Esteban-Martínez Lorena, Boya Patricia
Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Methods. 2015 Mar;75:79-86. doi: 10.1016/j.ymeth.2015.01.008. Epub 2015 Jan 30.
Autophagy is a highly dynamic process that mediates the degradation of cellular constituents inside lysosomes. It is characterized by the formation of autophagosomes, double membrane organelles that engulf cytosolic components and organelles and degrade their contents upon fusion with lysosomes. Upregulation of autophagy in response to specific stimuli can be determined by evaluating autophagic flux. This is achieved by comparing the number of autophagosomes in the absence and presence of lysosomal inhibitors. While the determination of autophagic flux in isolated cells is well-documented, few studies have described its determination in tissues or in vivo. Here, we describe the evaluation of autophagic flux both in vivo and ex vivo in several tissues, after treatment with lysosomal inhibitors and exposure to classical autophagy-inducing stimuli. This method uses LC3 lipidation, as determined by Western blot, fluorescence microscopy and flow cytometry. Our findings demonstrate that autophagic flux can be evaluated in vivo and ex vivo in several tissues.
自噬是一个高度动态的过程，介导溶酶体内细胞成分的降解。其特征是自噬体的形成，自噬体是一种双膜细胞器，可吞噬胞质成分和细胞器，并在与溶酶体融合后降解其内容物。通过评估自噬通量可以确定对特定刺激的自噬上调。这是通过比较在不存在和存在溶酶体抑制剂的情况下自噬体的数量来实现的。虽然在分离细胞中自噬通量的测定已有充分记录，但很少有研究描述其在组织或体内的测定。在这里，我们描述了在用溶酶体抑制剂处理并暴露于经典自噬诱导刺激后，在几种组织中体内和体外自噬通量的评估。该方法使用通过蛋白质免疫印迹、荧光显微镜和流式细胞术测定的LC3脂化。我们的研究结果表明，可以在几种组织中体内和体外评估自噬通量。
