FLOW CYTOMETRIC ESTIMATION OF DNA AND RNA-CONTENT IN INTACT-CELLS STAINED WITH HOECHST-33342 AND PYRONIN-Y
FLOW CYTOMETRIC ESTIMATION OF DNA AND RNA-CONTENT IN INTACT-CELLS STAINED WITH HOECHST-33342 AND PYRONIN-Y
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DOI：
10.1002/cyto.990020302
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发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
SHAPIRO, HM
中科院分区：
文献类型：
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作者：
SHAPIRO, HM
将 RNA 含量估计添加到 DNA 含量的流式细胞仪测量中，可以提供有关细胞在静止状态和增殖状态之间转换的有价值的信息。采用吖啶橙的平衡染色方法用于 DNA/RNA 含量测量，但难以应用于完整细胞，并且与荧光抗体或配体结合使用以展示细胞表面结构是不切实际的。使用 Hoechst 33342 (HO342) 和 Pyronin Y (PY) 的组合对完整细胞进行染色，使用双源流式细胞仪使用 UV 和蓝绿或绿色激发来估计 DNA/RNA 含量，测量 HO342 在 430-470 nm 处的荧光和 PY 在 590-650 nm 处的荧光。培养细胞[人T淋巴细胞CCRF-CEM细胞]和刺激的[大鼠]淋巴细胞获得的结果与使用吖啶橙进行DNA/RNA染色获得的结果非常一致； .apprx。通过 RNAse 消化，可以从用 HO342 和 PY 染色的乙醇固定细胞中去除 1/2 的 PY 荧光。 HO342/PY方法可与荧光素免疫荧光结合用于细胞表面标志物的检测。 HO342可以与其他三环杂芳族染料结合用于DNA/RNA估计； HO342 和恶嗪 1 的组合可以在双源仪器中使用汞弧灯和氦氖激光器进行激发。染色过程简单；将培养基中的细胞与5μM HO342在37℃下孵育。 C下45分钟，然后添加5μM PY(或恶嗪1)，并在另外45分钟温育后分析细胞，无需洗涤。这些染料组合对于活细胞染色和分选的适用性仍有待确定。
The addition of RNA content estimation to flow cytometric measurement of DNA content provides valuable information concerning cells'' transitions between quiescent and proliferative states. Equilibrium staining methods employing acridine orange were used for DNA/RNA content measurement but are difficult to apply to intact cells and impractical for use in conjunction with fluorescent antibodies or ligands for demonstration of cell surface structures. A combination of Hoechst 33342 (HO342) and pyronin Y (PY) were used to stain intact cells for DNA/RNA content estimation with a dual source flow cytometer using UV and blue-green or green excitation, measuring HO342 fluorescence at 430-470 nm and PY fluorescence at 590-650 nm. Results obtained with cultured cells [human T-lymphoblastoid CCRF-CEM cell] and stimulated [rat] lymphocytes are in good agreement with those obtained using acridine orange for DNA/RNA staining; .apprx. 1/2 of the PY fluorescence can be removed from ethanol-fixed cells stained with HO342 and PY by RNAse digestion. The HO342/PY method can be combined with fluorescein immunofluorescence for detection of cell surface markers. HO342 can be combined with other tricyclic heteroaromatic dyes for DNA/RNA estimation; the combination of HO342 and oxazine 1 can be excited in a dual source instrument using a Hg arc lamp and a He-Ne laser. The staining procedure is simple; cells in medium are incubated with 5 .mu.M HO342 at 37.degree. C for 45 min, 5 .mu.M PY (or oxazine 1) is then added and cells are analyzed without washing after an additional 45 min incubation. Suitability of these dye combinations for vital cell staining and sorting remains to be determined.