一种用于实时监测小鼠胚胎成纤维细胞饥饿诱导自噬的高通量方案。
A high-throughput protocol for monitoring starvation-induced autophagy in real time in mouse embryonic fibroblasts.
机构信息
Molecular, Cellular and Developmental Biology Department (MCD), Centre de Biologie Intégrative (CBI), University of Toulouse, CNRS, UPS, 31062 Toulouse, France.
Laboratory of Molecular Cancer Biology, VIB Center for Cancer Biology, KU Leuven, Leuven, Belgium.
出版信息
STAR Protoc. 2021 Nov 17;2(4):100966. doi: 10.1016/j.xpro.2021.100966. eCollection 2021 Dec 17.
Autophagy measurement has been challenging due to the transient nature of autophagy vesicles, in which degradation of cargo occurs. Here, we present a protocol to monitor starvation-induced autophagy using a live high-throughput microscopy system in a fast and automated manner without the need for sample preparation. We provide a detailed protocol describing the generation of turboGFP-LC3B expressing mouse embryonic fibroblasts (MEFs), the measurement of autophagy over time and the analysis of data. For complete details on the use and execution of this protocol, please refer to Nowosad et al. (2020, 2021).
自噬的测量具有挑战性，因为自噬囊泡的寿命很短，其内容物会被降解。在这里，我们提出了一种使用活细胞高通量显微镜系统以快速自动化的方式监测饥饿诱导自噬的方法，而无需进行样品制备。我们提供了一个详细的方案，描述了表达 turboGFP-LC3B 的小鼠胚胎成纤维细胞（MEFs）的生成、随时间测量自噬以及数据分析的过程。如需了解此方案的详细使用和执行情况，请参考 Nowosad 等人（2020 年，2021 年）的文献。
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