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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
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1465)

Abstract

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 

Notes

Acknowledgment

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.

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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

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