DNA FLOW CYTOMETRY OF ISOLATED KERATINIZED EPITHELIA: A METHODOLOGICAL STUDY BASED ON ULTRASONIC TISSUE DISAGGREGATION

Ultrasonication of keratinized, stratified, squamous epithelium, which had been separated from underlying tissue by means of acetic acid, resulted in disaggregation of all cellular layers in the epithelium, giving a suspension of single nuclei with mitoses preserved. This suspension was treated with RNAse and ethidium bromide for analysis by flow cytometry. From the resulting DNA histogram the G₁, S and G₂+ M fractions were estimated using the computer program of Fried (1976). Treatment with dithiothreitol before sonication increased the yield of nuclei in suspension and decreased the amount of debris and clumps, thereby suppressing overestimation of small S fractions. This method of preparation prior to DNA flow cytometry was useful for the study of the hamster cheek pouch epithelium and of normal and pathological human epidermis.     

The circadian variations in the epithelial growth of the hamster cheek pouch: quantitative analysis of DNA distributions

The pronounced diurnal rhythm in DNA distributions of the hamster cheek pouch epithelium both in the S fraction and in the (G2 + M) fraction was compared with previous studies of the changes in tritiated thymidine labelling index and mitotic activity. The DNA distributions were obtained by flow cytometry after ultrasonic disaggregation of the isolated epithelium into a suspension of single nuclei. The DNA distributions were analysed with the computer program of J. Fried (1976) and by planimetry. The S fraction was higher than the autoradiographic labelling index during the whole 24 hr period. Only the computer fitted S fraction and the labelling index had the same difference between maximal and minimal values, and maxima at the same time of day. The DNA distributions showed a diurnal release of G1 cells into S phase proceeding through (G2 + M) phase and returning to G1 phase within a 24 hr period.     

The Circadian Variations In the Eithelial Growth of the Hamster Cheek Pouch: Quantitative Analysis of Dna Distributions*

The pronounced diurnal rhythm in DNA distribution of the hamster check pouch epithelium both in the S fraction and in the (G₂+ M) fraction was compared with previous studies of the changes in tritiated thymidine labelling index and mitotic activity. the DNA distributions were obtained by flow cytometry after ultrasonic disaggregation of the isolated epithelium into a suspension of single nuclei. the DNA distributions were analysed with the computer program of J. Fried (1976) and by planimetry. the S fraction was higher than the autoradiographic labelling index during the whole 24 hr period. Only the computer fitted S fraction and the labelling index had the same difference between maximal and minimal values, and maxima at the same time of day. the DNA distributions showed a diurnal release of G₁ cells into S phase proceeding through (G₂+ M) phase and returning to G₁ phase within a 24 hr period.     

Nuclear DNA content of Solanum species grown in vitro, as determined by flow cytometry

The DNA relative content in nuclei from several Solanum species, which were used as partners for somatic hybridization, were determined using a flow cytometry method. The nuclei were isolated mechanically or via protoplasts from leaves of in vitro grown plants. In the case of S. nigrum as well as S. tuberosum cv. Bzura and dihaploid clone H8105, the nuclei were also obtained from suspension cultured cells by lysis of protoplasts. The source and the method of nuclei isolation affected the pattern of nuclear DNA in the genotypes studied. The mesophyll nuclei showed two distinct peaks on the DNA histograms, whereas the DNA peaks produced by cell suspension nuclei were broad and less distinct. The DNA content in the nuclei, calculated from the DNA histograms of the samples and a DNA standard historgam (Trout Red Blood Cells, having DNA content of 5.05 pg per nucleus), were much lower in mesophyll nuclei than in those obtained from the cell suspension for the same genotypes. The results are discussed in respect of the genetic instability of Solanum genotypes. The usefulness of a flow cytometry approach in somatic hybridization research is also discussed.     

Isolation of plant nuclei suitable for flow cytometry from recalcitrant tissue by use of a filtration column

Flow cytometry is widely applied in the determination of nuclear DNA content and ploidy level in many organisms. However, a difficulty with flow cytometry is the method's intrinsic inability to tolerate large particles that associate with the isolated nuclei. A suspension of plant nuclei can often contain a high level of crystalline calcium oxalate, which blocks the fluidics system of the flow cytometer. We designed a cotton column and added polyvinylpyrrolidone-40 to the buffer to remove phenolic impurities and cytoplasmic compounds from plant nuclei, making the suspension suitable for flow cytometry. This simple and highly efficient protocol enables isolation of intact nuclei from plant tissues containing high levels of polysaccharides, calcium oxalate crystals and other metabolites. Our protocol resulted in the isolation of intact nuclei from mature orchid leaves. This method can be used on recalcitrant tissues and is particularly effective on plants containing calcium oxalate crystals.     

Computer-aided S-phase fraction determination in DNA static cytometry in breast cancer. A preliminary methodologic study on cytologic material.

This methodologic study was performed on a single-cell-cycle breast carcinoma to evaluate the feasibility of computer-aided S-phase fraction determination in DNA static cytometry. The investigation was performed on Feulgen-stained cytologic material in which the total optical density values of 1,000 consecutive, randomly selected nuclei were analyzed (MultiCycle software). A good correlation in the S-phase fraction value with flow cytometry was obtained when the G2/G1 ratio was fixed at 1.95, when the histogram data points were smoothed at least once and the coefficient of variation of the G2 peak was the same as that of G0-G1 or when a first-order S-phase polynomial model was used. The percentages of nuclei in G0-G1 and G2 were somewhat similar to those obtained with flow cytometry. The greatest discrepancy with flow cytometry was observed in the value of the coefficient of variation of the G0-G1 peak of the static cytometric data: it was at least twice as great. It always remained high despite the software options used. As for the influence of the sample size in the S-phase calculation, the software was also run on samples of 600 and 200 nuclei. When the G2/G1 ratio was fixed at 1.95, the data obtained from 600 nuclei did not differ from those obtained with 1,000 nuclei, whereas an analysis on 200 nuclei showed a substantial variation. The software also allowed calculation of the ratio of the G0-G1 peak of the neoplastic population against that of the diploid reference (DNA index), the value of which in flow cytometry was 1.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mimosine arrests proliferating human cells before onset of DNA replication in a dose-dependent manner

The synchronization effects of the plant amino acid mimosine on proliferating higher eukaryotic cells are still controversial. Here, I show that 0.5 mM mimosine can induce a cell cycle arrest of human somatic cells in late G1 phase, before establishment of active DNA replication forks. The DNA content of nuclei isolated from mimosine-treated cells was determined by flow cytometry. The presence or absence of DNA replication forks in these isolated nuclei was then detected by DNA replication run-on assays in vitro. Treatment of asynchronously proliferating HeLa or EJ30 cells for 24 h with 0.5 mM mimosine resulted in a population synchronized in late G1 phase. S phase entry was inhibited by 0.5 mM mimosine in cells released from a block in mitosis or from quiescence. When added to early S phase cells, 0.5 mM mimosine did not prevent S phase transit, but delayed progression through late stages of S phase after a lag of 4 h, eventually resulting in a G1 phase population by preventing entry into the subsequent S phase. In contrast, lower concentrations of mimosine (0.1-0.2 mM) failed to prevent S phase entry, resulting in cells containing active DNA replication foci. The G1 phase arrest by 0.5 mM mimosine was reversible upon mimosine withdrawal. This synchronization protocol using 0.5 mM mimosine can be exploited for studying the initiation of human DNA replication in vitro.     

A method for flow cytometric cell cycle analysis of normal and psoriatic human epidermis based on a detergent/citric acid technique for suspension of nuclei

A new method is described for flow cytometric cell cycle analysis of normal and psoriatic human epidermis, based on non-enzymatic tissue disaggregation. The epidermis was isolated by treatment with acetic acid and stored by freezing. After thawing, the epidermis was disintegrated into a nuclear suspension by 3 steps: incubation with dithiotreitol, whirling in a buffer (pH 7.4) with the non-ionic detergent Nonidet P40, EGTA, RNase and spermine, and whirling after addition of citric acid to a final concentration of 1% (pH 2.4). The suspension was stained with propidium iodide and filtered before flow cytometry. The yield of suspended nuclei was approximately 70% of the original number of cells in the tissue. The detergent/citric acid method was found to be preferable to an ultrasonication method previously used on human epidermis. All cell cycle and cell maturation stages were represented in the detergent/citric acid suspension, in contrast to the selection of immature G1, S and G2 stages with enzymatic methods. In the analysis of psoriatic epidermis inadequately matured (parakeratotic) cells were present in the suspension and had to be discriminated by gating on light scattering intensity, as they were not susceptible to lysis and did not stain properly. The fraction of S phase nuclei was on average 1.9% in normal and 7.7% in psoriatic epidermis, thus confirming the results of other investigators using enzymes. The presence of mitotic figures in the suspension was demonstrated by flow sorting. In this way the mitotic fraction was estimated to 0.06% in normal and 0.22% in psoriatic epidermis, confirming histological data of other investigators.     

Flow cytometric discrimination of mitotic cells: resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde

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.     

Flow cytometric quantification of human chromosome specific repetitive DNA sequences by single and bicolor fluorescent in situ hybridization to lymphocyte interphase nuclei

Fluorescent in situ hybridization allows for rapid and precise detection of specific nucleic acid sequences in interphase and metaphase cells. We applied fluorescent in situ hybridization to human lymphocyte interphase nuclei in suspension to determine differences in amounts of chromosome specific target sequences amongst individuals by dual beam flow cytometry. Biotinylated chromosome 1 and Y specific repetitive satellite DNA probes were used to measure chromosome 1 and Y polymorphism amongst eight healthy volunteers. The Y probe fluorescence was found to vary considerably in male volunteers (mean fluorescence 169, S.D. 35.6). It was also detectable in female volunteers (mean fluorescence 81, S.D. 10.7), because 5-10% of this repetitive sequence is located on autosomes. The Y probe fluorescence in males was correlated with the position of the Y chromosome cluster in bivariate flow karyotypes. When chromosome 1 polymorphism was studied, one person out of the group of eight appeared to be highly polymorphic, with a probe fluorescence 26% below the average. By means of fluorescent in situ hybridization on a glass slide and bivariate flow karyotyping, this 26% difference was found to be caused by a reduction of the centromere associated satellite DNA on one of the homologues of chromosome 1. The simultaneous hybridization to human lymphocyte interphase nuclei of biotinylated chromosome 1 specific repetitive DNA plus AAF-modified chromosome Y specific DNA was detected by triple beam flow cytometry. The bicolor double hybridized nuclei could be easily distinguished from the controls. When the sensitivity of this bicolor hybridization is improved, this approach could be useful for automatic detection of numerical chromosome aberrations, using one of the two probes as an internal control.     

Fluorescence in situ hybridization to interphase cell nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization

Summary Fluorescence hybridization to interphase nuclei in liquid suspension allows quantification of chromosome-specific DNA sequences using flow cytometry and the analysis of the three-dimensional positions of these sequences in the nucleus using fluorescence microscopy. The three-dimensional structure of nuclei is substantially intact after fluorescence hybridization in suspension, permitting the study of nuclear organization by optical sectioning. Images of the distribution of probe and total DNA fluroescence within a nucleus are collected at several focal planes by quantitative fluorescence microscopy and image processing. These images can be used to reconstruct the three-dimensional organization of the target sequences in the nucleus. We demonstrate here the simultaneous localization of two human chromosomes in an interphase nucleus using two probe labeling schemes (AAF and biotin). Alternatively, dual-beam flow cytometry is used to quantify the amount of bound probe and total DNA content. We demonstrate that the intensity of probe-linked fluorescence following hybridization is proportional to the amount of target DNA over a 100-fold range in target content. This was shown using four human/hamster somatic cell hybrids carrying different numbers of human chromosomes and diploid and tetraploid human cell lines hybridized with human genomic DNA. We also show that populations of male, female, and XYY nuclei can be discriminated by measuring their fluores-cence intensity following hybridization with a Y-chromosome-specific repetitive probe. The delay in the increase in Y-specific fluorescence until the end of S-phase is consistent with the results recorded in previous studies indicating that these sequences are among the last to replicate in the genome. A chromosome-17-specific repetitive probe is used to demonstrate that target sequences as small as one megabase (Mb) can be detected using fluorescence hybridization and flow cytometry.     

Analysis of Nuclear DNA content in plant cells by Flow cytometry

Flow cytometry was used to analyse the DNA content of nuclei isolated from intact plant tissues and from callus and cell suspension cultures invitro. Cell nuclei were isolated either mechanically (chopping, syringing) or by a hypotonic lysis of isolated protoplasts. Although both methods gave similar results, a slight shift to lower ploidy levels was observed after protoplast isolation from intact tissues and calli. No differences were observed if the two methods were compared using cell suspension cultures. The results showed that flow cytometry is a rapid method of nuclear DNA content analysis in intact plant tissues and variousin vitro cultures.     

Flow cytometric DNA analysis of corneal epithelium

We have modified an existing technique in order to perform DNA analysis by flow cytometry (FCM) of corneal epithelium from the mouse, rat, chicken, rabbit, and human. This protocol permitted an investigation of human corneal scrapings from several categories: normal, aphakic bullous keratopathy (ABK), keratoconus (KC), Fuch's dystrophy, edema, epithelial dysplasia, and lipid degeneration. No abnormal characteristic cell-kinetic profile was detected when averaged DNA histograms were compared statistically between the normal and either ABK, KC, edema, or Fuch's dystrophy groups. Abnormal DNA histograms were recorded for cell samples that were taken 1) from three individuals who had epithelial dysplasia and 2) from one individual diagnosed with lipid degeneration. The former condition was characterized by histograms that had a subpopulation of cells with an aneuploid amount of DNA or had higher than normal percentages of cells in the S and G2 + M phases of the cell cycle. Corneal cells from the patient who had lipid degeneration had an abnormally high percentage of cells in the G2 + M phases of the cell cycle. The availability of accurate DNA flow cytometric analysis of corneal epithelium allows further studies on this issue from both experimental and clinical situations.     

Simultaneous detection of cyclin B1, p105, and DNA content provides complete cell cycle phase fraction analysis of cells that endoreduplicate

BACKGROUND: DNA analysis of endoreduplicating cells is difficult because of the overlap between stem-line G2 + M cells and 4C G1 cells. Simultaneous flow cytometry of DNA and cyclin B1 analytically separates these populations. The objective here was to develop simultaneous flow cytometry of DNA, cyclin B1, and p105 (highly expressed in mitosis) for improved, complete cell cycle phase fraction analysis of endoreduplicating cell populations. METHODS: Monoclonal antibody, GNS-1, reactive with human cyclin B1, was conjugated with fluorescein at three different fluorochrome-to-protein (F/P) ratios and tested for optimal sensitivity in a flow cytometric assay. A formaldehyde-methanol fixation procedure was optimized for retention of p105 within mitotic cells by analytic titration of formaldehyde. p105 was stained indirectly with Cy5-conjugated secondary antibody, followed by GNS-1, and DNA was stained with Hoechst 33342. The specificity of p105 in this assay was tested by comparison of manual and flow cytometric mitotic indices and by sorting and microscopic inspection. RESULTS: F/P 4.1 provided optimal fluorescein labeling of GNS-1. Formaldehyde (0.5%), followed by methanol permeabilization, fixed cells sufficiently to quantify stem-line and endoreduplicated G1, S, G2, and M phase fractions. Kinetic measurements of these fractions for both populations were demonstrated. CONCLUSIONS: The fluorochrome-to-protein ratio is important and can be optimized objectively for these assays. A permeabilization-sensitive antigen (p105), previously requiring formaldehyde/detergent-fixed cell preparations, was shown to work equally well with formaldehyde/ methanol fixation. Three-laser, two-parameter intracellular antigen analysis can be successfully coupled with DNA content analysis. Cell cycle kinetic analysis of endoreduplicating populations should be improved.     

Flow cytometric analysis and sorting of human endometrial cells after immunocytochemical labeling for cytokeratin using a monoclonal antibody

Endometrial cells in suspension were stained with propidium iodide and a monoclonal antibody against a cytokeratin intermediate filament protein specific for glandular and columnar cells (RGE 53). In this way columnar epithelial cells of the normal endometrium and of adenocarcinomas can be distinguished and separated by flow cytometry from non-epithelial cells (fibroblasts and inflammatory cells) and squamous epithelial cells, all of which are negative for RGE 53. This makes it possible to analyse and also sort pure fractions of this particular tissue type for further studies. The use of propidium iodide allows simultaneous DNA flow cytometry of these columnar epithelial cells. Therefore, the use of antibodies to cytokeratin in combination with propidium iodide can be of help in analyzing and sorting pure fractions of both normal and malignant cells. This allows a more refined examination of complex cell mixtures using flow cytometry.     

Growth factor for cardiac hypertrophy

Cardiac size can be regulated by the balance in activity between cardiac growth factors and inhibiting factors, chalones. This study was undertaken to verify the role of the cardiac growth factor and its purification from hypertrophied hearts. For this propose the hypertrophied hearts of renovascular hypertensive rats were used. The purification was made by using an isoelectric focusing chromatography and the HPLC method. We examined the cardiac growth effect of the isolated fractions with cultured chicken embryonic cardiac myocytes. Simultaneously, the influence of these fractions on the cardiac cell cycle was examined by DNA analysis with the flow cytometric method. If the hearts were overloaded due to hypertension, the growth of the cardiac size could be induced by increased the level of five proteins with different molecular weight and with an isoelectric point of 8.3. The significant growth activities were observed at these five proteins compared to the absence of the fractions. For the appearance of these growth effect, it is necessary that the structure of the protein is there fundamentally as a form with a molecular weight of 27 k dalton. After addition of these isolated fractions, BrdU content is S and G2 phases by flow cytometry was increased. This change indicates that the cardiac myocytes are stimulated in form DNA synthesis.     

Synchronization of 9L rat brain tumor cells by centrifugal elutriation

Asynchronous 9L cells were separated into relatively homogeneously-sized populations using centrifugal elutriation with both a conventional collection method and a long collection method. A substantial increase in the homogeneity of the volume distributions and in the degree of synchrony of the separated fractions was obtained using the long collection method. Autoradiographic data indicated that fractions containing greater than or equal to 97% G1 cells, greater than or equal to 80% S cells, and 70-75% G2 cells could be routinely recovered with this procedure. Recovery in these fractions varied from 5 to 8% of the total number of cells elutriated. The colony forming efficiency (CFE) of cells from fractions representing each phase of the cell cycle was a constant 60-70%, which was comparable to the 60-80% usually found for asynchronous 9L cells. The percentage of cells in the G1, S, and G2 phases in the elutriated fractions was more accurately determined from the volume distribution than from computer fits of the DNA histogram obtained from flow cytometry. In genereal, the degree of synchrony was related to the coefficient of variation (CV) of the volume distributions of the elutriated fractions. The CV was about 14% for all elutriated fractions. When the greater than or equal to 97% G1 population was allowed to progress to S and G2, the CVs were about 17 and 20.2%, respectively. Thus, the best nonperturbing method for obtaining synchronous 9L cells in the S or G2 phases was direct elutriation with the long collection method.     

Flow cytometric investigation of heterogeneous copper-sensitivity in asynchronously grown Saccharomyces cerevisiae

The variable stress-sensitivity of individual cells within pure cultures is widely noted but generally unexplained. Here, factors determining the heterogeneous susceptibility to copper toxicity in Saccharomyces cerevisiae were examined with a rapid non-perturbing approach based on flow cytometry. By determination of the DNA content (with propidium iodide) in cell fractions gated by forward angle light scatter (an indicator of the cell volume), it was shown that forward angle light scatter measurements gave an approximation of the cell cycle stage. Thus, our observation that cells in different forward angle light scatter fractions displayed differing Cu-sensitivities indicated that heterogeneous Cu-sensitivity is a function of the cell cycle stage. Furthermore, cells sorted by their Cu-sensitivity and-resistance and subsequently analyzed for DNA content were found predominantly to occupy G1/S and G2/M cell cycle stages, respectively. The oxidant-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate was used to show that the Cu-sensitivity of G2/M phase S. cerevisiae was correlated with greater levels of pre-existing reactive oxygen species in these cells. The results indicate that differential Cu-sensitivity in a S. cerevisiae culture is linked to the cell cycle stage and this link may be determined partly by cell cycle-dependent fluctuations in basal reactive oxygen species generation.     

Bromodeoxyuridine labeling and flow cytometric identification of replicating Saccharomyces cerevisiae cells: lengths of cell cycle phases and population variability at specific cell cycle positions.

An immunofluorescent staining procedure has been developed to identify, with flow cytometry, replicating cells of Saccharomyces cerevisiae after incorporation of bromodeoxyuridine (BrdUrd) into the DNA. Incorporation of BrdUrd is made possible by using yeast strains with a cloned thymidine kinase gene from the herpes simplex virus. An exposure time of 4 min to BrdUrd results in detectable labeling of the DNA. The BrdUrd/DNA double staining procedure has been optimized and the flow cytometry measurements yield histograms comparable to data typically obtained for mammalian cells. On the basis of the accurate assessment of cell fractions in individual cell cycle phases of the asynchronously growing cell population, the average duration of the cell cycle phases has been evaluated. For a population doubling time of 100 min it was found that cells spend in average 41 min in the replicating phase and 24 min in the G2+M cell cycle period. Assuming that mother cells immediately reenter the S phase after cell division, daughter cells spend 65 min in the G1 cell cycle phase. Together with the single cell fluorescence parameters, the forward-angle light scattering intensity (FALS) has been determined as an indicator of cell size. Comparing different temporal positions within the cell cycle, the determined FALS distributions show the lowest variability at the beginning of the S phase. The developed procedure in combination with multiparameter flow cytometry should be useful for studying the kinetics and regulation of the budding yeast cell cycle.     

Flow cytometric determination of cell cycle parameters of V79 cells by continuous labeling with bromodeoxyuridine

A simple method with which to determine the cell cycle parameters, TG1, TS and TG2M (the durations of the G1, S and G2 + M phases) is described. V79 Chinese hamster lung cells were used to evaluate the method. After continuous labeling with bromodeoxyuridine (BrdU), V79 cells were stained with anti BrdU-monoclonal antibody with FITC (fluorescein isothiocyanate) and with PI (propidium iodide). The individual cells were checked by flow cytometry for green and red fluorescences whose signal intensities corresponded to the BrdU and cellular DNA contents. The durations of G1, S and G2 + M phases of V79 cells were determined by measuring the cell fractions containing the nonlabeled G1, labeled S and nonlabeled G2 + M phases. The reliability of this method is discussed.