Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y

The addition of RNA content estimation to flow cytometric measurement of DNA content provides valuable information concerning cells' transitions between quiescent and proliferative states. Equilibrium staining methods employing acridine orange have been used for DNA/RNA content measurement but are difficult to apply to intact cells and impractical for use in conjunction with fluorescent antibodies or ligands for demonstration of cell surface structures. I have used a combination of Hoechst 33342 (HO342) and pyronin Y (PY) to stain intact cells for DNA/RNA content estimation with a dual source flow cytometer using UV and blue-green or green excitation, measuring HO342 fluorescence at 430--470 nm and PY fluorescence at 590--650 nm. Results obtained with cultured cells and stimulated lymphocytes are in good agreement with those obtained using acridine orange for DNA/RNA staining; about half of the PY fluorescence can be removed from ethanol-fixed cells stained with HO342 and PY by RNAse digestion. The HO342/PY method can be combined with fluorescein immunofluorescence for detection of cell surface markers. HO342 can be combined with other tricyclic heteroaromatic dyes for DNA/RNA estimation; the combination of HO342 and oxazine 1 can be excited in a dual source instrument using a mercury arc lamp and a helium-neon laser. The staining procedure is simple; cells in medium are incubated with 5 microM HO342 at 37 degrees C for 45 min, 5 microM PY (or oxazine 1) is then added and cells are analyzed without washing after an additional 45 min incubation. Suitability of these dye combinations for vital cell staining and sorting remains to be determined.     

Three-color flow cytometry analysis of tricistronic expression of eBFP, eGFP, and eYFP using EMCV-IRES linkages.

BACKGROUND: The ability to quickly analyze and sort double or triple fluorescent reporter constructs using simultaneous analysis provides significant flexibility in the solution of analytical and process-related questions in biotechnology. METHODS: Bicistronic eBFP/eGFP and eBFP/eYFP constructs were made on two mammalian episomal plasmids using an internal ribosomal entry sequence from encephalomyocarditis virus (EMCV-IRES) to link two GFP expressions. Simultaneous two-color flow cytometry (FCM) analysis was accomplished using a dual Argon-laser multi-line configuration set at excitation wavelengths of 360 and 488 nm. Blue fluorescence emission (440 nm) and green fluorescence emission (507 nm) were detected using 405/20 (FL4) and 510/20 (FL1) bandpass filters. Dual eBFP/eYFP and three-color simultaneous analysis of eBFP/eGFP/eYFP was accomplished using the dual-laser configuration but also using a short-pass (525-nm) dichroic mirror and 550/30 bandpass filter configuration to detect yellow fluorescence emission (527 nm) in a third channel (FL2). RESULTS: Human 293 cells transfected with the bicistronic construct of eBFP-IRES-eGFP were easily detected using simultaneous analysis, and the signals were well separated with a mean blue fluorescent intensity (MFI) in the 2nd-log decade (FL4) and green MFI in the 4th-log decade (FL1). Likewise, eBFP-IRES-eYFP transfected cells were as easily detected and also demonstrated very good signal separation. A tricistronic construct of eBFP-IRES-eGFP-IRES-eYFP was also made and transfected into 293 cells. Triple-color fluorescent cells were easily detected using the cytometer configuration for simultaneous analysis. All three signals separated with only moderate compensation required for green and yellow emission spectra. The respective MFI for each of the fluorescent proteins was correlative to what had been observed with the separate bicistronic constructs. CONCLUSIONS: Our results demonstrate that we have developed a novel fluorescent flow cytometry method that can be used as a powerful tool to differentiate and analyze three colors simultaneously from either a dual or a triple cistronic construct which has been transfected into living cells.     

High resolution chromosome analysis: one and two parameter flow cytometry

Isolated mammalian chromosomes have been quantitatively classified by high resolution flow cytometry. Chinese hamster chromosomes stained with 33258 Hoechst and excited in the UV showed a fluorescence distribution in which the 14 types of Chinese hamster chromosomes were resolved into 16 groups seen as distinct peaks in the distributions. Chinese hamster chromosomes were also stained with both 33258 Hoechst (HO) and chromomycin A3 (CA3); the two dye contents were measured by selective excitation in the UV and at 458 nm in a dual beam flow cytometer. The resulting two parameter distribution (HO versus CA3) showed 10 chromosome groups¹. Human strain LLL 761 chromosomes stained with HO and excited in the UV showed a fluorescence distribution in which the 23 types of human chromosomes were resolved into 12 groups. Human chromosomes stained with both HO and CA3 and measured in the dual beam flow cytometer produced two parameter fluorescence distributions which showed 20 groups. The chromosomes associated with each group were determined by quinacrine banding analysis of sorted chromosomes and by DNA cytophotometry of preidentified metaphase chromosomes. The relative HO and CA3 stain content and frequency of occurrence of chromosomes in each group were determined from the fluorescence distributions and compared to the results from DNA cytophotometry. The chromosome to chromosome variations in HO and CA3 staining are attributed to variations in chromosomal base composition.     

The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem

Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4′,6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an apparent photoconversion of DAPI that results in detection of a DAPI signal using a standard filter set for detection of green emission due to blue excitation. When a sample stained with DAPI only was first imaged with the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence was observed. Next, we imaged the sample with a DAPI filter set, obtaining a strong nuclear DAPI signal as expected. Upon reimaging the same samples with a FITC/GFP filter set, robust nuclear fluorescence was observed. We conclude that excitation with UV results in a photoconversion of DAPI that leads to detection of DAPI due to excitation and emission in the FITC/GFP channel. This phenomenon can affect data interpretation and lead to false-positive results when used together with fluorochrome-labeled nuclear proteins detected with blue excitation and green emission. In order to avoid misinterpretations, extra precaution should be taken to prepare staining solutions with low DAPI concentration and DAPI (UV excitation) images should be acquired after all other higher wavelength images. Of various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion while that for DAPI and Hoechst 33258 was much stronger. Different fixation methods did not substantially affect the strength of photoconversion. We also suggest avoiding the use of mounting medium with high glycerol concentrations since glycerol showed the strongest impact on photoconversion. This photoconversion effect cannot be avoided even when using narrow bandpass filter sets.     

Violet laser diodes as light sources for cytometry

BACKGROUND: Violet laser diodes have recently become commercially available. These devices emit 5-25 mW in the range of 395-415 nm, and are available in systems that incorporate the diodes with collimating optics and regulated power supplies in housing incorporating thermoelectric coolers, which are necessary to maintain stable output. Such systems now cost several thousand dollars, but are expected to drop substantially in price. Materials and Methods A 4-mW, 397-nm violet diode system was used in a laboratory-built flow cytometer to excite fluorescence of DAPI and Hoechst dyes in permeabilized and intact cells. Forward and orthogonal light scattering were also measured. RESULTS: DNA content histograms with good precision (G(0)/G(1) coefficient of variation 1.7%) were obtained with DAPI staining; precision was lower using Hoechst 33342. Hoechst 34580, with an excitation maximum nearer 400 nm, yielded the highest fluorescence intensity, but appeared to decompose after a short time in solution. Scatter signals exhibited relatively broad distributions. CONCLUSIONS: Violet laser diodes are relatively inexpensive, compact, efficient, and quiet light sources for DNA fluorescence measurement using DAPI and Hoechst dyes; they can also excite several other fluorescent probes.     

DAPI staining improved for quantitative cytofluorometry

DNA-DAPI complexes emit strong bluish white fluorescence when excited by ultraviolet light so that even very small amounts of DNA such as those in mitochondria, chloroplasts, and virus particles can be visualized. Moreover, the staining procedure with DAPI is very simple and requires no hydrolysis. However, DAPI staining was considered unsuitable for quantitative purpose; nonspecific cytoplasmic fluorescence, scattering of strong emission light, and fading of the fluorescence under UV excitation were major problems of DAPI staining in quantitative cytofluorometry. We found that (1) nonspecific cytoplasmic fluorescence could be eliminated by reducing the DAPI concentration to 50 ng/ml, (2) fluorescence decay was markedly decreased by adding electron donors and molecules containing SH radicals in the mounting media, and (3) light scattering became negligible after reducing the intensity of the excitation light. Thus satisfactory precision could be obtained in DNA quantification by epifluorescent cytophotometry on DAPI stained specimens.     

"Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: potential application at microgravity conditions in space

BACKGROUND: Conventional staining of cells or tissue sections on microscope slides involves immersing the slides into solutions of dyes then rinsing to remove the unbound dye. There are instances, however, when use of stain solutions is undesirable-e.g., at microgravity conditions in space, where the possibility of accidental spill (many dyes are known carcinogens) introduces health hazard. Likewise, transporting bulk of liquid stains and rinses may be burdensome in certain situations such as field expeditions or combat. METHODS: The "liquidless" staining procedure is proposed in which the dyes are contained in thin strips of hydrated polyacrylamide or gelatin gels that have been presoaked in the stain solutions. Fluorochromes that have affinity to DNA (propidium iodide, PI; 4,6-diamidino-2-phenylindole, DAPI, Hoechst 33342) or to protein (sulforhodamine 101) were used to saturate the gels. The gel strips were placed over the prefixed cells or tissue sections deposited on microscope slides and relatively low (20 g/cm2) pressure was applied to ensure the contact. The cells were also stained by using commercially available mounting media into which DAPI or PI were admixed. Intensity of fluorescence of the PI stained cells was measured by laser scanning cytometry (LSC). RESULTS: Satisfactory cell and tissue staining, with minimal background, was achieved after 10-20 min contact between the cells and gels. Optimal concentrations of the dyes in the solutions used to presoak the gels was found to be 2-4-fold higher than the concentrations used routinely in cytometry. The measurements of intensity of cellular fluorescence by LSC revealed that the staining of DNA was stoichiometric as reflected by the characteristic cellular DNA content frequency histograms with distinct G1, S, and G2/M cell populations and 2:1 ratio of G2/M to G1 peak fluorescence. Individual gels can be saturated with more than a single dye-e.g., to obtain differential DNA and protein staining. Cell staining with DAPI or PI in the gelatin-based mounting media led to high fluorescence background while staining with DAPI in "aqueous" medium was satisfactory. CONCLUSIONS: Relatively fast staining of cells or tissue sections on microscope slides can be achieved by nonconvective dye diffusion using hydrated gels permeated with the dyes, applied to cells at low pressure. The quality of the staining provided by this methodology is comparable to conventional cell staining in dye solutions.     

Mapping of antibody responses to the protective antigen of Bacillus anthracis by flow cytometric analysis

BACKGROUND: Different plant species vary as to the ratio of nucleotide base pairs of genomic DNA. A correlation between genome size and base pair ratio has been claimed. Base composition can be analyzed by base-specific dyes. METHODS: Genome size is determined by flow cytometry of suspensions of nuclei stained by the base independent dye, PI. For estimation of the AT frequency, the AT-specific dyes 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI) and Hoechst 33342 (HO) were used. We define a dye factor (DF) as the ratio of the two estimates (peak ratios) of nuclear fluorescence intensities of sample relative to reference plant nuclei using a given dye and an intercalating fluorochrome. RESULTS: No significant correlation between genome size and the DF for DAPI was found when 54 plant species were investigated. However, similarities within and differences among the plant families were shown. The comparison of DAPI and HO DFs gave no consistent differences as would be predicted from the model of different binding site length of dyes. This result may be explained by the nonrandom distribution of base pairs. CONCLUSIONS: There is no general correlation between genome size and AT/GC ratio in higher plants. Similar AT/GC ratios within a plant family result from the general similarity of the DNA sequences within a family. The fluorescence of base-specific dyes is influenced by the nonrandom distribution of bases in the DNA molecule.     

A flow cytometric study of chromosomes from rat kangaroo and chinese hamster cells

Summary Chromosomes from rat kangaroo (PTK) and chinese hamster (CHV 79) cells have been prepared for quantitative flow-cytometric analysis. The preparation time was optimized down to 30 (PTK) and 40 min (CHV 79). DAPI was used as a AT-sensitive fluorescent dye to stain for monoparameter DNA measurements. Simultaneous two-parameter DNA-protein analysis was carried out with DAPI and SR 101 (as a general protein fluorochrome) in combination. The karyotype of the PTK cells with 13 (14) chromosomes was separated into 10 DNA peaks. The X-chromosome bearing the nucleolus organizer region generates a distinct peak. The karyotype of the CHV 79 cells with 22 chromosomes was separated into 15 peaks. The DNA profile obtained indicates a geometric grading of the chromosomal amount of AT components in the karyotype of this particular cell line. The simultaneous DNA-protein analysis performed show enough sensitivity of the instrument utilizing high power UV excitation illumination to discriminate the two color emission consisting of blue (DAPI) and red (SR 101) fluorescence. Color overlapping could be completely avoided. Additionally, the quality (number, location, and resolution of peaks) of the DNA distribution was not influenced by the simultaneous application of a second fluorescent stain. Fluorescence activated electronic sorting applied on chromosomal fluorescence distributions providing purified fractions of chromosomes for subsequent biochemical and biological determinations is discussed.     

Flow cytometric analysis of fluorescence in situ hybridization with dye dilution and DNA staining (flow-FISH-DDD) to determine telomere length dynamics in proliferating cells.

BACKGROUND: Telomeres shorten during DNA replication; extensive erosion of telomeres likely promotes replicative senescence and chromosomal instability. Telomere length in individual cells has been quantified by flow cytometric analysis of fluorescence in situ hybridization (flow-FISH). To determine the rate of telomere attrition (telomere erosion per cell division), we combined flow-FISH with dye dilution and DNA staining (flow-FISH-DDD) and measured telomere-specific fluorescence in proliferating cells identified by cell generation and cell cycle phase. METHODS: Peripheral blood mononuclear cells (PBMC) were stained with the cell division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE), stimulated with phytohemagglutinin (PHA), grown for 5-6 days, hybridized with a telomere sequence-specific peptide nucleic acid fluorescent probe (PNA-Cy5), counterstained with DAPI, and analyzed by flow cytometry. The cell cycle distribution and cell division generations were respectively identified by analysis of DAPI emission and deconvolution of CFSE emission, and Cy5 emission was used to determine telomere-specific fluorescence, an indicator of telomere length, in each cell. RESULTS: In stimulated PBMC, in each cell cycle phase, the telomere-specific fluorescence diminished with increasing cell generation. The rate of decline of the telomere-specific fluorescence per cell generation did not significantly differ between cell cycle phases. CONCLUSIONS: Application of flow-FISH-DDD to measure mean telomere length and the rate of telomere attrition in proliferating cells may find use in studies of ageing and disease, the effects of telomere-modifying agents, and variability between individuals.     

Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy

BACKGROUND: The multiparameter fluorometric analysis of intact and fixed cells often requires the use of a nuclear DNA discrimination signal with spectral separation from visible range fluorochromes. We have developed a novel deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex(lambdamax) 646 nm; Em(lambdamax) 681 nm; Em(lambdarange) 665->800 nm), with high affinity for DNA and a high capacity to enter living cells. We describe here the spectral characteristics and applications of this synthetic compound, particularly in relation to cytometric analysis of the cell cycle. METHODS: Cultured human tumor cells were examined for the ability to nuclear locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM) and multiparameter flow cytometry. RESULTS: Multiparameter flow cytometry shows that the dye can rapidly report the cellular DNA content of live and fixed cells at a resolution level adequate for cell cycle analysis and the cycle-specific expression of cellular proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser red lines (633/647 nm) was found to offer a means of fluorescence signal discrimination by selective excitation, with greatly reduced emission overlap with UV-excitable and visible range fluophors as compared with propidium iodide. LSM reveals nuclear architecture and clearly defines chromosomal elements in live cells. DRAQ5 was found to permit multiphoton imaging of nuclei using a 1,047-nm emitting mode-locked YLF laser. The unusual spectral properties of DRAQ5 also permit live cell DNA analysis using conventional 488 nm excitation and the single-photon imaging of nuclear fluorescence using laser excitation between 488 nm and low infrared (IR; 780 nm) wavelengths. Single and multiphoton microscopy studies revealed the ability of DRAQ5 to report three-dimensional nuclear structure and location in live cells expressing endoplasmic reticulum targeted-GFP, MitoTracker-stained mitochondria, or a vital cell probe for free zinc (Zinquin). CONCLUSION: The fluorescence excitation and emission characteristics of DRAQ5 in living and fixed cells permit the incorporation of the measurement of cellular DNA content into a variety of multiparameter cytometric analyses.     

A comparison of low-power helium-cadmium and argon ultraviolet lasers in commercial flow cytometers

The feasibility of installing a low power ultraviolet (UV) laser in a commercial flow cytometer was evaluated by testing an Ortho Cytofluorograf 50HH and a Coulter Epics V. Both instruments were equipped with two argon ion lasers, one emitting at 488 nm and the other in the UV region and were tested by measuring the DNA content of cells stained with Hoechst 33342 or DAPI. The coefficient of variation (CV) of the G0/G1 peak of the DNA histograms produced by each instrument did not deteriorate markedly when results obtained at 100-125 mW were compared to those obtained at 10 mW. These tests indicated that a helium-cadmium laser (He-Cd) which can produce 10 mW at 325 nm should work well as a UV laser in these instruments. An Ortho Cytofluorograf IIs was purchased with a 10 mW He-Cd laser installed in the forward position. Studies of DNA content have confirmed that this low power UV laser can produce CVs of 2.2% with DAPI stained fixed cells and 3.6% with Hoechst 33342 stained viable lymphocytes. Thus, the He-Cd laser should provide a reasonable alternative as a UV source for flow cytometers.     

Observations of mitochondria and mitochondrial nuclei by double staining with rhodamine-123 and DAPI in synchronous cultures ofCatharanthus roseus

Mitochondria in cells ofCatharanthus roseus (L.) G. Don in synchronous cell division cultures were observed by double staining using fluorescence microscopy. The cells were stained with 4′-6-diamidino-2-phenylindole (DAPI) first and subsequently stained with rhodamine 123 (r-123). Immediately after staining with r-123, yellowishgreen, elongated and moving mitochondria were observed upon excitation at 485 nm. When the excitation filters were replaced by a UV filter (360 nm), 1 to 7 mitochondrial nucleoids were visible in each mitochondrion in the same field. Changes in the lengths of mitochondria during the cell cycle obtained from the observations under fluorescence microscopy by this staining method suggest the occurrence of multiplication of mitochondria concurrent with the cell cycle ofC. roseus.     

Counterstaining human chromosomes for flow karyology

Isolated human metaphase chromosomes stained with the fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) and chromomycin A3(CA3), and counterstained with nonfluorescent netropsin (NTR), have been analyzed by dual-laser flow cytometry. Counterstaining with NTR reduces DAPI fluorescence except at regions on chromosomes 1,9,15,16, and Y, corresponding to C-band heterochromatin. Bivariate flow karyology of human chromosomes treated with this triple-stain combination resolves chromosomes 1,9, and Y distinctly from the remaining chromosomes and resolves variations between chromosome homologues not detected by staining with propidium iodide (PI) or with the double stain combination Hoechst 33258(HO) and CA3.     

Flow Cytometric Analysis of Marine Bacteria with Hoechst 33342

We investigated the accuracy and precision of flow cytometric (FCM) estimates of bacterial abundances using 4′, 6-diamidino-2-phenylindole (DAPI) and Hoechst 33342 (HO342, a bisbenzamide derivative) on paraformaldehyde-fixed seawater samples collected from two stations near Oahu, Hawaii. The accuracy of FCM estimates was assessed against direct counts by using epifluorescence microscopy. DAPI and HO342 differ in two aspects of their chemistry that make HO342 better suited for staining marine heterotrophic bacteria for FCM analysis. These differences are most important in studies of open-ocean ecosystems that require dual-beam FCM analysis to clearly separate heterotrophic bacterial populations from populations of photosynthetic Prochlorococcus spp. Bacterial populations were easier to distinguish from background fluorescence when stained with HO342 than when stained with DAPI, because HO342 has a higher relative fluorescence quantum yield. A substantially higher coefficient of variation of blue fluorescence, which was probably due to fluorescent complexes formed by DAPI with double-stranded RNA, was observed for DAPI-stained populations. FCM estimates averaged 2.0 and 12% higher than corresponding epifluorescence microscopy direct counts for HO342 and DAPI-stained samples, respectively. A paired-sample t test between FCM estimates and direct counts found no significant difference for HO342-stained samples but a significant difference for DAPI-stained samples. Coefficients of variation of replicate FCM abundance estimates ranged from 0.63 to 2.9% (average, 1.5%) for natural bacterial concentrations of 6 × 10⁵ to 15 × 10⁵ cells ml^-1.     

Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation

The fluorescent dye 4′-6-Diamidino-2-phenylindole (DAPI) is frequently used in fluorescence microscopy as a chromosome and nuclear stain because of its high specificity for DNA. Normally, DAPI bound to DNA is maximally excited by ultraviolet (UV) light at 358 nm, and emits maximally in the blue range, at 461 nm. Hoechst dyes 33258 and 33342 have similar excitation and emission spectra and are also used to stain nuclei and chromosomes. It has been reported that exposure to UV can convert DAPI and Hoechst dyes to forms that are excited by blue light and emit green fluorescence, potentially confusing the interpretation of experiments that use more than one fluorochrome. The work reported here shows that these dyes can also be converted to forms that are excited by green light and emit red fluorescence. This was observed both in whole tissues and in mitotic chromosome spreads, and could be seen with less than 10-s exposure to UV. In most cases, the red form of fluorescence was more intense than the green form. Therefore, appropriate care should be exercised when examining tissues, capturing images, or interpreting images in experiments that use these dyes in combination with other fluorochromes.     

Establishment of a monoclonal antibody recognizing ultraviolet light-induced (6-4) photoproducts

We obtained a monoclonal antibody directed against UV-induced DNA damage. Analysis of the antigenic determinant in UV-irradiated DNA recognized by this antibody, 64M-1, revealed that it bound UV-irradiated oligo- or poly-nucleotides containing thymine-thymine or thymine-cytosine sequences. The antibody failed to bind DNA irradiated with 313 nm UV in the presence of acetophenone, which contained predominantly thymine dimers as DNA damage. The binding activity of this antibody to 254-nm UV-irradiated DNA decreased with 313-nm UV irradiation, and the decrease of this binding activity correlated with the decrease of fluorescence corresponding to (6-4) photoproducts. These results suggest that the antigenic determinant recognized by this monoclonal antibody is a (6-4) photoproduct. Using autoradiography with 3H-antibody, we could detect the formation of the (6-4) photoproduct in individual human cells irradiated with 254-nm UV doses as low as 20 J/m2.     

Flow cytometric discrimination between Acinetobacter calcoaceticus 69-V and Alcaligenes eutrophus JMP134 by fluorescently labelled rRNA-targeted oligonucleotide probes and DNA staining

In situ hybridization with fluorescently monolabelled rRNA-targeted oligonucleotide probes (17 to 18 nucleotides) was used to discriminate between Alcaligenes eutrophus JMP 134 and Acinetobacter calcoaceticus 69-V by flow cytometry. The strains were grown in batch experiments in a mixed population. The forward light scatter and fluorescence of each bacterial cell were measured with a single laser cytometer. The intensity of fluorescence after rRNA staining depended on the content of ribosomes, which correlated with the growth rate of bacteria. Therefore exponentially growing cells could be clearly detected. For other growth phases, signal amplification was necessary using multiple probes. The two bacterial strains were identified with differently labelled probes under an epifluorescent microscope. Using a single laser cytometer, rRNA based identification was possible nut not ideal. Better discrimination between the two strains of the mixed population was achieved by DNA staining, combined with the different forward light scatter signals. Due to the significantly different cellular DNA and GC content of both strains, the fluorescent dye DAPI (4′, 6-diamidino-2-phenylindole), preferring AT-rich regions of DNA, was found to be a supplementary tool for population analysis. The abundance ratios of the two strains in mixed culture determined by DNA or rRNA staining were similar.     

Light-induced permeabilization and merocyanine 540 staining of mouse spleen cells

Merocyanine 540 (M540) is a potential-sensitive, hydrophobic dye that preferentially incorporates into the 'fluid' domains of cellular membranes, distinguishing between hemopoietic cells according to their differentiation state. A bright staining with M540 is usually achieved by UV illumination of the cells during staining. We show by flow cytometric analysis that: (1) staining is greatly enhanced by UV illumination of mouse spleen cells before addition of the dye; (2) UV treatment causes an increased permeability toward propidium iodide and intracellular fluorescein as well; (3) the increment in M540 fluorescence precedes permeabilization to propidium iodide, while the latter precedes leakage of fluorescein. We also describe an overshoot and accelerated recovery of M540 fluorescence after photobleaching by a 514 nm laser beam. It is suggested that penetration of M540 to the more fluid inner membrane structures explains the fluorescence increment in both experiments.     

Development of a mechanism-based, DNA staining protocol using SYTOX orange nucleic acid stain and DNA fragment sizing flow cytometry

Accurate measurement of single DNA fragments by DNA fragment sizing flow cytometry (FSFC) depends upon precise, stoichiometric DNA staining by the intercalating dye molecules. In this study, we determined the binding characteristics of a commercially available 532 nm wavelength-excitable dye and used this information to develop a universal DNA staining protocol for DNA FSFC using a compact frequency-doubled Nd:YAG laser excitation source. Among twelve 532 nm wavelength-excitable nucleic acid staining dyes tested, SYTOX Orange stain showed the highest fluorescence intensity along with a large fluorescence enhancement upon binding to double-stranded DNA ( approximately 450-fold). Furthermore, using SYTOX Orange stain, accurate fragment-size-distribution histograms were consistently obtained without regard to the staining dye to base pair (dye/bp) ratio. A model describing two binding modes, intercalation (primary, yielding fluorescence) and external binding (secondary, involving fluorescence quenching), was proposed to interpret the performance of the dyes under different dye/bp ratios. The secondary equilibrium dissociation constant was found to be the most critical parameter in determining the sensitivity of each fluorophore to the staining dye/bp ratio. The measurements of both equilibrium dissociation constants provided us with a theoretical framework for developing a universal protocol which was successfully demonstrated over a wide range of DNA concentrations on a compact flow cytometer equipped with a frequency-doubled, diode-pumped, solid-state Nd:YAG laser for rapid and sensitive DNA fragment sizing.