诱导和检测哺乳动物细胞培养中的细胞坏死性死亡。

Induction and Detection of Necroptotic Cell Death in Mammalian Cell Culture.

机构信息

The Hormel Institute, University of Minnesota, Austin, MN, USA.

Masonic Cancer Center, Minneapolis, MN, USA.

出版信息

Methods Mol Biol. 2021;2255:119-134. doi: 10.1007/978-1-0716-1162-3_11.

DOI:10.1007/978-1-0716-1162-3_11

PMID:34033099

Abstract

The study of necroptosis is a rapidly growing field in current research of cell death mechanisms and cancer treatment strategies. While apoptotic cells can be reliably identified via annexin V assay, necroptosis is not associated with exposure of easily detectable markers. The most reliable way to identify necroptotic events is immunochemical detection of active phosphorylated RIPK1, RIPK3, and MLKL proteins facilitating necroptosis execution. This chapter describes a detailed protocol on necroptosis induction in human colon adenocarcinoma HT-29 cells, preparation of various positive and negative controls, detection of necroptosis mediator proteins via Western Blot analysis, and interpretation of results. This protocol allows reliable and specific detection of necroptosis in cell culture or tissue samples, and it provides a well-established model suitable for detailed studies of necroptosis molecular mechanisms in vitro.

摘要

细胞坏死的研究是当前细胞死亡机制和癌症治疗策略研究中一个快速发展的领域。虽然凋亡细胞可以通过 Annexin V 检测可靠地识别，但坏死并不与易于检测的标记物的暴露相关联。识别细胞坏死的最可靠方法是通过免疫化学检测活性磷酸化的 RIPK1、RIPK3 和 MLKL 蛋白，这些蛋白有助于细胞坏死的执行。本章描述了在人结肠腺癌 HT-29 细胞中诱导细胞坏死的详细方案，包括各种阳性和阴性对照的制备、通过 Western Blot 分析检测细胞坏死介体蛋白，以及结果的解释。该方案允许在细胞培养物或组织样本中可靠和特异性地检测细胞坏死，并提供了一个经过良好验证的模型，适用于体外研究细胞坏死分子机制的详细研究。