Detecting Autophagy and Autophagy Flux in Chronic Myeloid Leukemia Cells Using a Cyto-ID Fluorescence Spectrophotometric Assay

  • Sujuan Guo
  • Kevin J. Pridham
  • Zhi ShengEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1465)


Autophagy is a catabolic process whereby cellular components are degraded to fuel cells for longer survival during stress. Hence, autophagy plays a vital role in determining cell fate and is central for homeostasis and pathogenesis of many human diseases including chronic myeloid leukemia (CML). It has been well established that autophagy is important for the leukemogenesis as well as drug resistance in CML. Thus, autophagy is an intriguing therapeutic target. However, current approaches that detect autophagy lack reliability and often fail to provide quantitative measurements. To overcome this hurdle and facilitate the development of autophagy-related therapies, we have recently developed an autophagy assay termed as the Cyto-ID fluorescence spectrophotometric assay. This method uses a cationic fluorescence dye, Cyto-ID, which specifically labels autophagic compartments and is detected by a spectrophotometer to permit a large-scale and quantitative analysis. As such, it allows rapid, reliable, and quantitative detection of autophagy and estimation of autophagy flux. In this chapter, we further provide technical details of this method and step-by-step protocols for measuring autophagy or autophagy flux in CML cell lines as well as primary hematopoietic cells.

Key words

Autophagy Chronic myeloid leukemia Cyto-ID fluorescence spectrophotometric assay Cyto-ID autophagy assay 



We thank Yanping Liang and Susan Murphy in assisting us with our experiments. This work was supported by the start-up funds from the Virginia Tech Carilion Research Institute to Z.S.


  1. 1.
    Parzych KR, Klionsky DJ (2014) An overview of autophagy: morphology, mechanism, and regulation. Antioxid Redox Signal 20:460–473CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Kroemer G (2015) Autophagy: a druggable process that is deregulated in aging and human disease. J Clin Invest 125:1–4CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Jiang P, Mizushima N (2014) Autophagy and human diseases. Cell Res 24:69–79CrossRefPubMedGoogle Scholar
  4. 4.
    Green DR, Levine B (2014) To be or not to be? How selective autophagy and cell death govern cell fate. Cell 157:65–75CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Choi AM, Ryter SW, Levine B (2013) Autophagy in human health and disease. N Engl J Med 368:651–662CrossRefPubMedGoogle Scholar
  6. 6.
    Mathew R, White E (2011) Autophagy in tumorigenesis and energy metabolism: friend by day, foe by night. Curr Opin Genet Dev 21:113–119CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Rubinsztein DC, Codogno P, Levine B (2012) Autophagy modulation as a potential therapeutic target for diverse diseases. Nat Rev Drug Discov 11:709–730CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Nowell PCHD (1960) Chromosome studies on normal and leukemic human leukocytes. J Natl Cancer Inst 25:85–109PubMedGoogle Scholar
  9. 9.
    Rowley JD (1973) Letter: a new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature 243:290–293CrossRefPubMedGoogle Scholar
  10. 10.
    Shtivelman E, Lifshitz B, Gale RP, Canaani E (1985) Fused transcript of abl and bcr genes in chronic myelogenous leukaemia. Nature 315:550–554CrossRefPubMedGoogle Scholar
  11. 11.
    Bartram CR, de Klein A, Hagemeijer A, van Agthoven T, Geurts van Kessel A, Bootsma D et al (1983) Translocation of c-ab1 oncogene correlates with the presence of a Philadelphia chromosome in chronic myelocytic leukaemia. Nature 306:277–280CrossRefPubMedGoogle Scholar
  12. 12.
    Groffen J, Stephenson JR, Heisterkamp N, de Klein A, Bartram CR, Grosveld G (1984) Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell 36:93–99CrossRefPubMedGoogle Scholar
  13. 13.
    Zhang H, Li S (2013) Molecular mechanisms for survival regulation of chronic myeloid leukemia stem cells. Protein Cell 4:186–196CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Sinclair A, Latif AL, Holyoake TL (2013) Targeting survival pathways in chronic myeloid leukaemia stem cells. Br J Pharmacol 169:1693–707CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, Zimmermann J, Lydon NB (1996) Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 2:561–566CrossRefPubMedGoogle Scholar
  16. 16.
    Quentmeier H, Eberth S, Romani J, Zaborski M, Drexler HG (2011) BCR-ABL1-independent PI3Kinase activation causing imatinib-resistance. J Hematol Oncol 4:6CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Vakana E, Sassano A, Platanias LC (2010) Induction of autophagy by dual mTORC1-mTORC2 inhibition in BCR-ABL-expressing leukemic cells. Autophagy 6:966–967CrossRefPubMedGoogle Scholar
  18. 18.
    Osborn M, Hughes T (2010) Managing imatinib resistance in chronic myeloid leukaemia. Curr Opin Hematol 17:97–103CrossRefPubMedGoogle Scholar
  19. 19.
    Volpe G, Panuzzo C, Ulisciani S, Cilloni D (2009) Imatinib resistance in CML. Cancer Lett 274:1–9CrossRefPubMedGoogle Scholar
  20. 20.
    Sheng Z, Ma L, Sun JE, Zhu LJ, Green MR (2011) BCR-ABL suppresses autophagy through ATF5-mediated regulation of mTOR transcription. Blood 118:2840–2848CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Mortensen M, Soilleux EJ, Djordjevic G, Tripp R, Lutteropp M, Sadighi-Akha E, Stranks AJ, Glanville J, Knight S, Jacobsen SE, Kranc KR, Simon AK (2011) The autophagy protein Atg7 is essential for hematopoietic stem cell maintenance. J Exp Med 208:455–467CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Helgason GV, Karvela M, Holyoake TL (2011) Kill one bird with two stones: potential efficacy of BCR-ABL and autophagy inhibition in CML. Blood 118:2035–2043CrossRefPubMedGoogle Scholar
  23. 23.
    Donato NJ (2011) Bcr-Abl adds another twist to cell fate. Blood 118:2646–2647CrossRefPubMedGoogle Scholar
  24. 24.
    Crowley LC, Elzinga BM, O’Sullivan GC, McKenna SL (2011) Autophagy induction by Bcr-Abl-expressing cells facilitates their recovery from a targeted or nontargeted treatment. Am J Hematol 86:38–47CrossRefPubMedGoogle Scholar
  25. 25.
    Altman BJ, Jacobs SR, Mason EF, Michalek RD, MacIntyre AN, Coloff JL, Ilkayeva O, Jia W, He YW, Rathmell JC (2011) Autophagy is essential to suppress cell stress and to allow BCR-Abl-mediated leukemogenesis. Oncogene 30:1855–1867CrossRefPubMedGoogle Scholar
  26. 26.
    Puissant A, Robert G, Auberger P (2010) Targeting autophagy to fight hematopoietic malignancies. Cell Cycle 9:3470–3478CrossRefPubMedGoogle Scholar
  27. 27.
    Salomoni P, Calabretta B (2009) Targeted therapies and autophagy: new insights from chronic myeloid leukemia. Autophagy 5:1050–1051CrossRefPubMedGoogle Scholar
  28. 28.
    Bellodi C, Lidonnici MR, Hamilton A, Helgason GV, Soliera AR, Ronchetti M, Galavotti S, Young KW, Selmi T, Yacobi R, Van Etten RA, Donato N, Hunter A, Dinsdale D, Tirrò E, Vigneri P, Nicotera P, Dyer MJ, Holyoake T, Salomoni P, Calabretta B (2009) Targeting autophagy potentiates tyrosine kinase inhibitor-induced cell death in Philadelphia chromosome-positive cells, including primary CML stem cells. J Clin Invest 119:1109–1123CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Guo S, Liang Y, Murphy SF, Huang A, Shen H, Kelly DF, Sobrado P, Sheng Z (2015) A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications. Autophagy 11:560–572CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Chan LL, 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 8:1371–1382CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K et al (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8:445–544CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140:313–326CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Gottlieb RA, Andres AM, Sin J, Taylor DP (2015) Untangling autophagy measurements: all fluxed up. Circ Res 116:504–514CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Trout JJ, Stauber WT, Schottelius BA (1981) Increased autophagy in chloroquine-treated tonic and phasic muscles: an alternative view. Tissue Cell 13:393–401CrossRefPubMedGoogle Scholar
  35. 35.
    Sheng Z, Wang SZ, Green MR (2009) Transcription and signalling pathways involved in BCR-ABL-mediated misregulation of 24p3 and 24p3R. Embo J 28:866–876CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Sheng Z, Li L, Zhu LJ, Smith TW, Demers A, Ross AH, Moser RP, Green MR (2010) A genome-wide RNA interference screen reveals an essential CREB3L2-ATF5-MCL1 survival pathway in malignant glioma with therapeutic implications. Nat Med 16:671–677CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Virginia Tech Carilion Research InstituteRoanokeUSA
  2. 2.Graduate Program in Translational Biology, Medicine, and HealthVirginia TechBlacksburgUSA
  3. 3.Department of Biological Sciences and PathobiologyVirginia-Maryland College of Veterinary Medicine, Virginia TechBlacksburgUSA
  4. 4.Department of Internal MedicineVirginia Tech Carilion School of MedicineRoanokeUSA
  5. 5.Faculty of Health ScienceVirginia TechBlacksburgUSA

Personalised recommendations