Gump Jacob M, Thorburn Andrew
Department of Pharmacology; University of Colorado Anschutz Medical Campus; Aurora, CO USA.
Autophagy. 2014 Jul;10(7):1327-34. doi: 10.4161/auto.29394. Epub 2014 Jun 5.
We detail here a protocol using tandem-tagged mCherry-EGFP-LC3 (C-G-LC3) to quantify autophagic flux in single cells by ratiometric flow cytometry and to isolate subpopulations of cells based on their relative levels of autophagic flux. This robust and sensitive method measures autophagic flux rather than autophagosome number and is an important addition to the autophagy researcher's array of tools for measuring autophagy. Two crucial steps in this protocol are i) generate cells constitutively expressing C-G-LC3 with low to medium fluorescence and low fluorescence variability, and ii) correctly set up gates and voltage/gain on a properly equipped flow cytometer. We have used this method to measure autophagic flux in a variety of cell types and experimental systems using many different autophagy stimuli. On a sorting flow cytometer, this technique can be used to isolate cells with different levels of basal autophagic flux, or cells with variable induction of flux in response to a given stimulus for further analysis or experimentation. We have also combined quantification of autophagic flux with methods to measure apoptosis and cell surface proteins, demonstrating the usefulness of this protocol in combination with other flow cytometry labels and markers.
我们在此详细介绍一种使用串联标记的mCherry-EGFP-LC3（C-G-LC3）的方法，通过比率流式细胞术对单细胞中的自噬通量进行定量，并根据细胞的自噬通量相对水平分离细胞亚群。这种强大且灵敏的方法测量的是自噬通量而非自噬体数量，是自噬研究人员用于测量自噬的工具阵列中的一项重要补充。该方法的两个关键步骤是：i）生成组成型表达C-G-LC3且荧光强度低至中等、荧光变异性低的细胞；ii）在配备适当的流式细胞仪上正确设置门控和电压/增益。我们已使用此方法在多种细胞类型和实验系统中，利用许多不同的自噬刺激来测量自噬通量。在分选流式细胞仪上，该技术可用于分离具有不同基础自噬通量水平的细胞，或对给定刺激有不同通量诱导的细胞，以进行进一步分析或实验。我们还将自噬通量的定量与测量细胞凋亡和细胞表面蛋白的方法相结合，证明了该方法与其他流式细胞术标记和标志物联合使用的实用性。
