Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
Autophagy. 2010 Jul;6(5):634-41. doi: 10.4161/auto.6.5.12112. Epub 2010 Jul 1.
Autophagy is a cellular degradation process with an increasingly recognised importance in many biological pathways such as nutrient sensing, stress responses and development. We present a straightforward assay for autophagy which combines the sensitivity of the EGFP-LC3 reporter protein with the throughput capacity and quantitative power of flow cytometry. Because saponin extraction is specific for the non-autophagosome associated EGFP-LC3-I form of the protein, flow cytometry can be used to measure total fluorescence of saponin extracted HOS-EGFP-LC3 cells as a measure of the levels of autophagosome associated EGFP-LC3-II. Combined with inhibitors of degradation, we have adapted this assay to differentiate between constitutive and induced autophagy and to quantify the changes in flux of the system. Moreover, using direct antibody staining for the endogenous LC3 protein, we have extended this assay to the detection of autophagosome formation in non-transfected cells.
自噬是一种细胞降解过程，在许多生物学途径中具有越来越重要的作用，如营养感应、应激反应和发育。我们提出了一种简单的自噬测定方法，该方法将 EGFP-LC3 报告蛋白的灵敏度与流式细胞术的高通量能力和定量能力相结合。由于皂苷提取是针对非自噬体相关的 EGFP-LC3-I 形式的蛋白质特异性的，因此可以使用流式细胞术来测量皂苷提取的 HOS-EGFP-LC3 细胞的总荧光强度，作为自噬体相关的 EGFP-LC3-II 水平的测量。结合降解抑制剂，我们已经将该测定方法进行了改编，以区分组成性和诱导性自噬，并定量该系统通量的变化。此外，使用针对内源性 LC3 蛋白的直接抗体染色，我们已经将该测定方法扩展到检测非转染细胞中自噬体的形成。
