Sauvat Allan, Wang Yidan, Segura Florian, Spaggiari Sabrina, Müller Kevin, Zhou Heng, Galluzzi Lorenzo, Kepp Oliver, Kroemer Guido
Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France.
INSERM, U1138, Paris, France.
Oncotarget. 2015 Apr 20;6(11):9467-75. doi: 10.18632/oncotarget.3266.
Cellular viability is usually determined by measuring the capacity of cells to exclude vital dyes such as 4',6-diamidino-2-phenylindole (DAPI), or by assessing nuclear morphology with chromatinophilic plasma membrane-permeant dyes, such as Hoechst 33342. However, a fraction of cells that exclude DAPI or exhibit normal nuclear morphology have already lost mitochondrial functions and/or manifest massive activation of apoptotic caspases, and hence are irremediably committed to death. Here, we developed a protocol for the simultaneous detection of plasma membrane integrity (based on DAPI) or nuclear morphology (based on Hoechst 33342), mitochondrial functions (based on the mitochondrial transmembrane potential probe DiOC6(3)) and caspase activation (based on YO-PRO®-3, which can enter cells exclusively upon the caspase-mediated activation of pannexin 1 channels). This method, which allows for the precise quantification of dead, dying and healthy cells, can be implemented on epifluorescence microscopy or flow cytometry platforms and is compatible with a robotized, high-throughput workflow.
细胞活力通常通过测量细胞排斥诸如4',6-二脒基-2-苯基吲哚（DAPI）等活性染料的能力来确定，或者通过使用亲染色质的质膜渗透性染料（如Hoechst 33342）评估细胞核形态来确定。然而，一部分排斥DAPI或表现出正常细胞核形态的细胞已经失去了线粒体功能和/或表现出凋亡半胱天冬酶的大量激活，因此不可避免地走向死亡。在此，我们开发了一种方案，用于同时检测质膜完整性（基于DAPI）或细胞核形态（基于Hoechst 33342）、线粒体功能（基于线粒体跨膜电位探针DiOC6(3)）和半胱天冬酶激活（基于YO-PRO®-3，其仅在半胱天冬酶介导的泛连接蛋白1通道激活时才能进入细胞）。这种方法能够精确量化死亡、濒死和健康细胞，可在落射荧光显微镜或流式细胞术平台上实施，并且与自动化的高通量工作流程兼容。
