Park Joon Tae, Lee Young-Sam, Park Sang Chul
Division of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Incheon, South Korea.
Well Aging Research Center, DGIST, Daegu, South Korea.
Methods Mol Biol. 2019;1896:149-157. doi: 10.1007/978-1-4939-8931-7_14.
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.
自噬是一种进化上保守的分解代谢过程，有助于在各种应激条件下清除受损的细胞成分。此外，它在维持细胞稳态中发挥着多种生理和病理生理作用。最近，自噬在细胞衰老过程中的关键作用得到了证据支持，这些证据表明通过重新建立自噬可逆转衰老。由于相当多的注意力已转向理解衰老和自噬背后的分子机制，一种在衰老过程中准确量化自噬的方法对于理解其在衰老和衰老相关疾病中的作用至关重要。在本章中，我们描述了使用CYTO-ID绿色染料和DQ™ Red BSA来监测自噬通量，作为一种准确量化自噬活性的方法。该技术依赖于CYTO-ID绿色染料对自噬体染色的特异性以及自淬灭的DQ™ Red BSA蛋白酶底物在酸性区室中的裂解。特别是，在此我们描述了在衰老过程中量化自噬的方案。
