Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA.
Anal Bioanal Chem. 2010 Feb;396(3):1177-85. doi: 10.1007/s00216-009-3298-3. Epub 2009 Nov 25.
Early detection of apoptotic cells via caspase activity is demonstrated with fast response time. Fluorescence correlation spectroscopy (FCS) is used to identify the presence of a cleaved fluorogenic probe based on the fluorescence of rhodamine 110 in Jurkat cells. FCS curves are shown to be markedly different for autofluorescent (non-apoptotic) cells, whereas cells with cleaved probe showed diffusion and molecular brightness characteristic of rhodamine 110. Using FCS measurements, cells were identified as apoptotic on the basis of the presence of autocorrelated fluorescence, average molecular brightness (eta), and molecular dwell time (tau (D)). Apoptotic cells identified in this manner were detected as early as 45 min after induction. Unlike other methods with similar identification times, such as western blotting and electron microscopy, cells remain viable for further analysis. This multi-parameter approach is rapid, flexible, and does not require transfection of the cells prior to analysis, enabling apoptosis to be identified early in a wide variety of cell types.
通过 caspase 活性对凋亡细胞进行早期检测，其响应时间快。荧光相关光谱（FCS）用于识别基于 Jurkat 细胞中罗丹明 110 的荧光的切割荧光探针的存在。FCS 曲线对于自发荧光（非凋亡）细胞明显不同，而具有切割探针的细胞显示出罗丹明 110 的扩散和分子亮度特征。使用 FCS 测量，根据自相关荧光、平均分子亮度（eta）和分子停留时间（tau（D）），可以鉴定细胞是否发生凋亡。以这种方式鉴定的凋亡细胞在诱导后 45 分钟即可检测到。与其他具有相似鉴定时间的方法（如 Western blot 和电子显微镜）不同，细胞仍然保持活力，可用于进一步分析。这种多参数方法快速、灵活，并且在分析之前不需要转染细胞，从而能够在多种细胞类型中早期识别凋亡。
