Interactions of intercalating fluorochromes with DNA analyzed by conventional and fluorescence lifetime flow cytometry utilizing deuterium oxide

Sailer, Brian L.; Nastasi, Anthony; Valdez, J.G.; Steinkamp, John A.; Crissman, Harry A.

doi:10.1002/(sici)1097-0320(19961001)25:2<164::aid-cyto5>3.0.co;2-h

Cited by 27 publications

(23 citation statements)

References 20 publications

(11 reference statements)

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“…Differences in the structure of fluorochrome molecules and in their mode of DNA binding lead to differences in D 2 O-induced enhancement in the intensity and lifetime. Prior studies in our laboratory showed that D 2 O had a more pronounced effect on the fluorescence intensity and lifetime of PI bound to chromatin of fixed CHO cells compared to EB (Sailer et al 1996). Enhancement of the fluorescence intensity of DNA-bound PI in D 2 O significantly improved the G 0 /G 1 peak CV values for PI-stained CHO cells when these were analyzed on a commercial flow cytometer with limited laser power (Sailer et al 1996).…”

mentioning

confidence: 86%

“…intensity (Mazzini et al 1983;Mazzini and Giordano 1980;Olmsted and Kearns 1977) as well as fluorescence lifetime (Sailer et al 1996) in flow cytometry. Szabo et al (1986) also demonstrated spectrophotometrically that the lifetime of DAPI bound to DNA is increased in D 2 O.…”

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confidence: 98%

“…Phase-sensitive flow cytometry provides the capability to quantify fluorescence lifetimes and to resolve the emissions of multiple fluorochrome labels that have different lifetime values (Steinkamp and Crissman 1993). Quantitation of the lifetime of DNAbound fluorochromes provides information on the molecular interactions of a fluorochrome within the target molecule (Sailer et al 1996;Heller and Greenstock 1994). The lifetimes of DNA-bound fluorochromes are altered by physical and chemical factors such as solvents, cations, pH, energy transfer, and quenching.…”

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confidence: 99%

“…Measurement of the lifetime of the DNAbound fluorochrome by phase-sensitive flow cytometry coupled with simultaneous DNA content analysis allows discrimination of subpopulations of cells with discrete lifetime alterations. Application of this new flow cytometric technology showed that subpopulations of apoptotic HL-60 cells with altered chromatin structure had a shorter lifetime compared to the nonapoptotic portion of the population after staining with PI or EB (Sailer et al 1996).…”

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confidence: 99%

“…Alterations in chromatin structure change the accessibility of the DNA-bound fluorochromes and influence both the intensity (Evenson et al 1986;Darzynkiewicz et al 1984) and the lifetime of the bound dye (Sailer et al 1996;Heller and Greenstock 1994;Lakowicz 1992;Dewey 1991;Jameson and Reinhart 1989). Differences in the structure of fluorochrome molecules and in their mode of DNA binding lead to differences in D 2 O-induced enhancement in the intensity and lifetime.…”

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confidence: 99%

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Differential Effects of Deuterium Oxide on the Fluorescence Lifetimes and Intensities of Dyes with Different Modes of Binding to DNA

Sailer

Nastasi

Valdez

et al. 1997

J Histochem Cytochem.

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SUMMARY Deuterium oxide (D 2 O) increases both the fluorescence lifetime and the fluorescence intensity of the intercalating dyes propidium iodide (PI) and ethidium bromide (EB) when bound to nucleic acid structures. We have used spectroscopic analysis coupled with conventional and phase-sensitive flow cytometry to compare the alterations in intensity and lifetime of various DNA-binding fluorochromes bound to DNA and Chinese hamster ovary (CHO) cells in the presence of D 2 O vs phosphate-buffered saline (PBS). Spectroscopic and flow cytometric studies showed a differential enhancement of intensity and lifetime based on the mode of fluorochrome-DNA interaction. The fluorescence properties of intercalating probes, such as 7-aminoactinomycin D (7-AAD) and ethidium homodimer II (EthD II) were enhanced to the greatest degree, followed by the probes TOTO and YOYO, and the non-intercalating probes Hoechst 33342 (HO) and 4 Ј ,6-diamidino-2-phenylindole (DAPI). The non-intercalating probe mithramycin (MI) gave unexpected results, showing a great enhancement of fluorescence intensity and lifetime in D 2 O, indicating that when staining is performed in PBS, much of the MI fluorescence is quenched by the solvent environment. Apoptotic subpopulations of HL-60 cells had a shorter lifetime compared to nonapoptotic subpopulations when stained with EthD II. These results indicate that accessibility of the dye molecules to the solvent environment, once bound to DNA, leads to the differential enhancement effects of D 2 O on fluorescence intensity and lifetime of these probes. D na-binding fluorochromes have been used extensively in many applications, including flow cytometry and gel and capillary electrophoresis. Fluorochromes utilized in these applications must satisfy several criteria. They must bind specifically to nucleic acids, there must be a low quantum yield as free dye in solution, and there must be enhancement of quantum yield on binding to nucleic acid structures. Several classes of DNA-specific fluorochromes with different modes of base pair ( bp ) binding are available: (a) the DNA intercalators propidium iodide (PI) and ethidium bromide (EB), which lack appreciable bp specificity; (b) DNA intercalators with A-T bp preference, such as ethidium homodimer II (EthD II) or with G-C preference, such as 7-amino actinomycin D (7-AAD); and (c) non-intercalating probes with either A-T bp specificity, such as Hoechst 33342 (HO) or 4 Ј ,6-diamidino-2-phenylindole (DAPI) or with G-C specificity, such as mithramycin (MI). Two high quantum yield cyanine fluorochromes, TOTO and YOYO, are dimers of the dyes thiazole orange and oxazole yellow, respectively (Lee et al. 1986). These fluorochromes are believed to bind to DNA by intercalation, without base specificity (Haugland 1992) but with some base sequence preference (Netzel et al. 1995).Enhancement of the fluorescence intensity of DNAbinding probes improves the signal-to-noise ratio, allowing the use of lower dye concentrations and thereby reducing potential self-quenching between d...

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confidence: 86%

mentioning

confidence: 98%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Differential Effects of Deuterium Oxide on the Fluorescence Lifetimes and Intensities of Dyes with Different Modes of Binding to DNA

Sailer

Nastasi

Valdez

et al. 1997

J Histochem Cytochem.

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Genome size and GC-percent in vertebrates as determined by flow cytometry: The triangular relationship

1998

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Genome size and GC‐percent were determined by means of a special method of DNA flow cytometry in 154 vertebrate species. For the total dataset, a highly significant positive correlation was found between both parameters. The overall distribution of points is not linear but triangular: a wide range of GC‐percent values is observed at the lower end of genome size range, whereas with an increase in genome size the lower limit for GC‐percent is elevated, gradually approaching the upper limit (about 46%). In teleost fishes, which occupy the lower part of genome size range, the negative relationship between both parameters was observed. Two positive linear relationships were found between mean genome size and GC‐percent of the main vertebrate groups (one includes fishes, amphibians, and mammals, the other consists of reptiles and birds, which show the higher GC‐percent for their genome sizes). Distribution of variance between taxonomic levels indicates that GC‐percent is more evolutionarily conservative than genome size in anamniotes. Anuran amphibians show the greatest part of genome size variability at the lower taxonomic levels as compared to other vertebrates (with no additional variance already above the genus level). The data obtained with different methods are compared. It is shown that the proposed method can provide useful data for studies on genome evolution and biodiversity. Cytometry 31:100–109, 1998. © 1998 Wiley‐Liss, Inc.

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Chromatin signal in genome size measurements

Vinogradov

1999

Cytometry

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Interactions of intercalating fluorochromes with DNA analyzed by conventional and fluorescence lifetime flow cytometry utilizing deuterium oxide

Cited by 27 publications

References 20 publications

Differential Effects of Deuterium Oxide on the Fluorescence Lifetimes and Intensities of Dyes with Different Modes of Binding to DNA

Differential Effects of Deuterium Oxide on the Fluorescence Lifetimes and Intensities of Dyes with Different Modes of Binding to DNA

Genome size and GC-percent in vertebrates as determined by flow cytometry: The triangular relationship

Chromatin signal in genome size measurements

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