TOTO and YOYO: new very bright fluorochromes for DNA content analyses by flow cytometry

Cytometry
G T HironsH A Crissman

Abstract

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 G1 and S phase cells, but decreased significantly for cells in G2/M. These results indicate that the cyanine dyes may be useful in examining specific changes in chromatin structure duri...Continue Reading

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