DNA fluorometric assay in 96-well tissue culture plates using Hoechst 33258 after cell lysis by freezing in distilled water.
DNA fluorometric assay in 96-well tissue culture plates using Hoechst 33258 after cell lysis by freezing in distilled water.
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DOI：
10.1016/0003-2697(90)90382-j
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发表时间：
1990-11
影响因子：
2.9
通讯作者：
R. Rago;J. Mitchen;G. Wilding
中科院分区：
文献类型：
--
作者：
R. Rago;J. Mitchen;G. Wilding
描述了一种使用双苯并咪唑（Hoechst 33258）测定96孔组织培养板中细胞DNA含量的简单测定法。在感兴趣的时间点，将板中的培养基清空并冷冻储存。当进行测定时，将培养物在蒸馏水中短暂孵育并再次冷冻。这个过程裂解细胞，并允许荧光染料和细胞DNA的快速和彻底的混合。冷冻可方便地储存培养物直至测定时。实验可以分批进行，进一步减少处理时间，并提供更好的实验内和实验间标准化。该测定生成DNA荧光与细胞数量的线性标准曲线，其范围适用于微量培养威尔斯孔。这使得能够快速准确地测量细胞数量，涉及最少的处理时间，使得该测定非常适合于细胞增殖研究。
A simple assay is described using bisbenzimidazole (Hoechst 33258) to determine cellular DNA content in 96-well tissue cultures plates. At time points of interest, the plates are emptied of media and stored frozen. When the assay is to be performed, cultures are briefly incubated in distilled water and frozen again. This process lyses the cells and allows rapid and thorough mixing of the fluorochrome and cellular DNA. Freezing permits convenient storage of cultures until the time of assay. Experiments can be batched, further reducing processing time and giving better intra- and interexperimental standardization. The assay generates a linear standard curve for DNA fluorescence versus cell number, the range of which is appropriate for microculture wells. This enables the rapid and accurate measurement of cell number involving minimal processing time, making this assay well suited for cell proliferation studies.