Molecular Signaling and Cell Death Unit, VIB Inflammation Research Center, Ghent, Belgium.
Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
Nat Protoc. 2016 Aug;11(8):1444-54. doi: 10.1038/nprot.2016.085. Epub 2016 Jul 14.
Several cell death assays have been developed based on a single biochemical parameter such as caspase activation or plasma membrane permeabilization. Our fluorescent apoptosis/necrosis (FAN) assay directly measures cell death and distinguishes between caspase-dependent apoptosis and caspase-independent necrosis of cells grown in any multiwell plate. Cell death is monitored in standard growth medium as an increase in fluorescence intensity of a cell-impermeable dye (SYTOX Green) after plasma membrane disintegration, whereas apoptosis is detected through caspase-mediated release of a fluorophore from its quencher (DEVD-amc). The assay determines the normalized percentage of dead cells and caspase activation per condition as an end-point measurement or in real time (automated). The protocol can be applied to screen drugs, proteins or siRNAs for interference with cell death while simultaneously detecting cell death modality switching between apoptosis and necrosis. Initial preparation may take up to 5 d, but the typical hands-on time is ∼2 h.
已经开发了几种基于单一生化参数的细胞死亡测定法，例如半胱天冬酶激活或质膜通透性。我们的荧光细胞凋亡/坏死（FAN）测定法直接测量细胞死亡，并区分在任何多孔板中生长的细胞中依赖于半胱天冬酶的凋亡和不依赖于半胱天冬酶的坏死。在标准生长培养基中监测细胞死亡，这是在质膜崩解后细胞不可渗透染料（SYTOX Green）的荧光强度增加的结果，而凋亡则通过半胱天冬酶介导的荧光团从猝灭剂（DEVD-amc）中的释放来检测。该测定法可用于筛选药物、蛋白质或 siRNA，以干扰细胞死亡，同时检测细胞死亡模式在凋亡和坏死之间的切换。初始准备可能需要长达 5 天，但实际操作时间通常约为 2 小时。
