Department of Pathology, University of Miami Miller School of Medicine, Miami 33101, Florida, USA.
Cytometry A. 2009 Oct;75(10):862-5. doi: 10.1002/cyto.a.20780.
The Click-iT Assay developed and commercialized by Invitrogen is based on incorporation of a new 5-bromo-2'-deoxyuridine analog, 5-ethynyl-2'-deoxyuridine (EdU) into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. This relatively convenient and useful procedure depends on fixation of cells with paraformaldehyde and staining of the DNA with 7-aminoactinomycin-D (7-AAD). Both of these procedures result in DNA histograms with broad coefficients of variation (CV's). In this report, we have shown that after EdU incorporation, nuclei isolated by lysis can be incubated with the Click-iT Assay and stained with propidium iodide for generation of DNA histograms with low CV's. This modified procedure results in better DNA histograms by replacing 7-AAD with propidium iodide and also saves processing time by eliminating the fixation and permeabilization steps.
Invitrogen 公司开发并商业化的 Click-iT 检测法基于将一种新型的 5-溴-2'-脱氧尿苷类似物，5-乙炔基-2'-脱氧尿苷（EdU）掺入新合成的 DNA 中，并通过铜介导的“点击”反应与叠氮染料结合。这种相对方便和有用的方法依赖于使用多聚甲醛固定细胞，并使用 7-氨基放线菌素 D（7-AAD）对 DNA 进行染色。这两个步骤都会导致 DNA 直方图的变异系数（CV）较宽。在本报告中，我们已经表明，在 EdU 掺入后，通过裂解分离的核可以与 Click-iT 检测法孵育，并使用碘化丙啶染色以生成 CV 值较低的 DNA 直方图。通过用碘化丙啶替代 7-AAD，此改良方法可以生成更好的 DNA 直方图，并且通过消除固定和通透步骤也节省了处理时间。
