A tool for enhancement and scoring of DNA repair foci.
A tool for enhancement and scoring of DNA repair foci.
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DNA 修复焦点增强和评分的工具。
DOI：
10.1002/cyto.a.20653
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Dynlacht,JosephR
中科院分区：
文献类型：
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作者：
Gerashchenko,BogdanI;Dynlacht,JosephR
DNA双链断裂（DSBs）发生后，Mre11 DNA修复复合体的Mre11和Rad50蛋白聚集在DSBs位点，形成离散的核病灶。对含有Mre11/Rad50蛋白的病灶进行精确评分取决于对这些病灶的检测，其中一些病灶的荧光染色强度与未被纳入病灶的剩余Mre11和Rad50蛋白的荧光染色强度过于接近。用不同剂量的X射线（0 ~ 12 Gy）照射指数生长的人类U‐1黑色素瘤细胞，诱导其形成修复病灶。照射4小时后，使用双色免疫荧光染色技术同时标记细胞中的Mre11和Rad50蛋白。采用激光扫描共聚焦显微镜采集随机选取的细胞核的合成图像。对Mre11和Rad50蛋白的等强荧光信号进行强度相关分析（ICA），得到具有相关像元的区域。ICA可以增强对Mre11/Rad50病灶（“隐藏”病灶）的低水平荧光检测，仅对一种蛋白质成像几乎无法检测到这种病灶。例如，在6 Gy‐辐照的细胞核中，仅成像一种蛋白（Mre11或Rad50）显示9个病灶，而用ICA成像两种蛋白显示11个病灶。ICA允许评估不同剂量X射线照射下细胞核灶的剂量依赖性，病灶形成增加至6 Gy剂量。我们利用Mre11和Rad50蛋白的双色免疫荧光染色和这两种靶蛋白的ICA积累的数据为提高DNA修复病灶的检测和准确性提供了基础。©2008国际细胞术进步学会
Upon induction of DNA double‐strand breaks (DSBs), Mre11 and Rad50 proteins of the Mre11 DNA repair complex accumulate at the sites of DSBs and form discrete nuclear foci. Precision in scoring of Mre11/Rad50‐containing foci depends upon detection of those foci, some of which have a fluorescence staining intensity that is too close to the fluorescence staining intensity of the remaining Mre11 and Rad50 proteins that have not been incorporated into foci. Human U‐1 melanoma cells in exponential growth were irradiated with various doses of X‐rays (0‐12 Gy) to induce the formation of repair foci. Four hours after irradiation, cells were simultaneously labeled for Mre11 and Rad50 proteins, using a two‐color immunofluorescence staining technique. Laser scanning confocal microscopy was used to collect the composite images of randomly selected cell nuclei. Intensity correlation analysis (ICA) of equally intense fluorescence signals from Mre11 and Rad50 proteins was performed to obtain the regions with correlated pixels. ICA permitted enhanced detection of low level fluorescence of Mre11/Rad50 foci (“hidden” foci) that can be barely detected upon imaging of only one protein. For example, while imaging of only one protein (either Mre11 or Rad50) in the nucleus of a 6 Gy‐irradiated cell revealed 9 foci, imaging of two proteins with ICA revealed 11 foci. ICA permitted an evaluation of the dose dependence of nuclear foci in cells irradiated with various doses of X‐rays, with focus formation increasing up to a dose of 6 Gy. Our data accumulated using two‐color immunofluorescence staining of Mre11 and Rad50 proteins and ICA of these two target proteins provide a basis for enhanced detection and accuracy in the scoring of DNA repair foci. © 2008 International Society for Advancement of Cytometry