Detecting Autophagy and Autophagy Flux in Chronic Myeloid Leukemia Cells Using a Cyto-ID Fluorescence Spectrophotometric Assay
Detecting Autophagy and Autophagy Flux in Chronic Myeloid Leukemia Cells Using a Cyto-ID Fluorescence Spectrophotometric Assay
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DOI：
10.1007/978-1-4939-4011-0_9
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发表时间：
2016-01-01
期刊：
影响因子：
--
通讯作者：
Sheng, Zhi
中科院分区：
文献类型：
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作者：
Guo, Sujuan;Pridham, Kevin J.;Sheng, Zhi
自噬是一种分解代谢过程，其中细胞组分被降解为燃料电池，以便在应激期间存活更长时间。因此，自噬在决定细胞命运中起着至关重要的作用，并且是许多人类疾病（包括慢性髓性白血病（CML））的稳态和发病机制的中心。自噬在CML的白血病发生和耐药中起重要作用。因此，自噬是一个有趣的治疗靶点。然而，目前检测自噬的方法缺乏可靠性，并且通常无法提供定量测量。为了克服这一障碍，促进自噬相关疗法的发展，我们最近开发了一种自噬测定法，称为Cyto-ID荧光分光光度测定法。该方法使用阳离子荧光染料Cyto-ID，其特异性地标记自噬区室，并通过分光光度计检测以允许大规模和定量分析。因此，它允许快速，可靠和定量检测自噬和估计自噬通量。在本章中，我们进一步提供了这种方法的技术细节和一步一步的协议，用于测量自噬或自噬流量在CML细胞系以及原代造血细胞。
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.