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Bromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, respectively, can be discriminated from interphase cells using a new method, based on immunocytochemical staining of BrdUrd and flow cytometric four-parameter analysis of DNA content, BrdUrd incorporation, and forward and orthogonal light scatter. The method was optimized using the human leukemia cell lines HL-60 and K-562. Samples of 10(5) ethanol-fixed cells were treated with pepsin/HCl and stained as a nuclear suspension with anti-BrdUrd antibody, FITC-conjugated secondary antibody, and propidium iodide. Labelled mitoses could be discriminated from unlabelled mitoses, and from labelled and unlabelled G2 cells, by their intermediate log FITC fluorescence intensity. In addition, mitoses and G2 nuclei differed in forward and orthogonal light scattering, but had equal intensity of propidium iodide fluorescence. This method for discrimination of labelled mitoses was also tested on cultured normal adult human keratinocytes labelled with iododeoxyuridine (IdUrd). In keratinocytes, where the cell structure was preserved after pepsin/HCl, IdUrd labelled mitotic cells were similarly discriminated in the log FITC/propidium iodide fluorescence distribution. This interpretation was supported by experiments using mitotic arrest, fluorescence activated cell sorting and microscopy, and comparison with an alternative flow cytometric method for discrimination of mitoses.

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.

A novel procedure for calculating base-pair frequencies in whole genomes is reported. This has been developed during a study of the role of heterochromatin in microevolution. Closely related species of the Crepis praemorsa complex have similar karyotypes but for their heterochromatin. The changes in relative AT frequency between species have been attributed to heterochromatin sequences by in situ banding of chromosomes with two base-specific fluorochromes. The absolute genome size of species, measured by cytofluorometry, correlated positively with increased karyotypic heterochromatin, as did the proportion of AT bases in the DNA. However, the determination of base content has called for a curvilinear interpretation of data obtained with two base-specific fluorochromes (bisbenzimide Hoechst 33342 and mithramycin), in contrast to the commonly assumed but erroneous direct relationship between fluorescence intensity and base content. Essentially, the fluorochromes' requirements for a sequence of certain base-pairs lead to the notion of Coefficients of Overspecificity: the result is a simple formula for calculating the AT proportion in a genome relative to a reference species from cytometric data, taking account of ligand binding statistics. These statistics and probabilities of oligonucleotide binding are essentially the same.

DNA content determination requires the use of standards. Vindelov has shown the need to use two standards. Chicken and trout erythrocytes are commonly used, but they are not ideal standards. On the one hand, their DNA contents rarely frame the studied sample DNA content, and, on the other hand, as their base compositions are different in terms of A + T/G + C, their relative indices change according to the stains used. Use of triploid trout erythrocytes instead of chicken erythrocytes allows elimination of these two drawbacks; however, diploid trout must be differentiated from triploid trout. The present paper shows that an anatomic malformation is found with the triploid trout and so justifies the use of paired diploid and triploid trout as standards to measure nuclear DNA content.

We have examined, by flow cytometry, the apparent DNA content of frog blood cells that had been fixed with either 50% ethanol, 70% ethanol, or 66% methanol, before being stained with either mithramycin, propidium iodide, or Hoechst 33258. After 50% ethanol fixation, regardless of the dye used, the DNA content of the hemopoietic cells appeared unimodal, but after either 70% ethanol or 66% methanol fixation, it appeared bimodal. Cell sorting revealed that the lower and upper modes are represented by erythrocytes (RBCs) and leukocytes (WBCs), respectively. In amphibians, the chromatin of metabolically inactive RBCs is highly condensed relative to the chromatin of metabolically active WBCs. The bimodal distribution of DNA contents seen with 66% methanol and 70% ethanol, but not 50% ethanol, seems to reflect this disparity in the degree of chromatin condensation existing between the RBCs and WBCs. This, in turn, implies that the accessibility of fluorescent DNA dyes to the chromatin of fixed frog hemopoietic cells, especially of RBCs, can be affected by the concentration of alcohol used for their fixation.

Accessibility of mouse testicular and vas deferens (vas) sperm cell DNA to acridine orange, propidium iodide, ellipticine, Hoechst 33342, mithramycin, chromomycin A3, 4'6-diamidino-2-phenylindole (DAPI), and 7-amino-actinomycin D (7-amino-AMD) was determined by flow cytometry. Permeabilized cells were either stained directly or after pretreatment with 0.06 N HCl. For histone-containing tetraploid, diploid, and round spermatid cells, HCl extraction of nuclear proteins caused an approximately sixfold increase of 7-amino-AMD stainability but had no significant effect on DAPI stainability. For these same cell types, the stainability with other intercalating (acridine orange, propidium iodide, ellipticine) and externally binding (Hoechst 33342, mithramycin, chromomycin A3) dyes was increased by 1.6- to 4.0-fold after HCl treatment. In sharp contrast, HCl treatment of vas sperm did not increase the staining level of 7-amino-AMD, DAPI, or propidium iodide but did increase the staining level for the other intercalating dyes (1.3- to 1.5-fold) and external dyes (1.3- to 1.9-fold). Elongated spermatids that contain a mixture of protein types including histones, transition proteins, and protamines demonstrated the greatest variability of staining with respect to type of stain and effect of acid extraction of proteins. In general, for nearly all dyes, the round spermatids had an increased level and tetraploid cells had a decreased level of stainability relative to the same unit DNA content of diploid cells. The observed differential staining is discussed in the context of chromatin alterations related to the unique events of meiosis and protein displacement and replacement during sperm differentiation.

It is expected that the time sequence of cells passing through the sensing region of a flow cytometer should follow the statistics of a Poisson process. Thus, measurement of time intervals between passages of fluorescent microspheres and cells stained with mithramycin demonstrated the negative exponential distribution characteristic of such a process. However, analysis of cells stained with fluorescein-iso-thiocyanate showed marked deviations from a negative exponential distribution of time intervals. The distribution had an additional component due to time intervals with mean value about 60 times shorter than the population mean, and in certain cases more than 50% of the cells were registered with such short time intervals. This percentage could, however, be reduced to acceptable levels by modifications of the sample preparation procedures. Measurement of the distribution of time intervals between cell passages is proposed as a method for the evaluation of possible improvements in sample preparation procedures.

Highly synchronized cultures of HSF-55 human diploid fibroblasts contain subpopulations of cells with intact plasma membranes that do not participate in the parasynchronous division wave. To determine the fate of these laggard cells, cultures were incubated with BrdU for variable periods to label newly replicated DNA in both the readily synchronizable and nonsynchronizable subpopulations. The kinetics of labeling with BrdU were determined with a two-laser flow cytometric technique that did not employ antibody to BrdU, but instead monitored emission of fluorescence from DNA-specific stains that differed in the degree of BrdU-induced quenching of their fluorescence signals. Approximately 90% of the cells rapidly incorporated BrdU and later divided within a 3 hr period. The remaining 10% of the cells, however, were found to reside within a minority subpopulation that maintained the capacity to traverse the cell cycle, but at a greatly reduced rate relative to the progression capacity of the majority of cells. Cells were viably sorted from these cohorts within the synchronized culture, and their kinetic behavior was determined through direct measurement of their growth rates and plating efficiencies. As predicted by the BrdU labeling studies, the sorted cells from the minority, slowly traversing subpopulation divided at a rate that was 30 to 50% lower than that obtained with cells sorted from the readily synchronizable subpopulation. From consideration of the kinetics of entry into S-phase of the majority and minority subpopulations, protocols are described that should allow preparation of relatively pure populations of both early- and late-replicating species of human DNA.

The principle of a multiple wavelength illumination method for flow cytometers, based upon a combination of a helium-neon laser and an arc lamp as illumination sources is described. By using a prism, the light from the arc lamp is dispersed and the different colors are imaged at different places on the sample stream. The small angle light scattering from the helium-neon laser light is measured as a relevant parameter and serves as a trigger signal for subsequent measurements of fluorescence or scattering of light from the arc lamp. Two experimental systems are described utilizing this principle: a system where the emission is detected orthogonally with respect to the direction of the illumination beams, and an epi-illumination system. With the orthogonal set-up multiple wave-length right angle scattering measurements are possible. This is illustrated by showing that the orthogonal scattering from erythrocytes is strongly dependent on the illumination wavelength. It is further shown that the apparatus is suitable for the measurement of intracellular pH using the pH dependence of the excitation spectrum of fluorescein. The epi-illumination system allows excitation of two (or more) fluorescent dyes with different excitation spectra. In this case the emission spectra of the fluorescent dyes may overlap substantially. This is shown by simultaneous measurement of DNA and protein of Chinese hamster lung cells using mitramycin and tetramethyl rhodamin isothiocyanate (TRITC).

In cytometric clinical application, it is important to obtain cell suspensions rapidly with as little cytological alteration as possible. A procedure has been achieved to prepare cell suspensions for flow cytometric analysis. The leucoconcentration technique, first described by Herbeuval for cytologic analysis, has been modified to be applied in cytometry. This technique involves Saponin lysis of red cells of peripheral blood or bone marrow samples that have been previously fixed with picric acid alcohol solution. Cells in suspension are not shifted and tinctorial affinity is not modified. Then cells have been stained with Mithramycin. Each parameter defined by Crissman has been analyzed to define the best staining conditions. The availability of Leucoconcentration with Mithramycin-DNA-staining permits determination of cell cycle with a fine resolution.

Bromodeoxyuridine (BrdU) incorporation into cellular DNA has a differential effect on the cell-associated fluorescence of several DNA-specific dyes. After cells were treated with BrdU, flow microfluorometry was used to study the relative increase or decrease influorescence of stained cells. Bromodeoxyuridine incorporation into CHO cells increased the fluorescence of mithramycin-, olivomycin-, or chromomycin-stained cells, decreased that of propidium iodide-stained cells, and had little, if any, effect on the fluorescence of acriflavine Feulgen-stained cells. Changes in relative fluorescence of cell associated dyes are due to changes in the amounts of dye bound to cells with BrdU-substituted DNA. Colorimetric and absorbance measurement of DNA content showed that BrdU does not alter the diploid DNA content of CHO cells; however, BrdU induces perturbations in the distribution of cells about the cell cycle which cause an increase in average DNA content.

The leucoconcentration technique allows rapid obtainment of cellular suspensions from total blood or bone marrow for flow cytometric analysis. The technique is based on picric acid in ethyl alcohol fixation and saponin red cell lysis, followed by mithramycin staining for DNA. It gives a good resolution of DNA distributions that allow detection of slight variations in DNA content. These results were obtained with cellular suspensions differing only in one X or Y chromosome (male, female, Klinefelter and Turner syndromes). In these studies the ratio of the DNA content of X and Y chromosomes agrees with the chromosomal mass ratio already reported by other authors, but the "absolute values" are 10-fold more compared to these same works. Our conclusion is that leucoconcentration technique followed by DNA staining with mithramycin increases the difference in the dye's penetration and binding between X and Y chromosomes.

Detailed, simplified techniques are described for simultaneous staining and analysis of DNA and protein in a number of mammalian cell types. Cell staining involves the addition of appropriate dye mixtures to unfixed or ethanol-fixed cells and subsequent analysis of cell populations in the staining reagents generally within 10 to 20 minutes. The approach is novel in that no centrifugation steps are involved during the staining procedure, thus, eliminating cell clumping and cell loss and making the procedures appropriate for samples containing limited numbers of cells. For single wavelength analysis, staining of DNA and protein in ethanol-fixed cells was accomplished with a dye solution containing propidium iodide, fluorescein isothiocyanate and RNase. After 20 minutes at room temperature cells were analyzed using the 488 nanometer (nm) laser excitation line. For dual laser analysis the following dye combinations were employed without RNase: mithramycin-rhodamine 640, mithramycin-substituted rhodamine isothiocyanate, Hoechst 33342-rhodamine 640 and Hoechst 33342-rhodamine isothiocyanate. Unfixed cells were also stained with the Hoechst 33342-rhodamine 640 dye combination. Mithramycin was excited at 457.9 nm, Hoechst 33342 at 333-363 nm, and the rhodamine dyes at 568 nm. Cell types analyzed included Chinese hamster ovary cells, cultured mouse colon 26 cells, mouse embryo forelimb bud cells, and rat cell samples obtained by lung lavage.

The high condensation and flat shape of the mammalian sperm nucleus present unique difficulties to flow cytometric measurement of DNA content. Chromatin compactness makes quantitative fluorescent staining for DNA difficult and causes a high index of refraction. The refractive index makes optical measurements sensitive to sperm head orientation. We demonstrate that the optical problems can be overcome using the commercial ICP22 epiillumination flow cytometer (Ortho Instruments, Westwood, MA) or a specially built cell orientating flow cytometer (OFCM). The design and operation of the OFCM are described. Measurements of the angular dependence of fluorescence from acriflavine stained rabbit sperm show that it is capable of orienting flat sperm with a tolerance of +/- 7 degrees. Differences in the angular dependence for the similarly shaped bull and rabbit sperm allow discrimination of these cells. We show that DNA staining with 4-6 diamidino-2-phenylindole (DAPI) or an ethidium bromide mithramycin combination allows resolution of the X and Y populations in mouse sperm. They have also been successful with sperm from the bull, ram, rabbit, and boar. Reliable results with human sperm are not obtained. The accuracy of the staining and measurement techniques are verified by the correct determination of the relative DNA content of these two populations in sperm from normal mice and those with the Cattanach [7 to X] translocation. Among the potential uses of these techniques are measurement of DNA content errors induced in sperm due to mutagen exposure, and assessment of the fractions of X and Y sperm in semen that may have one population artifically enriched.

Cells in mitosis can be flow cytometrically discriminated from G1, S, and G2 cells by analysis of a nuclear suspension prepared with nonionic detergent, fixed with formaldehyde, and stained with mithramycin, propidium iodide, or ethidium bromide. With these DNA-fluorochromes, the fluorescence is quenched by formaldehyde less in mitotic nuclei than in interphase nuclei. Mitotic nuclei have a 20-40% increased mithramycin fluorescence and 30-60% decreased light scatter in comparison to those of G2 nuclei. There is a high correlation (r = 0.95; P less than 0.001) between microscope counts of mitotic figures in smear preparations of the initial cell suspension and the flow cytometrically estimated fraction of nuclei with increased mithramycin fluorescence. Flow sorting (FACS) demonstrates that the mitotic nuclei are confined to the peak of increased mithramycin fluorescence and decreased light scatter. The method has been applied to cultures of Yoshida ascites tumor cells, JB-1 reticulosarcoma cells, and PHA-stimulated human lymphocytes, incubated in the presence or absence of vinblastine for mitotic arrest. In a heteroploid mixture of fixed Yoshida (near-diploid) and JB-1 (hypotetraploid) nuclei, the mitotic fractions of the two cell lines could be estimated separately when analyzed with mithramycin fluorescence versus light scatter or with mithramycin fluorescence versus propidium iodide fluorescence.