Krylov S N, Zhang Z, Chan N W, Arriaga E, Palcic M M, Dovichi N J
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
Cytometry. 1999 Sep 1;37(1):14-20. doi: 10.1002/(sici)1097-0320(19990901)37:1<14::aid-cyto2>3.0.co;2-j.
We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell.
A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence.
Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells.
This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.
我们创造了两个术语：第一，化学细胞术描述了使用高灵敏度化学分析技术来研究单个细胞。第二，代谢细胞术是化学细胞术的一种形式，它监测单个细胞中产生的一系列生物合成和生物降解产物。在本文中，我们描述了代谢细胞术与图像细胞术的结合，以将寡糖代谢活性与细胞周期相关联。我们使用该技术来测量单个细胞中的DNA倍性、荧光二糖的摄取以及代谢产物的量。
将结肠腺癌细胞系（HT29）与荧光二糖一起孵育，该二糖被细胞摄取并转化为一系列生物合成和生物降解产物。细胞还用YOYO - 3和Hoechst 33342处理。YOYO - 3信号用作活死检测，而Hoechst 33342信号用于通过荧光图像细胞术估计活细胞的倍性。在倍性分析后，将一个细胞注入熔融石英毛细管中，在那里细胞被裂解。然后通过毛细管电泳分离荧光代谢产物并通过激光诱导荧光检测。
用代谢细胞术测量的底物摄取产生的结果与使用激光扫描共聚焦显微镜测量的结果相似。用我们简单的图像细胞术技术获得的DNA倍性直方图与使用流式细胞术获得的相似。G(1)期的细胞在底物方面未显示任何生物合成活性。在G(2)/M期细胞中区分出几组具有独特生物合成模式的细胞。
这是第一篇将代谢细胞术和图像细胞术结合起来以将代谢产物的形成与细胞周期相关联的报告。在逐个细胞的基础上监测完整的酶促级联反应并与细胞周期相关联。
