Single molecule fluorescence correlation spectroscopy of single apoptotic cells using a red-fluorescent caspase probe.
Single molecule fluorescence correlation spectroscopy of single apoptotic cells using a red-fluorescent caspase probe.
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DOI：
10.1039/c2an16173g
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发表时间：
2012-07-07
期刊：
影响因子：
--
通讯作者：
Pappas D
中科院分区：
文献类型：
--
作者：
Dong M;Martinez MM;Mayer MF;Pappas D
单细胞中单分子的检测使得能够以高灵敏度和高时间分辨率进行生化分析。本研究应用单分子荧光相关光谱技术（FCS）对单个活细胞凋亡进行了检测。使用一种新的红色荧光探针测定半胱天冬酶活性，该探针避免了绿色荧光探针和细胞自发荧光的光谱重叠。这种新的探针，2SPBO-Casp，通过将水溶性尼罗蓝衍生物（2SBPO）偶联到天冬氨酸残基上而合成。在凋亡诱导和半胱天冬酶活化后，游离2SBPO染料显示在探针切割后在细胞内积累。在我们实验室先前的工作中，使用基于罗丹明110的探针在诱导后45分钟检测单个凋亡细胞中的单分子荧光。然而，需要进行显著的统计分析以排除假阳性。2SBPO-Casp的使用克服了自发荧光问题，并提供了稳定的荧光信号。在我们的单分子FCS测量中，基于它们的相关系数值（R2）确定拉莫斯细胞凋亡。含有R2 ≥ 0.65的细胞被鉴定为高度相关，因此确定为凋亡。通过这种方式鉴定的单个凋亡细胞早在诱导后30分钟就被鉴定，并且凋亡细胞的数量在第3小时达到峰值，这与其他技术一致。使用单分子技术和一种新的凋亡探针，时间动力学阐明了更好的灵敏度和分辨率比以前的研究。
The detection of single molecules in single cells has enabled biochemical analyses to be conducted with high sensitivity and high temporal resolution. In this work, detection of apoptosis was studied by single molecule fluorescence correlation spectroscopy (FCS) in single living cells. Caspase activity was assayed using a new red fluorogenic probe that avoids the spectral overlap of green fluorescent probes and cell autofluorescence. This new probe, 2SPBO-Casp, was synthesized by coupling a water-soluble Nile Blue derivative (2SBPO) to an aspartic acid residue. Upon apoptosis induction and caspase activation, free 2SBPO dye is shown to accumulate inside the cell after probe cleavage. In previous work in our lab, single molecule fluorescence in single apoptotic cells was detected 45 minutes after induction using a rhodamine 110 based probe. However, significant statistical analysis was needed to exclude false positives. The use of 2SBPO-Casp overcomes the autofluorescence problem and offers a steady fluorescence signal. In our single molecule FCS measurements, Ramos cells were determined apoptotic on the basis of their correlation coefficient value (R2). Cells that contain an R2 ≥ 0.65 were identified as highly correlated and therefore determined to be apoptotic. Single apoptotic cells identified by this manner were identified as early as 30 minutes after induction and the number of apoptotic cells reached a peak value at the 3rd hour, which is consistent with other techniques. Using single molecule techniques and a new apoptosis probe, the temporal dynamics were elucidated with better sensitivity and resolution than in previous studies.
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DOI：
10.1039/c0an00845a
发表时间：
2011-09-07
期刊：
The Analyst
影响因子：
--
作者：
Khanal G;Chung K;Solis-Wever X;Johnson B;Pappas D
通讯作者：
Pappas D
影响因子：
2.1
作者：
Ho, NH;Weissleder, R;Tung, CS
通讯作者：
Tung, CS
影响因子：
4.3
作者：
Reif, Randall D.;Aguas, Charmaine;Pappas, Dimitri
通讯作者：
Pappas, Dimitri
微芯片电泳-激光诱导荧光同时测定凋亡细胞线粒体区室中谷胱甘肽和过氧化氢的有效方法
影响因子：
7.4
作者：
Chen, Zhenzhen;Li, Qingling;Tang, Bo
通讯作者：
Tang, Bo
迈向治疗效果的早期评估：超灵敏检测凋亡细胞的电化学平台
影响因子：
7.4
作者：
Zhang, Jing-Jing;Zheng, Ting-Ting;Zhu, Jun-Jie
通讯作者：
Zhu, Jun-Jie