Shen Yuqing, Vignali Paolo, Wang Ruoning
Center for Childhood Cancer & Blood Diseases, The Research Institute at Nationwide Children's Hospital, Ohio State University, Columbus, OH, USA.
Department of Microbiology and Immunology, Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Medical School, Southeast University, Nanjing, China.
Bio Protoc. 2017 Aug 20;7(16). doi: 10.21769/BioProtoc.2517.
The flow cytometric quantitation of DNA content by DNA-binding fluorochrome, propidium iodide (PI) is the most widely used method for cell cycle analysis. However, the commonly used methods are time-consuming and labor-intensive and are incompatible with staining of mitotic markers by fluorescent-labeled antibodies. Here, we report an optimized simple protocol for rapid and simultaneous analysis of characteristic mitotic phosphorylated proteins and DNA content, permitting the quantification of cells in mitosis, G, S and G stage in a single assay. The protocol detailed here employs detergent-based hypotonic solution to rapidly permeabilize cells and allows simultaneous staining of DNA with PI and mitotic marker, phospho-Histone H3, with specific antibody within 20 min. The protocol requires only inexpensive and commercial available reagents and also enables a rapid and complete analysis of cell cycle profile.
通过与DNA结合的荧光染料碘化丙啶（PI）进行流式细胞术定量DNA含量，是细胞周期分析中使用最广泛的方法。然而，常用方法耗时且费力，并且与用荧光标记抗体对有丝分裂标记物进行染色不兼容。在此，我们报告了一种优化的简单方案，用于快速同时分析特征性有丝分裂磷酸化蛋白和DNA含量，允许在一次检测中对有丝分裂期、G期、S期和G期的细胞进行定量。此处详述的方案采用基于去污剂的低渗溶液快速使细胞通透，并允许在20分钟内用PI同时对DNA进行染色，并用特异性抗体对有丝分裂标记物磷酸化组蛋白H3进行染色。该方案仅需要廉价的市售试剂，还能够对细胞周期谱进行快速且完整的分析。
