Kubbies M, Schindler D, Hoehn H, Rabinovitch P S
Cell Tissue Kinet. 1985 Sep;18(5):551-62. doi: 10.1111/j.1365-2184.1985.tb00696.x.
Human lymphocyte cell cycle kinetics was studied in parallel in whole blood and in isolated lymphocyte cultures by differential metaphase labelling and by flow cytometry, employing the principle of quenching of Hoechst fluorescence by BrdU substituted DNA. The BrdU-Hoechst flow technique yields information on the kinetics of cell recruitment and cell cycle progression superior to the differential metaphase staining, since it provides data from interphase cells, including cycle compartment durations, non-cycling cell fractions and transition probabilities. The Smith and Martin model, modified to include a fraction of non-cycling cells, yields excellent correspondence to the experimental data. We show that lymphocytes isolated from Ficoll gradients respond to PHA stimulation with a 4-6 hr delay compared to whole blood cultures or to cultures with autologous serum supplementation. A detailed study of the effects of such culture supplements on lag phase duration, cell cycle compartment length, non-cycling cell fractions and transition probabilities illustrates the application and reproducibility of the flow assay. The potential of the method is further documented with two examples showing the dependence of lymphocyte proliferation on donor age and donor genotype.
通过差异中期标记和流式细胞术，利用BrdU取代DNA淬灭Hoechst荧光的原理，在全血和分离的淋巴细胞培养物中并行研究了人类淋巴细胞的细胞周期动力学。BrdU-Hoechst流式技术提供的关于细胞募集和细胞周期进程动力学的信息优于差异中期染色，因为它能提供来自间期细胞的数据，包括周期阶段持续时间、非循环细胞比例和转换概率。修改后的Smith和Martin模型纳入了一部分非循环细胞，与实验数据具有很好的一致性。我们发现，与全血培养物或添加自体血清的培养物相比，从Ficoll梯度分离的淋巴细胞对PHA刺激的反应延迟4-6小时。对这类培养补充剂对延迟期持续时间、细胞周期阶段长度、非循环细胞比例和转换概率影响的详细研究，说明了流式检测的应用和可重复性。该方法的潜力通过两个例子进一步得到证明，这两个例子显示了淋巴细胞增殖对供体年龄和供体基因型的依赖性。
