Kaminskyy Vitaliy O
Department of Physiology and Pharmacology, Biomedicum, Karolinska Institutet, Stockholm, Sweden.
Methods Mol Biol. 2022;2445:65-74. doi: 10.1007/978-1-0716-2071-7_5.
Autophagy is deregulated in cancer cells and often activated as a cellular stress response to anticancer therapies. Flow cytometry-based assays enable detection and quantification of various cellular markers in live or fixed cells. Here, a flow cytometry-based assay to characterize autophagy across the cell cycle is described. This method is based on selective plasma membrane permeabilization with digitonin and extraction of membrane-unbound LC3 protein followed by staining of the autophagosome-bound LC3 protein with antibody and labeling of DNA with propidium iodide. Staining with the LC3 antibody described here can be also combined with the staining of other cellular markers, allowing to quantitatively assess autophagy in relation to different cellular processes by flow cytometry.
自噬在癌细胞中失调，并且常作为对抗癌疗法的细胞应激反应而被激活。基于流式细胞术的检测方法能够检测和定量活细胞或固定细胞中的各种细胞标志物。在此，描述了一种基于流式细胞术的检测方法，用于表征整个细胞周期中的自噬。该方法基于用洋地黄皂苷选择性通透质膜并提取膜结合的LC3蛋白，然后用抗体对自噬体结合的LC3蛋白进行染色，并用碘化丙啶标记DNA。此处所述的LC3抗体染色也可与其他细胞标志物的染色相结合，从而通过流式细胞术定量评估自噬与不同细胞过程的关系。
