Gillies R J, Didier N, Denton M
Anal Biochem. 1986 Nov 15;159(1):109-13. doi: 10.1016/0003-2697(86)90314-3.
Determining the cytostatic or cytotoxic effects of various conditions on monolayer cells requires techniques that are rapid, reproducible, and able to monitor these effects as a function of time. Methods currently used to monitor cytostasis or cytotoxicity are either static or indirect; that is, they are designed to test effects of various treatments either at single time points or on associated cellular processes, such as membrane integrity. Because of these limitations in extant techniques, we undertook this study to improve methods for the rapid determination of cell number in monolayer cultures. We have arrived at conditions of staining cell nuclei with crystal violet under fixed regimens which allow rapid and reproducible quantification of cell number in cultures grown in 24-well miniwells. Quantification is possible by solubilizing the adsorbed dye into a solution of Triton X-100 and determining optical density (O.D.) using spectrophotometry. The present communication documents that O.D. is linearly related to cell number with a sensitivity of ca. 500 cells and that the technique is applicable to study agents which affect cell proliferation.
确定各种条件对单层细胞的细胞生长抑制或细胞毒性作用需要快速、可重复且能够随时间监测这些作用的技术。目前用于监测细胞生长抑制或细胞毒性的方法要么是静态的，要么是间接的；也就是说，它们旨在在单个时间点或相关细胞过程（如膜完整性）上测试各种处理的效果。由于现有技术存在这些局限性，我们开展了这项研究以改进单层培养物中细胞数量的快速测定方法。我们已经确定了在固定方案下用结晶紫对细胞核进行染色的条件，这使得在24孔微孔板中生长的培养物中的细胞数量能够快速且可重复地定量。通过将吸附的染料溶解到Triton X - 100溶液中并使用分光光度法测定光密度（O.D.）来进行定量。本通讯证明，光密度与细胞数量呈线性关系，灵敏度约为500个细胞，并且该技术适用于研究影响细胞增殖的试剂。
