Gordon L K, Parker C W
J Immunol. 1981 Oct;127(4):1634-9.
A microfluorometric assay has been developed for the quantitation of de novo DNA synthesis. The assay reliably detects newly synthesized DNA in individual cells and provides a relative measure of the proportion of new DNA in each cell. When combined with microscopic techniques for cell identification, selected subpopulations can be examined in samples containing a complex mixture of interacting cell types. The assay employs the quenching of Hoechst-33258 fluorescence by bromodeoxyuridine incorporated into newly synthesized DNA. The ability of BUdR to quench H-33258 fluorescence is lost after a brief exposure of ultraviolet fluorescence excitation. Therefore, quenched and unquenched fluorescence intensity measurements from the same cell can be compared. Increases in fluorescence intensity occur only in cells that have synthesized DNA after the addition of BUdR. In addition the change in fluorescence intensity is proportional to the degree of BUdR substitution within the range of the assay and provides a relative measure of DNA synthesis.
已开发出一种微量荧光测定法用于定量从头合成DNA。该测定法能可靠地检测单个细胞中新合成的DNA，并提供每个细胞中新DNA比例的相对测量值。当与用于细胞鉴定的显微镜技术相结合时，可在含有相互作用细胞类型复杂混合物的样本中检查选定的亚群。该测定法利用掺入新合成DNA中的溴脱氧尿苷对Hoechst-33258荧光的猝灭作用。在短暂暴露于紫外荧光激发后，溴脱氧尿苷猝灭H-33258荧光的能力丧失。因此，可以比较来自同一细胞的猝灭和未猝灭荧光强度测量值。荧光强度增加仅发生在添加溴脱氧尿苷后合成了DNA的细胞中。此外，在测定范围内，荧光强度的变化与溴脱氧尿苷取代程度成正比，并提供DNA合成的相对测量值。
