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首页> 外文期刊>Biological chemistry >iBH3: simple, fixable BH3 profiling to determine apoptotic priming in primary tissue by flow cytometry
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iBH3: simple, fixable BH3 profiling to determine apoptotic priming in primary tissue by flow cytometry

机译：iBH3：简单，可固定的BH3分析，可通过流式细胞仪确定原发组织中的细胞凋亡

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摘要

Dysregulation of the mitochondrial pathway of apoptosis, controlled by the BCL-2 family of proteins, leads to disease states including cancer. Rapid analysis of a cell's dependency on the BCL-2 family of proteins is hindered by the complex interactions of more than a dozen proteins. Transcript or even protein levels are therefore generally insufficient to predict a cell's response to perturbations like chemotherapy. Previously, we developed the JC-1 BH3 method to provide a same day functional assay to assess a cell's propensity to undergo apoptosis and demonstrated its utility in predicting response to chemotherapy. We have now improved upon these methods to create a robust assay amenable to high throughput platforms using cytochrome c retention in formaldehyde fixed cells to remove the time sensitivity of JC-1 potential measurements. BH3 profiling by intracellular staining (iBH3) is suitable for 96- and 384-well formats, and can be used to directly screen candidate BH3-mimetic compounds for activity. When used as the final component of dynamic BH3 profiling (DBP), which uses a drug pretreatment prior to iBH3 to assess the change in profile due to treatment, it can predict the response of cells to chemotherapy days before they show signs of death.
机译：由蛋白质的BCL-2家族控制的线粒体细胞凋亡途径的失调导致包括癌症在内的疾病状态。十几种蛋白质之间的复杂相互作用阻碍了细胞对BCL-2蛋白质家族依赖性的快速分析。因此，转录本甚至蛋白质水平通常不足以预测细胞对诸如化学疗法之类的扰动的反应。以前，我们开发了JC-1 BH3方法来提供当日功能测定，以评估细胞发生凋亡的倾向，并证明其可用于预测对化学疗法的反应。现在，我们对这些方法进行了改进，以创建一种适用于高通量平台的强大测定方法，该方法使用了将细胞色素c保留在甲醛固定细胞中，从而消除了JC-1电位测量的时间敏感性。通过细胞内染色（iBH3）进行的BH3分析适用于96和384孔形式，可用于直接筛选候选BH3模拟化合物的活性。当用作动态BH3轮廓分析（DBP）的最终组件时，该功能在iBH3之前使用药物预处理来评估由于治疗引起的轮廓变化，它可以预测细胞对化学疗法的反应，直到它们显示出死亡迹象。

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