Krishan Awtar, Hamelik Ronald M
University of Miami Miller School of Medicine, Miami, Florida, USA.
Curr Protoc Cytom. 2010 Apr;Chapter 7:Unit7.36. doi: 10.1002/0471142956.cy0736s52.
The Click-iT EdU cell proliferation assay (Invitrogen) for detection of replicating cells is based on incorporation of EdU into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. In the protocol provided by Invitrogen, cells are fixed with paraformaldehyde and stained with 7-aminoactinomycin D (7-AAD) for DNA content analysis. Both of these procedures result in DNA histograms with a broad coefficient of variation. We have modified this protocol and show that after EdU incorporation, nuclei isolated by hypotonic lysis of cells can be directly labeled using the Click-iT Alexa Fluor 488 Assay kit and stained with propidium iodide. This modified procedure using isolated nuclei and propidium iodide staining results in DNA histograms with better resolution (lower coefficient of variation of the G(1) peak) and shorter processing time by eliminating the fixation and permeabilization steps.
用于检测增殖细胞的Click-iT EdU细胞增殖检测法（英杰公司）是基于将EdU掺入新合成的DNA中，并通过铜介导的“点击”反应由叠氮化物染料对其进行识别。在英杰公司提供的方案中，细胞用多聚甲醛固定，并用7-氨基放线菌素D（7-AAD）染色以进行DNA含量分析。这两个步骤都会产生变异系数较大的DNA直方图。我们对该方案进行了改进，结果表明在掺入EdU后，通过低渗裂解细胞分离得到的细胞核可以直接使用Click-iT Alexa Fluor 488检测试剂盒进行标记，并用碘化丙啶染色。这种使用分离细胞核和碘化丙啶染色的改进方法通过省去固定和通透步骤，产生了分辨率更高（G(1)峰的变异系数更低）且处理时间更短的DNA直方图。
