Singh Bindu, Bhaskar Sangeeta
Product Development Cell, National Institute of Immunology, New Delhi, India.
Methods Mol Biol. 2019;2045:245-258. doi: 10.1007/7651_2018_190.
Autophagy is a conserved catabolic process that degrades cytoplasmic constituents in the lysosome and thus contributes to the maintenance of intracellular homeostasis. The process of autophagy has been involved in many physiological and pathological processes. Therefore, there is a developing need to identify, quantify, and manipulate the autophagic process accurately in the cells. As autophagy involves dynamic and complex processes, therefore various approaches are needed to study this process precisely. In this chapter, we have tried to elaborate the approaches and methods to monitor autophagy, with a primary focus on mammalian macroautophagy. Autophagy induction can be detected using Western blotting of LC3 (marker protein for autophagosomes) in which LC3-II levels represent the quantity of autophagosomes formed on induction to a particular stimulus. This can also be confirmed by puncta formation assay using confocal microscopy. Further, the autophagic flux can be examined using bafilomycin A1 as inhibitor of autophagosome-lysosome fusion and acidification of lysosomal compartments, thereby leading to accumulation of autophagosomes which is represented by high LC3-II levels. The autophagolysosomal degradation or proteolysis which is the last step of autophagy can be analyzed by DQ-BSA assay.
自噬是一种保守的分解代谢过程,可在溶酶体中降解细胞质成分,从而有助于维持细胞内稳态。自噬过程涉及许多生理和病理过程。因此,迫切需要在细胞中准确识别、量化和操纵自噬过程。由于自噬涉及动态和复杂的过程,因此需要各种方法来精确研究这一过程。在本章中,我们试图阐述监测自噬的方法,主要侧重于哺乳动物的巨自噬。可以使用LC3(自噬体的标记蛋白)的蛋白质印迹法检测自噬诱导,其中LC3-II水平代表在特定刺激诱导下形成的自噬体数量。这也可以通过共聚焦显微镜的斑点形成试验来证实。此外,可以使用巴弗洛霉素A1作为自噬体-溶酶体融合和溶酶体区室酸化的抑制剂来检测自噬通量,从而导致自噬体积累,这表现为高LC3-II水平。自噬溶酶体降解或蛋白水解是自噬的最后一步,可以通过DQ-牛血清白蛋白试验进行分析。
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