Temporal dynamics of receptor-induced apoptosis in an affinity microdevice
Temporal dynamics of receptor-induced apoptosis in an affinity microdevice
复制标题
DOI：
10.1007/s00216-010-3567-1
复制
发表时间：
2010-08-01
影响因子：
4.3
通讯作者：
Pappas, Dimitri
中科院分区：
文献类型：
--
作者：
Reif, Randall D.;Aguas, Charmaine;Pappas, Dimitri
阐明了 Fas 诱导的细胞凋亡的时间动态。 Jurkat 细胞被捕获在涂有抗 CD95 的微型装置的亲和表面上，CD95 是一种已知可通过外源性（半胱天冬酶 8）途径诱导细胞凋亡的抗体。细胞凋亡诱导的时间由细胞与表面的结合控制。结合后，细胞持续用半胱天冬酶探针 L-双天冬氨酸罗丹明 110 (D(2)R) 染色，并通过光学显微镜监测细胞的荧光 6 小时。这种方法使每个细胞的时间动态正常化，因为结合事件也是细胞凋亡的开始。除了提供随时间变化的凋亡细胞数量之外，还可以监测单个细胞的荧光，提供有关每个细胞中 caspase 活性时间的信息。还测量了每个细胞中 D(2)R 的半胱天冬酶裂解率，并显示给定群体中的细胞之间具有良好的一致性。半胱天冬酶抑制剂 z-VAD-FMK 对半胱天冬酶活性时间的影响也得到了研究，结果表明它可以显着减缓细胞凋亡过程。将来，其他半胱天冬酶探针可用于提供有关半胱天冬酶激活的时间动态的附加信息。荧光相关光谱等其他技术可以与这些方法结合使用，以提供更快的时间响应，并有助于阐明细胞凋亡过程的异质性。
The temporal dynamics of Fas-induced apoptosis is elucidated. Jurkat cells are captured on the affinity surface of a microdevice coated with anti-CD95, an antibody known to induce apoptosis in cells via the extrinsic (caspase 8) pathway. The timing of apoptosis induction is controlled by the binding of the cells to the surface. Once bound, the cells are continuously stained with the caspase probe, L-bisaspartic acid rhodamine 110 (D(2)R), and the fluorescence of the cells was monitored for 6 h by light microscopy. This approach normalizes the temporal dynamics for each cell, as the binding event is also the start of apoptosis. In addition to providing the number of apoptotic cells over time, the fluorescence of individual cells can be monitored, providing information about the timing of caspase activity in each cell. The rate of caspase cleavage of D(2)R in each cell is also measured and shows good agreement between the cells in a given population. The effects of the caspase inhibitor, z-VAD-FMK, on the timing of caspase activity are also investigated and are shown to dramatically slow the apoptotic process. In the future, other caspase probes could be used to provide additional information about the temporal dynamics of caspase activation. Additional techniques, such as fluorescence correlation spectroscopy, can be coupled to these methods to provide faster temporal response and help to elucidate the heterogeneity of the apoptosis process.