ARTICLE |
Correspondence to: Harry A. Crissman, Life Sciences Div., Los Alamos National Laboratory, LS-4 M888, Los Alamos, NM 87545.
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Summary |
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Deuterium oxide (D2O) 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 D2O 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',6-diamidino-2-phenylindole (DAPI). The non-intercalating probe mithramycin (MI) gave unexpected results, showing a great enhancement of fluorescence intensity and lifetime in D2O, 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 non-apoptotic 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 D2O on fluorescence intensity and lifetime of these probes. (J Histochem Cytochem 45:165-175, 1997)
Key Words: fluorescence lifetime, fluorescence intensity, flow cytometry, DNA-binding fluorochromes, deuterium oxide, apoptosis
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Introduction |
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Dna-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',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 (
Enhancement of the fluorescence intensity of DNA-binding probes improves the signal-to-noise ratio, allowing the use of lower dye concentrations and thereby reducing potential self-quenching between dye molecules bound in close proximity. Deuterium oxide (D2O; heavy water) increases EB and PI fluorescence intensity (
Measurement of fluorescence lifetime using phase-sensitive electronics has recently provided an additional parameter for flow cytometry analysis (
Alterations in chromatin structure change the accessibility of the DNA-bound fluorochromes and influence both the intensity (
In this study we used conventional and phase-sensitive flow cytometry to examine the effects of D2O on dyes with different modes of DNA binding. D2O enhanced the fluorescence intensity and fluorescence lifetime of all the dyes, but to an extent dependent on the mode of interaction of the dye with the DNA. Fluorescence intensity of non-intercalating, A-T base-specific dyes, such as HO and DAPI, was enhanced only slightly by D2O, whereas the intensity of intercalating dyes with base specificity, such as 7-AAD (G-C-specific) and EthD II (A-T-specific) was enhanced to a greater degree, although not to as great an extent as previously noted for PI and EB. Fluorescence lifetimes of stained cells were increased by D2O to an extent somewhat proportional to the enhanced fluorescence intensity. Variation in either dye concentrations or the relative proportion of D2O in the solvent provides the potential to preselect a lifetime value for a probe in studies utilizing phase-resolved measurements to separate the fluorescence of dyes on the basis of their respective lifetimes rather than of their spectral emission patterns. The interaction of EthD II with subpopulations of apoptotic cells also significantly reduced fluorescence lifetime compared to the non-apoptotic cells.
The results of this study indicate that the different modes of fluorochrome-DNA interaction, as well as differences in solvent accessibility of the fluorochromes, lead to a differential enhancement effect of D2O on intensity and lifetime. The use of D2O to study intensity and lifetime provides additional information on the microenvironment of the fluorochrome-DNA interaction. An example in this study was the unexpected D2O-induced enhancement noted for the non-intercalating G-C-specific MI. On the basis of the non-intercalating mode of MI-DNA binding, a minimal enhancement of fluorescence intensity and lifetime comparable to results noted for HO and DAPI would have been expected. The great enhancement of MI intensity and the lifetime values in D2O infer that when PBS is used as the solvent, a large portion of the MI fluorescence is potentially being lost due to solvent quenching. The results of this study will lead to further insight into the mechanisms and environmental influences on fluorochrome-DNA interactions.
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Materials and Methods |
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Cells and Culture Conditions
Chinese hamster ovary (CHO) cells were grown in suspension culture at 37C in spinner flasks containing Ham's F10 medium (Gibco BRL; Grand Island, NY) supplemented with 15% heat-inactivated bovine calf serum (Hyclone Laboratories; Logan, UT), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco). Cell densities were maintained at 0.25-4.5 x 105 cells/ml. The population doubling time was 13-14 hr.
Human promyelocytic (line HL-60) cells were maintained in suspension culture at 37C in T-150 flasks containing RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (Hyclone Laboratories), 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco), and 1.25 mM L-glutamine (Gibco). The population doubling time was 15-18 hr when cell densities were maintained at 0.2-1 x 106 cells/ml.
Induction of Apoptosis in HL-60 Cells and Extraction of Low Molecular Weight DNA
HL-60 cells in culture were treated with 0.15 µM campto-thecin (Sigma; St Louis, MO) for 3 hr before fixation. Cells were harvested from suspension culture by centrifugation at room temperature (RT) for 5 min at 200 x g and thoroughly resuspended in one volume of cold Puck's saline A, without Ca2+ and Mg2+ and containing 1.0 mM EDTA (Gibco). Three volumes of cold 95% ethanol were then added to yield a final cell concentration of 1.0 x 106/ml in 70% ethanol. After at least 24 hr of fixation, the cells were centrifuged at RT for 5 min at 200 x g and the fixative aspirated. Cell pellets were resuspended in 50 µl of phosphate-citrate buffer (192 parts 0.2 M Na2HPO4, 8 parts 0.1 M citric acid, pH 7.8) and incubated at RT for 15 min to extract low molecular weight DNA before staining (
Ethanol Fixation and Cell Staining
CHO cells were harvested from suspension culture by centrifugation at RT for 5 min at 200 x g and ethanol-fixed as described above to yield a final cell concentration of 1.0 x 106/ml in 70% ethanol. Cells were fixed for at least 24 h, before staining. For staining, cells were centrifuged at RT for 5 min at 200 x g and the fixative aspirated. Stock solutions of DAPI (Molecular Probes; Eugene, OR) were prepared at 1 mg/ml in distilled H2O or D2O, HO (Calbiochem; La Jolla, CA) or MI (Pfizer Laboratories; Groton, CT) was mixed at 1 mg/ml in either PBS, pH 7.0, or D2O with 0.15 M NaCl (D2O-saline, pH 7.1). Stock solutions of 7-AAD (Molecular Probes) and EthD II (Molecular Probes) were mixed at 1 mg/ml in DMSO and further diluted in PBS or D2O-saline before staining. TOTO (Molecular Probes) or YOYO (Molecular Probes) was obtained at 1 mM in DMSO and was diluted in PBS or D2O-saline before staining. Cells were resuspended in either PBS or D2O-saline at 1 x 106 cells/ml containing RNASE (Sigma) at a concentration of 50 µg/ml. For staining with MI, MgCl2 was added to 5 mM. For the fluorochromes used, the modes of fluorochrome-DNA interaction and BP binding preference are listed in Table 1.
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Spectrofluorometric Analysis
Spectrofluorometric scans of the fluorochromes were performed using a computer-driven SPEX (Edison, NJ) Fluorolog 1680 0.22 m double spectrometer with a 500-W xenon lamp source. All entrance and exit slits were set at 0.5 mm during sample runs. Free dye in solution, calf thymus DNA (Sigma) with fluorochromes in solution (50 µg/ml), or fixed and stained CHO cells were analyzed. Dye concentrations used for spectrophotometry were 0.5 µg/ml HO, 1.0 µg/ml DAPI, 20 µg/ml EthD II, 20 µg/ml 7-AAD, 5 µg/ml MI, 1 µg/ml TOTO, and 1 µg/ml YOYO.
Conventional Flow Cytometry
Flow cytometric measurements were performed on a Los Alamos flow cytometer (
Lifetime Flow Cytometry
Fluorescence lifetime measurements were performed on the Los Alamos phase-sensitive flow cytometer which has been described in detail (
Statistical Analysis
Descriptive statistics and t-tests were performed in Microsoft Excel (Microsoft; Redmond, WA). Results are expressed as the mean ± SEM of at least three experiments.
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Results |
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Comparison of Fluorescence Intensity and Spectral Alterations of Fluorochromes in PBS or D2O-Saline Bound to Calf Thymus DNA and Stained CHO Cells
The use of D2O-saline in place of PBS as the solvent in spectral studies resulted in increased fluorescence emission intensities for all of the free, unbound dyes in solution (except for TOTO and YOYO, which have no appreciable fluorescence except when DNA-bound), ranging from 20% to 177%. In order of increasing enhancement by D2O were DAPI<MI<HO<EthD II< 7-AAD (Table 2). When bound to calf thymus DNA, all of the dyes exhibited a 6-129% increase in fluorescence intensity in D2O-saline compared to PBS, with the dyes ordered by increasing enhancement: YOYO< TOTO<HO<DAPI<MI<7-AAD<EthD II (Table 2). When bound to fixed, RNASE-treated CHO cells the fluorescence enhancement in D2O-saline compared to PBS was 16-186%, with the dyes ranked as: DAPI< YOYO<TOTO<HO<MI<7-AAD<EthD II (Table 2).
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Changes in the excitation peaks shown in Table 3 were observed among the different substrates utilized (free dye, calf thymus DNA, fixed CHO cells, respectively) for DAPI (all p<0.05). HO, EthD II, and MI showed changes in excitation peak only between the free dye in solution and bound to DNA, either calf thymus DNA or within fixed CHO cells (all p<0.05). 7-AAD showed changes in excitation peak between either the free dye in solution or within fixed CHO cells and bound to calf thymus DNA (p<0.05). Although changes in the excitation spectra were noted for the different substrates, only minor changes were observed when the solvent used was either D2O-saline or PBS (all p>0.05). Changes were noted in the emission peak wavelength for 7-AAD (free dye or within fixed CHO cells vs calf thymus DNA; p<0.05), HO (all p<0.05), and EthD II (all p<0.05) among the different substrates. Only minor changes were noted in the emission spectra when the stained substrates in PBS or D2O-saline were compared, except for the change in emission peak for free DAPI in solution between PBS and D2O-saline (440 to 460 nm, respectively; p<0.01).
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Conventional Flow Cytometric Fluorescence Intensity Measurements Using Various Solvent Ratios of PBS to D2O-Saline
The effects of PBS on the fluorescence enhancement provided by D2O was determined using conventional flow cytometric analysis of fixed CHO cells stained in mixture ratios of PBS to D2O-saline with 10% volume increments from 100% PBS to 100% D2O-saline. Figure 1 illustrates the enhancement of fluorescence intensity when samples were stained in 100% D2O-saline vs 100% PBS for MI, DAPI, and 7-AAD. Fluorochrome intensity enhancement in D2O varied, with the increase of the mean G0/G1 peak value from 100% PBS to 100% D2O-saline ranging from 7.7 to 150.5% (Table 4). Figure 2 shows examples of the fluorochrome responses to various mixture ratios of PBS to D2O-s aline. The mean value of the G0/G1 peak was linearly correlated with the percent PBS for EthD II (r2 =0.952), MI (r2=0.986), and for TOTO (data not shown; r2=0.941) (all p<0.05). The probes 7-AAD, HO, and YOYO were not affected linearly by the addition of D2O-saline (data not shown). 7-AAD showed a constant mean G0/G1 peak value from 100% PBS until a ratio of 40% PBS:60% D2O-saline, at which point the mean increased (p<0.05). For HO, this shift occurred at 40% PBS:60% D2O-saline (p<0.05), and for YOYO occurred at 70% PBS:30% D2O-saline (p<0.05). G0/G1 peak mean values for DAPI (data not shown) were only slightly decreased over the range of mixture ratios (p>0.05). For dyes that changed linearly over the range of mixture ratios, the slope of the regression line is another indicator of how D2O-saline affects the fluorescence intensity. The slope for MI (-0.87 ±0.15) is larger than for TOTO (-0.03±0.04) or EthD II (-0.34±0.09) (both p<0.01), indicating that PBS has a greater quenching effect on MI fluorescence.
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Fluorescence Lifetime of DNA Fluorochromes
For phase-sensitive flow cytometric lifetime measurements, each of the fluorochromes was utilized to stain fixed CHO cells in PBS, and Figure 3 illustrates the fluorescence lifetime histograms obtained. Lifetime values for the probes were: 2.7 ± 0.05 nsec for DAPI, 2.3± 0.06 nsec for HO, 1.5 ± 0.02 nsec for MI, 0.8 ± 0.002 nsec for 7-AAD, 10.0 ± 0.45 nsec for EthD II, 2.2 ± 0.14 nsec for TOTO, and 2.1 ± 0.09 nsec for YOYO. PI and EB had lifetime values of 15 ± 0.5 and 19.5 ± 0.4 nsec, respectively.
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Fluorescence Lifetime Alterations in D2O
Figure 4 illustrates the effects of D2O on fluorescence lifetime for DAPI-, MI-, and EthD II-stained CHO cells. At dye concentrations somewhat routinely utilized for DNA content analysis by flow cytometry, all of the fluorochromes showed an increase in fluorescence lifetime when staining was performed in D2O-saline vs PBS (Table 5). Significant increases in fluorescence lifetime in D2O-saline were seen for all of the fluorochromes. The increase of fluorescence lifetime in D2O-saline for the fluorochromes parallels the spectral and conventional flow cytometric intensity observations, in which the intercalating fluorochromes 7-AAD and EthD II and the non-intercalating MI exhibit the largest increases.
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Fluorescence Lifetime Alterations with Increasing Dye Concentration
Figure 5 illustrates the change in fluorescence lifetime in D2O-saline vs PBS with increasing dye concentrations for MI, 7-AAD and TOTO. MI showed an increasing lifetime up to staining concentrations of 1 µg/ml, but lifetime values decreased at higher dye concentrations. All of the other probes showed the highest fluorescence lifetime at the lowest dye concentrations, with the lifetimes decreasing as the dye concentration increased. One exception was the sample stained with 100 µg/ml 7-AAD in D2O-saline, in which the lifetime increased compared to the sample stained with 10 µg/ml (p<0.05). In most cases, samples stained in D2O-saline had a higher lifetime than the corresponding sample in PBS. The only exception was for 7-AAD (Figure 5) at 0.01 µg/ml dye concentration, for which the lifetime for the sample in PBS was 30.8% higher (p<.05) than the sample in D2O-saline (1.7 ± 0.17 and 1.3 ± 0.56 nsec, respectively).
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Differential Stainability of Apoptotic Subpopulations in D2O-Saline Compared to PBS
Apoptotic cell subpopulations are apparent in a DNA content histogram as a sub-G0/G1 peak after treatment of HL-60 cells with CAM for 3 hr, ethanol fixation, and PC buffer extraction of low molecular weight DNA. Depending on the dye used for staining, the apoptotic subpopulations are shifted disproportionately from the non-apoptotic G0/G1 peak in D2O-saline compared to PBS (Table 6).
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Fluorescence Lifetime of Apoptotic Subpopulations
Figure 6 illustrates the change in fluorescence lifetime for apoptotic cells stained with EthD II in PBS (Figure 6A and Figure 6E) or D2O-saline (Figure 6B and Figure 6F). In PBS, the lifetime of the apoptotic subpopulation was 26% lower than that of the non-apoptotic cells (7.2 ± 0.18 and 9.8 ± 1.2 nsec, respectively; p<0.01), and in D2O-saline the apoptotic subpopulation was 25% lower (7.9 ± 0.3 and 10.5 ± 1.4 nsec, respectively; p<0.01). When stained with the other fluorochromes utilized in this study, such as MI (Figure 6C and Figure 6G, PBS; Figure 6D and Figure 6H, D2O-saline), the apoptotic subpopulation did not have a different lifetime from that of the non-apoptotic cells (all p>0.05). In Figure 6B, a shoulder appears next to the peak representing the non-apoptotic cells in the sample stained with EthD II in D2O-saline, and is apparently a portion of the G0/G1 sub-population (Figure 6F). It is possible that we are measuring a subset of the G0/G1 population, but further study will be required before this determination can be made.
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Discussion |
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The fluorochromes analyzed in this study have been used previously for the spectroscopic quantification of DNA (
Phase-sensitive flow cytometric analysis of the DNA-binding fluorochromes provided fluorescence lifetime values in accord with previously published results (
Flow cytometric analysis showed that the various fluorochromes are affected by D2O in a similar manner, but to a degree that is dependent on their mode of DNA binding. The fluorescence intensities of DAPI, HO, TOTO, and YOYO were not enhanced dramatically when they were bound to fixed CHO cells, as was the case for 7-AAD, EthD II, and MI in this study, or for PI and EB in our previous study (
In our studies utilizing a range of dye concentrations, fluorescence lifetime decreased concomitant with increasing dye concentrations. This decrease in fluorescence lifetime may be due to fluorescence quenching, a process that reduces the quantum yield of a probe without changing the fluorescence spectrum (
Apoptosis, or programmed cell death, is characterized by internucleosomal cleavage of nuclear DNA which can be extracted from the cells after permeabilization of the cell membrane (
It is interesting to note that smaller molecular weight intercalating dyes, such as PI and EB, have relatively longer lifetime values than the larger molecular weight fluorochromes. Intercalation stabilizes the fluorochrome molecule and shields it from the solvent environment, increasing its lifetime value relative to the unbound dye. In the presence of D2O, the fluorescence lifetimes are further increased, indicating that interaction of the fluorochromes with the D2O solvent environment leads to a further stabilization of the bound fluorochrome. The larger molecular weight fluorochromes may not be shielded from the solvent environment to the same extent once intercalated, leading to lower lifetime values and decreased enhancement by D2O. Also of note is the fact that the lifetime for EB bound to fixed cells (19.5) is almost twice as large as the lifetime of EthD II (10.53) when the cells are stained in PBS. These results appear to indicate that even though these two dyes have the same fluorochrome group, the size of the molecule and the interaction of the probe with DNA and the solvent alter the fluorescence properties.
As was noted in experiments with changing dye concentrations, the fluorescence lifetime of the DNA fluorochromes is dependent on the dye concentration as well as on the solvent. This effect leads to potential preselection of specific lifetime value for a probe through a combination of dye concentration and solvent, either PBS or D2O-saline. The use of multiple DNA fluorochromes has been used previously to elucidate changes throughout the cell cycle (
In summary, we have utilized D2O to illustrate differences in the binding of various DNA-specific fluorochromes measured by spectrophotometry, conventional flow cytometry, and phase-sensitive flow cytometry. We have shown that differences in chromatin structure, dye structure, dye-DNA interaction, and solvent accessibility lead to differences in the fluorescence intensity and fluorescence lifetime of the bound fluorochrome. These results, combined with future studies, will lead to further insight into the microenvironment of DNA-fluorochrome interaction and the effects of environmental influences on these interactions.
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Acknowledgments |
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Supported by the United States Department of Energy and the Los Alamos National Laboratory Flow Cytometry Resource (NIH grants p41-RR01315, RR06758, and R01 RR07855).
Received for publication May 13, 1996; accepted October 1, 1996.
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Literature Cited |
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Barcellona ML, Cardiel G, Gratton E (1990) Time-resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides. Biochem Biophys Res Commun 170:270-280[Medline]
Barcellona ML, Gratton E (1990) The fluorescence properties of a DNA probe: 4',6-diamidino-2-phenylindole (DAPI). Eur Biophys J 17:315-323[Medline]
Crissman HA, Hirons GT (1994) Staining of DNA in live and fixed cells. In Darzynkiewicz Z, Robinson JP, Crissman HA, eds. Methods in Cell Biology. Vol 41. San Diego, Academic Press, 195-209
Darzynkiewicz Z, Bruno S, Del Bino G, Gorczyca W, Hotz MA, Lassota P, Traganos F (1992) Features of apoptotic cells measured by flow cytometry. Cytometry 13:795-808[Medline]
Darzynkiewicz Z, Traganos F, Kapuscinski J, Staiano-Coico L, Melamed MR (1984) Accessibility of DNA in situ to various fluorochromes: relationship to chromatin changes during erythroid differentiation of Friend leukemia cells. Cytometry 5:355-363[Medline]
Deka C, Sklar LA, Steinkamp JA (1994) Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique. Cytometry 17:94-101[Medline]
Dewey TG (1991) Biophysical and Biochemical Aspects of Fluorescence Spectroscopy. New York, Plenum Press
Dörr F (1983) Mechanisms of energy transfer In Hoppe W, Loh-mann W, Markl H, Ziegler H, eds. Biophysics. Berlin, Springer-Verlag, 265-288
Evenson D, Darzynkiewicz Z, Jost L, Janca F, Ballachey B (1986) Changes in accessibility of DNA to various fluorochromes during spermatogenesis. Cytometry 7:45-53[Medline]
Glazer AN, Peck K, Mathies RA (1990) A stable double-stranded DNA-ethidium homodimer complex: application to picogram fluorescence detection of DNA in agarose gels. Proc Natl Acad Sci USA 87:3851-3855[Abstract]
Gong J, Traganos F, Darzynkiewicz Z (1994) A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal Biochem 218:314-319[Medline]
Haugland RO (1992) Molecular Probes Handbook of Fluorescent Probes and Research Chemicals. 5th ed. Eugene, OR, Molecular Probes, Inc.
Heller DP, Greenstock CL (1994) Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye. Biophys Chem 50:305-312[Medline]
Hirons GT, Fawcett JJ, Crissman HA (1994) TOTO and YOYO: new very bright fluorochromes for DNA content analyses by flow cytometry. Cytometry 15:129-140[Medline]
Hochstrasser RA, Millar DP (1992) Fluorescence self-quenching of ethidium bromide intercalated in DNA. SPIE 1640:599-605
Jameson DM, Reinhart GD (1989) Fluorescent Biomolecules: Methodologies and Applications. New York, Plenum Press
Kapuscinski J, Szer W (1979) Interactions of 4',6-diamidine-2-phenylindole with synthetic polynucleotides. Nucleic Acids Res 6:3519-3534[Abstract]
Lakowicz JR (1992) Time-resolved laser spectroscopy in biochemistry III. Proc SPIE 1640:807
Lakowicz JR (1983) Principles of Fluorescence Spectroscopy. New York, Plenum Press
Lee LG, Chen C-H, Chiu LA (1986) Thiazole orange: a new dye for reticulocyte analysis. Cytometry 7:508-517[Medline]
Mazzini G, Giordano P (1980) Effects of some solvents on the fluorescence intensity of phenantridinic derivatives-DNA complexes: flow cytofluorometric applications. In Laerum OD, Lindmo T, Thorud E, eds. Flow Cytometry IV. Oslo, Universitetsforlaget, 74-76
Mazzini G, Giordano P, Riccardi A, Montecucco CM (1983) A flow cytometric study of the propidium iodide staining kinetics of human leukocytes and its relationship with chromatin structure. Cytometry 3:443-448[Medline]
Nairn RS, Dodson ML, Humphrey RM (1982) Comparison of ethidium bromide and 4',6-diamidino-2-phenylindole as quantitative fluorescent stains for DNA in agarose gels. J Biochem Biophys Methods 6:95-103[Medline]
Netzel TL, Nafisi K, Zhao M, Lenhard JR, Johnson I (1995) Base-content dependence of emission enhancements, quantum yields, and lifetimes for cyanine dyes bound to double-strand DNA: photophysical properties of monomeric and bichromophoric DNA stains. J Phys Chem 99:17936-17947
Olmsted J, Kearns DR (1977) Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids. Biochemistry 16:3647-3654[Medline]
Pinsky BG, Ladasky JJ, Lakowicz JR, Berndt K, Hoffman RA (1993) Phase-resolved fluorescence lifetime measurements for flow cytometry. Cytometry 14:123-135[Medline]
Sailer BL, Nastasi AJ, Valdez JG, Steinkamp JA, Crissman HA (1996) Interactions of intercalating fluorochromes with DNA analyzed by conventional and fluorescence lifetime flow cytometry utilizing deuterium oxide. Cytometry 25:164-172[Medline]
Schmid IS, Krall WJ, Uittenbogaart CH, Braun J, Giorgi JV (1992) Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry. Cytometry 13:204-208[Medline]
Steinkamp JA, Crissman HA (1993) Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase-sensitive flow cytometry. Cytometry 14:210-216[Medline]
Steinkamp JA, Habbersett RC, Hiebert RD (1991) Improved multilaser/multiparameter flow cytometer for analysis and sorting of cells and particles. Rev Sci Instrum 62:2751-2764
Steinkamp JA, Stewart CC, Crissman HA (1982) Three-color fluorescence measurements on single cells excited at three laser wavelengths. Cytometry 2:226-231[Medline]
Steinkamp JA, Yoshida TM, Martin JC (1993) Flow cytometer for resolving signals from heterogeneous fluorescence emissions and quantifying lifetime in fluorochrome-labeled cells/particles by phase-sensitive detection. Rev Sci Instrum 64:3440-3450
Szabo AG, Krajcarski DI, Cavatorta P, Masotti L, Barcellona ML (1986) Excited state pKa behaviour of DAPI. A rationalization of the fluorescence enhancement of DAPI in DAPI-nucleic acid complexes. Photochem Photobiol 44:143-150
Van Lancker M, Gheyssens LC (1986) A comparison of four frequently used assays for quantitative determination of DNA. Anal Lett 19:615-623
Wadkins RM, Jovin TM (1991) Actinomycin D and 7-aminoactinomycin D binding to single-stranded DNA. Biochemistry 30:9469-9478[Medline]
Ward DC, Reich E, Goldberg IH (1965) Base specificity in the interaction of polynucleotides with antibiotic drugs. Science 149:1259-1263[Medline]