TOTO AND YOYO - NEW VERY BRIGHT FLUOROCHROMES FOR DNA CONTENT ANALYSES BY FLOW-CYTOMETRY
TOTO AND YOYO - NEW VERY BRIGHT FLUOROCHROMES FOR DNA CONTENT ANALYSES BY FLOW-CYTOMETRY
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DOI：
10.1002/cyto.990150206
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发表时间：
1994-02-01
期刊：
影响因子：
--
通讯作者：
CRISSMAN, HA
中科院分区：
文献类型：
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作者：
HIRONS, GT;FAWCETT, JJ;CRISSMAN, HA
对细胞核、乙醇固定的 CHO 细胞以及用两种新型花青染料 TOTO 和 YOYO 染色的分离人 GM130 染色体进行了流式细胞术 (FCM) 研究。这些荧光染料分别是噻唑橙和恶唑黄的二聚体，具有高量子效率并且对 DNA 和 RNA 均表现出特异性。 TOTO 和 YOYO 与溶液中的 dsDNA 结合，在 488 nm 和 457 nm（FCM 中使用的大多数激光器可用的波长）激发时分别发射 530 nm 和 510 nm 的光。经 RNase 处理的 CHO 细胞核，用 TOTO 或 YOYO 染色，提供的 DNA 直方图具有低变异系数，与用碘化丙啶 (PI) 或光神霉素 (MI) 染色的细胞核获得的结果一样好或更好。此外，通过等摩尔比较，用 YOYO 染色的细胞核发出的荧光比用 MI 染色的细胞核强 1,000 倍以上。对 YOYO 和 Hoechst 33258 染色的细胞核的荧光比率分析表明，G(1) 和 S 期细胞的 YOYO 与 Hoechst 荧光比率保持相对恒定，但 G(2)/M 期细胞显着下降。这些结果表明花青染料可用于检查细胞周期 G(2)/M 期期间染色质结构的特定变化。用 TOTO 或 YOYO 染色的乙醇固定的 CHO 细胞没有产生高质量的可重复 DNA 直方图，可能是因为 DNA 对这些大荧光染料的可及性较差。然而，对仅用 TOTO 或 YOYO 染色并依次用紫外和可见光波长激发的人类 GM130 染色体的双变量分析显示，许多单个染色体峰的分辨率与用 H2O 和染色霉素 A 染色的染色体获得的结果相似 (3)。总的来说，这些研究显示了在需要灵敏检测 DNA 的 FCM 研究中使用这些新型花青染料的潜在优势。 (C) 1994 Wiley-liss, Inc.
Flow cytometric (FCM) studies were performed on nuclei, ethanol-fixed CHO cells, and isolated human GM130 chromosomes stained with two new cyanine dyes, TOTO and YOYO. These fluorochromes, which are dimers of thiazole orange and oxazole yellow, respectively, have high quantum efficiencies and exhibit specificities for both DNA and RNA. Bound to dsDNA in solution, TOTO and YOYO emit at 530 and 510 nm, respectively, when excited at 488 nm and 457 nm, wavelengths available from most lasers employed in FCM. RNase-treated CHO nuclei, stained with either TOTO or YOYO, provided DNA histograms, with low coefficients of variation, that were as good as or better than those obtained with nuclei stained with propidium iodide (PI) or mithramycin (MI). In addition, by comparison on an equimolar basis, nuclei stained with YOYO fluoresced over 1,000 times more intensely than nuclei stained with MI. Fluorescence ratio analyses of nuclei stained with both YOYO and Hoechst 33258 showed that the ratio of YOYO to Hoechst fluorescence remained relatively constant for G(1) and S phase cells, but decreased significantly for cells in G(2)/M. These results indicate that the cyanine dyes may be useful in examining specific changes in chromatin structure during G(2)/M phases of the cell cycle. Ethanol-fixed CHO cells stained with TOTO or YOYO did not yield reproducible DNA histograms of good quality, presumably because of the poor accessibility of DNA to these large fluorochromes. However, bivariate analyses of human GM130 chromosomes stained with TOTO or YOYO alone and excited sequentially with uv and visible wavelengths showed resolution of many individual chromosome peaks similar to results obtained for chromosomes stained with HO and chromomycin A(3). Collectively, these studies show potential advantages for the use of these new cyanine dyes in FCM studies that require the sensitive detection of DNA. (C) 1994 Wiley-liss, Inc.