Aghamohammadi S Z, Savage J R
Division of Cell and Molecular Biology, MRC Radiobiology Unit, Didcot, Oxon, Great Britain.
Mutat Res. 1991 Nov;251(1):133-41. doi: 10.1016/0027-5107(91)90223-b.
We describe a pulse BrdU double-labelling method which we have used for a cohort-analysis study of a conventional adaptive response experiment in stimulated human lymphocytes. This method allows us to identify the cycle position occupied by each scored metaphase at the time of primer and at the time of challenge. Responses in cells at defined developmental stages can then be compared irrespective of mitotic perturbation. Using a primer X-ray dose of 1.0 cGy, and a challenge dose of 1.5 Gy 6 h later, we found a transitory depression in frequency of total aberrations present in cells sampled at 6 h but not at 9 h after challenge. When defined cohorts were summed for both sample times, no significant adaptive response was observed. A repeat experiment where terminal BrdU labelling was used after the challenge failed to provide any positive response at either 6 or 9 h. The technique presented not only ensures that similar cell mixtures from a heterogeneous, asynchronous population can be scored and compared, but it serves also to demonstrate the kinetic complexity of stimulated lymphocyte cultures.
我们描述了一种脉冲BrdU双标记方法，该方法已用于对受刺激的人淋巴细胞进行的传统适应性反应实验的队列分析研究。这种方法使我们能够确定每个计分中期在引物处理时和激发时所占据的细胞周期位置。然后，可以比较处于特定发育阶段的细胞中的反应，而无需考虑有丝分裂扰动。使用1.0 cGy的引物X射线剂量，并在6小时后使用1.5 Gy的激发剂量，我们发现在激发后6小时而非9小时采样的细胞中，总畸变频率出现短暂下降。当将两个采样时间的特定队列相加时，未观察到明显的适应性反应。在激发后使用终末BrdU标记的重复实验在6小时或9小时均未提供任何阳性反应。所提出的技术不仅确保可以对来自异质、异步群体的相似细胞混合物进行计分和比较，而且还用于证明受刺激淋巴细胞培养物的动力学复杂性。
