Zhao R, Natarajan A, Srienc F
Department of Chemical Engineering and Materials Science, Biological Process Technology Institute, University of Minnesota, 240 Gortner Lab, 1479 Gortner Avenue, St. Paul, MN 55108, USA.
Biotechnol Bioeng. 1999 Mar 5;62(5):609-17. doi: 10.1002/(sici)1097-0290(19990305)62:5<609::aid-bit13>3.0.co;2-c.
For direct and on-line study of the physiological states of cell cultures, a robust flow injection system has been designed and interfaced with flow cytometry (FI-FCM). The core of the flow injection system includes a microchamber designed for sample processing. The design of this microchamber allows not only an accurate on-line dilution but also on-line cell fixation, staining, and washing. The flow injection part of the system was tested by monitoring the optical density of a growing E.coli culture on-line using a spectrophotometer. The entire growth curve, from lag phase to stationary phase, was obtained with frequent sampling. The performance of the entire FI-FCM system is demonstrated in three applications. The first is the monitoring of green fluorescent protein fluorophore formation kinetics in E.coli by visualizing the fluorescence evolution after protein synthesis is inhibited. The data revealed a subpopulation of cells that do not become fluorescent. In addition, the data show that single-cell fluorescence is distributed over a wide range and that the fluorescent population contains cells that are capable of reaching significantly higher expression levels than that indicated by the population average. The second application is the detailed flow cytometric evaluation of the batch growth dynamics of E.coli expressing Gfp. The collected single-cell data visualize the batch growth phases and it is shown that a state of balanced growth is never reached by the culture. The third application is the determination of distribution of DNA content of a S. cerevisiae population by automatically staining cells using a DNA-specific stain. Reproducibility of the on-line staining reaction shows that the system is not restricted to measuring the native properties of cells; rather, a wider range of cellular components could be monitored after appropriate sample processing. The system is thus particularly useful because it operates automatically without direct operator supervision for extended time periods.
为了直接在线研究细胞培养物的生理状态，设计了一种强大的流动注射系统，并将其与流式细胞术（FI-FCM）连接。流动注射系统的核心包括一个用于样品处理的微腔室。该微腔室的设计不仅允许精确的在线稀释，还允许在线细胞固定、染色和洗涤。通过使用分光光度计在线监测生长中的大肠杆菌培养物的光密度来测试系统的流动注射部分。通过频繁采样获得了从延迟期到稳定期的完整生长曲线。在三个应用中展示了整个FI-FCM系统的性能。第一个应用是通过观察蛋白质合成被抑制后的荧光变化来监测大肠杆菌中绿色荧光蛋白荧光团的形成动力学。数据显示存在一个不发荧光的细胞亚群。此外，数据表明单细胞荧光分布在很宽的范围内，并且荧光群体中的细胞能够达到比群体平均值所表明的显著更高的表达水平。第二个应用是对表达绿色荧光蛋白的大肠杆菌分批生长动力学进行详细的流式细胞术评估。收集的单细胞数据可视化了分批生长阶段，结果表明培养物从未达到平衡生长状态。第三个应用是通过使用DNA特异性染料自动染色细胞来确定酿酒酵母群体的DNA含量分布。在线染色反应的可重复性表明该系统不仅限于测量细胞的天然特性；相反，经过适当的样品处理后，可以监测更广泛的细胞成分。因此，该系统特别有用，因为它可以在无需操作员直接监督的情况下长时间自动运行。
