Zinc Fixation Preserves Flow Cytometry Scatter and Fluorescence Parameters and Allows Simultaneous Analysis of DNA Content and Synthesis, and Intracellular and Surface Epitopes
Zinc Fixation Preserves Flow Cytometry Scatter and Fluorescence Parameters and Allows Simultaneous Analysis of DNA Content and Synthesis, and Intracellular and Surface Epitopes
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DOI：
10.1002/cyto.a.20914
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发表时间：
2010-08-01
期刊：
影响因子：
3.7
通讯作者：
Christensen, Rikke
中科院分区：
文献类型：
--
作者：
Jensen, Uffe Birk;Owens, David M.;Christensen, Rikke
锌盐固定(ZBF)已被证明在完整组织的组织化学分析中具有优势，但尚未被用于侧重于单个细胞定量分析的流式细胞术程序。在这里，我们展示了ZBF在保存表面表位标记和通过流式细胞术测量的前向和侧向散射参数方面与多聚甲醛一样好的表现。ZBF固定的小鼠上皮角质形成细胞表现出与活细胞高度相似的表面标记SCA-1、CD34和26整合素的染色模式。此外，ZBF还保留了DNA，允许随后的定量PCR分析或标记，以便在表面和细胞内表位染色后并入胸苷类似物EDU。最后，ZBF处理允许标记细胞的长期存储，而这些参数几乎没有变化。因此，我们提出了一种锌盐固定细胞的方案，允许通过流式细胞术同时分析DNA以及细胞内和细胞表面蛋白。(C)2010年国际细胞测量促进会
Zinc salt-based fixation (ZBF) has proved advantageous in histochemical analyses conducted on intact tissues but has not been exploited in flow cytometry procedures that focus on quantitative analysis of individual cells. Here, we show that ZBF performs equally well to paraformaldehyde in the preservation of surface epitope labeling and forward and side scatter parameters as measured by flow cytometry. ZBF-fixed mouse epithelial keratinocytes exhibit a staining pattern for the surface markers Sca-1, CD34 and 26 integrin that is highly analogous to live cells. Furthermore, ZBF also preserves DNA allowing subsequent quantitative PCR analysis or labeling for incorporation of the thymidine analog EdU following surface and intracellular epitope staining. Finally, ZBF treatment allows for long-term storage of labeled cells with little change in these parameters. Thus, we present a protocol for zinc salt fixation of cells that allows for the simultaneous analysis of DNA and intracellular and cell surface proteins by flow cytometry. (C) 2010 International Society for Advancement of Cytometry