Aten J A, Bakker P J, Stap J, Boschman G A, Veenhof C H
Laboratory for Radiobiology, University of Amsterdam, The Netherlands.
Histochem J. 1992 May;24(5):251-9. doi: 10.1007/BF01046839.
A procedure was developed that very effectively distinguishes between IdUrd and CldUrd incorporated in the DNA of cell nuclei and chromosomes. For double staining we used the rat anti-BrdUrd monoclonal antibody from Sera-lab that binds specifically to CldUrd and BrdUrd but not to IdUrd, in combination with the mouse anti-BrdUrd monoclonal antibody from Becton Dickinson. This antibody binds to all three halogenated deoxyuridines, but when the nuclei are washed in TRIS buffer with a high salt concentration the antibodies linked to CldUrd-labelled DNA are removed. When analysing the effect of the deoxyuridines on the cell cycle we found that the growth kinetics of Chinese hamster cells were not changed by adding IdUrd or CldUrd for 30 min at a concentration of 10 microM, whereas adequate double labelling required only 2 min pulses. The effectiveness of the technique was demonstrated in two model experiments. The first test concerned the assessment of cell recruitment in the central areas of slow-growing clones, after addition of fresh medium. The second experiment focussed on the spatial resolution of the method. Double-labelled metaphase chromosomes showed interspersed green and red replication bands with a spacing corresponding with medium resolution Giemsa banding patterns.
我们开发了一种程序，能非常有效地区分掺入细胞核和染色体DNA中的碘脱氧尿苷（IdUrd）和氯脱氧尿苷（CldUrd）。进行双重染色时，我们使用了Sera-lab公司的大鼠抗溴脱氧尿苷单克隆抗体，它能特异性结合CldUrd和BrdUrd，但不结合IdUrd，并将其与Becton Dickinson公司的小鼠抗溴脱氧尿苷单克隆抗体联合使用。这种抗体能结合所有三种卤代脱氧尿苷，但当细胞核在高盐浓度的TRIS缓冲液中洗涤时，与CldUrd标记的DNA相连的抗体就会被去除。在分析脱氧尿苷对细胞周期的影响时，我们发现，在10微摩尔浓度下添加IdUrd或CldUrd 30分钟，中国仓鼠细胞的生长动力学没有改变，而进行充分的双重标记仅需2分钟的脉冲。该技术的有效性在两个模型实验中得到了证明。第一个测试是在添加新鲜培养基后，评估生长缓慢的克隆中心区域的细胞募集情况。第二个实验聚焦于该方法的空间分辨率。双重标记的中期染色体显示出绿色和红色复制带相间分布，其间距与中等分辨率的吉姆萨带型相对应。
