Early detection of apoptosis in living cells by fluorescence correlation spectroscopy
Early detection of apoptosis in living cells by fluorescence correlation spectroscopy
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DOI：
10.1007/s00216-009-3298-3
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发表时间：
2010-02-01
影响因子：
4.3
通讯作者：
Pappas, Dimitri
中科院分区：
文献类型：
--
作者：
Martinez, Michelle M.;Reif, Randall D.;Pappas, Dimitri
通过半胱天冬酶活性的凋亡细胞的早期检测被证明具有快速的响应时间。荧光相关光谱法（FCS）是用来识别裂解的荧光探针的基础上Jurkat细胞中的罗丹明110的荧光的存在。FCS曲线显示出显着不同的自发荧光（非凋亡）的细胞，而细胞裂解探针显示扩散和分子亮度特性的罗丹明110。使用FCS测量，基于自相关荧光、平均分子亮度（eta）和分子停留时间（tau（D））的存在将细胞鉴定为凋亡的。早在诱导后45分钟就检测到以这种方式鉴定的凋亡细胞。不像其他方法具有类似的识别时间，如蛋白质印迹和电子显微镜，细胞保持活力，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细胞凋亡。
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.