Pei Jingqi, Panina Svetlana B, Kirienko Natalia V
Department of BioSciences, Rice University.
Department of BioSciences, Rice University;
J Vis Exp. 2020 May 12(159). doi: 10.3791/61295.
The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.
线粒体对致癌转化的作用是一个备受关注且正在积极研究的课题。随着癌症代谢领域变得愈发复杂，使用各种能造成线粒体损伤的化合物（即所谓的线粒体靶向剂）来靶向线粒体的目标正变得相当流行。不幸的是，许多现有的细胞毒性检测方法，比如基于四唑盐或刃天青的检测方法，其检测过程需要功能性线粒体酶。靶向线粒体的化合物所造成的损伤常常会影响这些检测方法的准确性。在此，我们描述了一种基于两种荧光染料进行差异染色的改良方案，其中一种染料可穿透细胞（Hoechst 33342），另一种则不能（碘化丙啶）。染色差异使得活细胞和死细胞得以区分。该检测方法适用于自动显微镜检查和图像分析，这提高了通量并减少了偏差。这也使得该检测方法能够以高通量方式用于96孔板，成为药物研发工作的一个可行选择，特别是当所研究的药物具有一定程度的线粒体毒性时。重要的是，与其他方法相比，通过Hoechst/PI染色检测获得的结果在台盼蓝排斥实验结果之间以及生物重复样本之间都显示出更高的一致性。
