Chul ParkEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1896)


Autophagy constitutes an evolutionarily conserved catabolic process that contributes to the clearance of damaged cellular components in response to a variety of stress conditions. Additionally, it plays a variety of physiological and pathophysiological roles in maintaining cell homeostasis. Recently, the critical role of autophagy during cellular senescence has been supported by evidences demonstrating the reversal of senescence by the reestablishment of autophagy. As considerable attention has been directed toward understanding the molecular mechanisms underlying senescence and autophagy, a method to accurately quantify autophagy during senescence is critical to understand its role in senescence and senescence-related diseases. In this chapter, we describe the use of CYTO-ID® green dye and DQ™ Red BSA to monitor the autophagic flux as an accurate method to quantify autophagic activity. This technique relies on the specificity of CYTO-ID® green dye in staining autophagosome and the cleavage of the self-quenched DQ™ Red BSA protease substrates in an acidic compartment. In particular, herein we describe protocols to quantify autophagy during senescence.

Key words

Autophagy Senescence CYTO-ID® green dye DQ™ Red BSA Autophagic flux 



This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2018R1D1A1B07040293), the DGIST R&D Program of the Ministry of Science, ICT and Technology of KOREA (2017010072 and 2017010115), and Chonnam National University R&D Program Grant for the Research Chair Professor.

Competing financial interests statement

The authors declare no competing financial interests.


  1. 1.
    Mizushima N, Komatsu M (2011) Autophagy: renovation of cells and tissues. Cell 147(4):728–741. Scholar
  2. 2.
    Singh R, Cuervo AM (2011) Autophagy in the cellular energetic balance. Cell Metab 13(5):495–504. Scholar
  3. 3.
    Yang Z, Klionsky DJ (2010) Eaten alive: a history of macroautophagy. Nat Cell Biol 12(9):814–822CrossRefGoogle Scholar
  4. 4.
    Kroemer G, Marino G, Levine B (2010) Autophagy and the integrated stress response. Mol Cell 40(2):280–293. Scholar
  5. 5.
    Hayflick L (1965) The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 37:614–636CrossRefGoogle Scholar
  6. 6.
    Ziegler DV, Wiley CD, Velarde MC (2015) Mitochondrial effectors of cellular senescence: beyond the free radical theory of aging. Aging Cell 14(1):1–7. Scholar
  7. 7.
    Madeo F, Zimmermann A, Maiuri MC, Kroemer G (2015) Essential role for autophagy in life span extension. J Clin Invest 125(1):85–93. Scholar
  8. 8.
    Terman A, Kurz T, Navratil M, Arriaga EA, Brunk UT (2010) Mitochondrial turnover and aging of long-lived postmitotic cells: the mitochondrial–lysosomal axis theory of aging. Antioxid Redox Signal 12(4):503–535. Scholar
  9. 9.
    Kang HT, Lee KB, Kim SY, Choi HR, Park SC (2011) Autophagy impairment induces premature senescence in primary human fibroblasts. PLoS One 6(8):e23367. Scholar
  10. 10.
    Xie Z, Klionsky DJ (2007) Autophagosome formation: core machinery and adaptations. Nat Cell Biol 9(10):1102–1109. Scholar
  11. 11.
    Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H, Levine B (1999) Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 402(6762):672–676. Scholar
  12. 12.
    Menzies FM, Moreau K, Puri C, Renna M, Rubinsztein DC (2012) Measurement of autophagic activity in mammalian cells. Curr Protoc Cell Biol Chapter 15:Unit15.16.
  13. 13.
    Mizushima N, Yoshimorim T, Levine B (2010) Methods in mammalian autophagy research. Cell 140(3):313–326. Scholar
  14. 14.
    Loos B, du Toit A, Hofmeyr JH (2014) Defining and measuring autophagosome flux-concept and reality. Autophagy 10(11):2087–2096. Scholar
  15. 15.
    Yoshii SR, Mizushima N (2017) Monitoring and measuring autophagy. Int J Mol Sci 18(9):1865. Scholar
  16. 16.
    Biederbick A, Kern HF, Elsasser HP (1995) Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol 66(1):3–14PubMedGoogle Scholar
  17. 17.
    Tan Q, Wang H, Hu Y, Hu M, Li X et al (2015) Src/STAT3-dependent heme oxygenase-1 induction mediates chemoresistance of breast cancer cells to doxorubicin by promoting autophagy. Cancer Sci 106(8):1023–1032. Scholar
  18. 18.
    Kozako T, Suzuki T, Yoshimitsu M, Uchida Y, Kuroki A, Aikawa A, Honda S-i, Arima N, Soeda S (2015) Novel small-molecule SIRT1 inhibitors induce cell death in adult T-cell leukaemia cells. Sci Rep 5:11345. Scholar
  19. 19.
    Chan LL-Y, Shen D, Wilkinson AR, Patton W, Lai N, Chan E, Kuksin D, Lin B, Qiu J (2012) A novel image-based cytometry method for autophagy detection in living cells. Autophagy 8(9):1371–1382. Scholar
  20. 20.
    Lee JS, Lee GM (2012) Monitoring of autophagy in Chinese hamster ovary cells using flow cytometry. Methods 56(3):375–382. Scholar
  21. 21.
    Klappan AK, Hones S, Mylonas I, Bruning A (2012) Proteasome inhibition by quercetin triggers macroautophagy and blocks mTOR activity. Histochem Cell Biol 137(1):25–36. Scholar
  22. 22.
    Warenius HM, Kilburn JD, Essex JW, Maurer RI, Blaydes JP, Agarwala U, Seabra LA (2011) Selective anticancer activity of a hexapeptide with sequence homology to a non-kinase domain of Cyclin Dependent Kinase 4. Mol Cancer 10:72. Scholar
  23. 23.
    Decker RS, Fuseler JF (1984) Methylated amino acids and lysosomal function in cultured heart cells. Exp Cell Res 154(1):304–309CrossRefGoogle Scholar
  24. 24.
    Hostetler KY, Reasor M, Yazaki PJ (1985) Chloroquine-induced phospholipid fatty liver. Measurement of drug and lipid concentrations in rat liver lysosomes. J Biol Chem 260(1):215–219PubMedGoogle Scholar
  25. 25.
    Ivy G, Schottler F, Wenzel J, Baudry M, Lynch G (1984) Inhibitors of lysosomal enzymes: accumulation of lipofuscin-like dense bodies in the brain. Science 226(4677):985–987. Scholar
  26. 26.
    Frost LS, Dhingra A, Reyes-Reveles J, Boesze-Battaglia K (2017) Chapter three—the use of DQ-BSA to monitor the turnover of autophagy-associated cargo. In: Galluzzi L, Bravo-San Pedro JM, Kroemer G (eds) Methods in enzymology, vol 587. Academic Press, New York, pp 43–54. Scholar
  27. 27.
    Goeritzer M, Vujic N, Schlager S, Chandak PG, Korbelius M, Gottschalk B, Leopold C, Obrowsky S, Rainer S, Doddapattar P, Aflaki E, Wegscheider M, Sachdev V, Graier WF, Kolb D, Radovic B, Kratky D (2015) Active autophagy but not lipophagy in macrophages with defective lipolysis. Biochim Biophys Acta 1851(10):1304–1316. Scholar
  28. 28.
    Authier F, Posner BI, Bergeron JJ (1996) Endosomal proteolysis of internalized proteins. FEBS Lett 389(1):55–60CrossRefGoogle Scholar
  29. 29.
    Goebeler V, Poeter M, Zeuschner D, Gerke V, Rescher U (2008) Annexin A8 regulates late endosome organization and function. Mol Biol Cell 19(12):5267–5278. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Joon Tae Park
    • 1
  • Young-Sam Lee
    • 2
    • 3
  • Sang Chul Park
    • 2
    • 4
    Email author
  1. 1.Division of Life Sciences, College of Life Sciences and BioengineeringIncheon National UniversityIncheonSouth Korea
  2. 2.Well Aging Research CenterDGISTDaeguSouth Korea
  3. 3.Department of New BiologyDGISTDaeguSouth Korea
  4. 4.The Future Life and Society Research CenterChonnam National UniversityGwangjuSouth Korea

Personalised recommendations