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

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 during G2/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 wave-lengths showed resolution of many individual chromosome peaks similar to results obtained for chromosomes stained with HO and chromomycin A3. Collectively, these studies show potential advantages for the use of these new cyanine dyes in FCM studies that require the sensitive detection of DNA.

TO‐PRO‐3 iodide: A novel HeNe laser‐excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry

“…It clearly shows a very good correlation between the percentage S-phase positive cells using either PI or TP3 under all our experimental conditions. This can easily be explained by the similarity of the two fluorochromes, both intercalating in ds DNA (9). An example of a triple labeling is demonstrated in Figure 3.…”

“…So others have chosen to use a dual-beam UV and 488 nm system and have used Hoechst 33342 as the DNA stain (1,141. Nevertheless, having a t our disposal an inexpensive low-power air-cooled HeNe laser (633 nm), we searched for a new red emitting fluorophore which could be excited with this laser. Recently, a family of new DNA probes with exceptional spectral properties has become available from Molecular Probes, Inc. (Eugene, OR) (7,9). TP3, which has peak absorbance at 642 nm and emission at 661 nm, is best excited by the HeNe laser at 633 nm.…”

Development and validation of flow cytometric measurement for parasitemia in cultures of P. falciparum vitally stained with YOYO‐1

“…Then DNA dye of each 20 ll of YOYO-1 solution was added into the 0.3 ml of sample to a final dye concentration of 500 ng/ml. The appropriate concentration of YOYO-1 was found to be optimal to discriminate infected erythrocytes from low to high counts (14).…”

“…The supernatant was removed by aspiration, and the cells resuspended in 0.3 ml PBS buffer supplemented with 0.25% Triton X-100 and left in this solution for 10 min at room temperature. The best concentration (0.25%) of Triton X-100 was selected from our previous study (14). After centrifugation, the permeabilized cells were re-suspended in 0.3 ml of RNase at 0.5 mg/ml and incubated for at least 2 h at 37°C for a complete digestion of reticulocyte RNA.…”

“…Therefore, the absence of reticulocytes and normocytes may reduce the interference in measurement of parasitemia (14). Present study proposes here use of a flow cytometer (Cell Lab QuantaTM, Beckman Coulter Inc., Fullerton, CA) with a 488 nm laser combined with and YOYO-1 nucleic acid dye.…”