Niles Andrew L, Moravec Richard A, Eric Hesselberth P, Scurria Michael A, Daily William J, Riss Terry L
Promega Corp., Madison, WI 53711, USA.
Anal Biochem. 2007 Jul 15;366(2):197-206. doi: 10.1016/j.ab.2007.04.007. Epub 2007 Apr 12.
A method to simultaneously determine the relative numbers of live and dead cells in culture by introducing a combination of two fluorogenic substrates or a fluorogenic and a luminogenic protease substrate into the sample is described. The method is based on detection of differential ubiquitous proteolytic activities associated with intact viable cells and cells that have lost membrane integrity. A cell-permeable peptide aminofluorocoumarin substrate detects protease activity restricted to intact viable cells. Upon cell death, the viable cell protease marker becomes inactive. An impermeable peptide rhodamine 110 (or aminoluciferin) conjugated substrate detects protease activity from nonviable cells that have lost membrane integrity. The multiplex assay can detect 200 dead cells in a population of 10,000 viable cells. The protease substrate reagents do not damage viable cells over the course of the assay, thus the method can be multiplexed further with other assays in a homogeneous format. Ratiometric measurement of viable and dead cells in the same sample provides an internal control that can be used to normalize data from other cell-based assays.
描述了一种通过将两种荧光底物或一种荧光和一种发光蛋白酶底物的组合引入样品中来同时测定培养物中活细胞和死细胞相对数量的方法。该方法基于检测与完整活细胞和失去膜完整性的细胞相关的差异普遍蛋白水解活性。一种可透过细胞的肽氨基荧光香豆素底物检测限于完整活细胞的蛋白酶活性。细胞死亡后，活细胞蛋白酶标记物失活。一种不可透过的肽罗丹明110（或氨基荧光素）偶联底物检测来自失去膜完整性的非活细胞的蛋白酶活性。多重测定可在10,000个活细胞群体中检测到200个死细胞。在测定过程中，蛋白酶底物试剂不会损害活细胞，因此该方法可以与其他测定以均相形式进一步多重化。对同一样品中活细胞和死细胞的比率测量提供了一种内部对照，可用于对来自其他基于细胞的测定的数据进行归一化。
