Flow cytometric analysis of chromosomes and cells using a modified BrdU-Hoechst method

Chromosomes and interphase cells were harvested from cultures of the Chinese hamster line B14 F28 grown in medium containing BrdU up to four cell cycles and stained with the fluorescent dye 33342 Hoechst for flow cytometry. The newly synthetized BrdU-DNA is not stainable by the Hoechst dye which is highly specific for thymidine. The temporal development of the DNA fluorescence after addition of BrdU to the growth medium has been investigated. The chromosomal fluorescence intensity is reduced one step per generation. The extent of the intensity decrease by BrdU incorporation is proportional to the amount of new DNA and it is realized by repeated measurement following an UV-exposure. This UV-illumination stops the quenching by BrdU of the Hoechst stain induced DNA fluorescence. Therefore, the entire DNA content of these chromosomes now becomes measurable. The obtained intensity gain serves as a measure of the extent of the previous BrdU caused intensity shift. In this way we could establish 3 successive mitoses. Principally, this method is suitable also for measurement of whole cells in order to obtain both the number of generations in the experimental period and the phase distribution of the cell cycle.     

Flow cytometric analysis of the chromosomes and stability of a wheat cell-culture line

A rapidly growing, long-term suspension culture derived from Triticum aestivum L. (wheat) was synchronized using hydroxyurea and colchicine, and a chromosome suspension with chromosomes was made. After staining with the DNA-specific fluorochromes Hoechst 33258 and Chromomycin univariate and bivariate flow-cytometry histograms showed 15 clearly resolved peaks corresponding to individual chromosome types or groups of chromosomes with similar DNA contents. The flow karyotype was closely similar to a histogram of DNA content measurements of Feulgen-stained chromosomes made by microdensitometry. We were able to show the stability of the flow karyotype of the cell line over a year, while a parallel subculture had a slightly different, stable, karyotype following different growth conditions. The data indicate that flow cytometric analysis of plant karyotypes enables accurate, statistically precise chromosome classification and karyotyping of cereals. There was little overlap between individual flow-histogram peaks, so the method is useful for flow sorting and the construction of chromosome specific-recombinant DNA libraries. Using bivariate analysis, the AT:GC ratio of all the chromosomes was remarkably similar, in striking contrast to mammalian flow karyotypes. We speculate about a fundamental difference in organization and homogenization of DNA sequences between chromosomes within mammalian and plant genomes. Received: 24 April 1996 / Accepted: 24 May 1996     

Flow cytometric analysis of antibody producing cells using double immunofluorescent staining

Summary Double fluorescent labeling, with fluorescein isothiocyanate (FITC)-labeled F(ab)₂ specific for the heavy chain and R-phycoerythrin (R-PE)-labeled F(ab)₂ specific for the light chain, was demonstrated as a convenient means for the accurate evaluation of a heterogeneous non-antibody-producing population. Furthermore, it could be used for monitoring the changes in each immunoglobulin (Ig) chain content of the cells during the batch culture, which will facilitate the study on antibody synthesis, assembly and secretion.     

Flow cytometric analysis and sorting of plant chromosomes from Petunia hybrida protoplasts

A method to obtain a high metaphase index and thereafter a plant chromosome suspension is described for Petunia hybrida (2n = 14). Mesophyll protoplast cultures have been used, giving easily disrupted cell walls and a high percentage of dividing cells after 42 h. On 2.5 mM colchicine-treated cells, metaphase indexes reaching 10% were routinely obtained. The lysis medium in which the protoplast-derived cells were disrupted was a simplified culture medium. After chromosome release, samples were stained with Hoechst 33342 dye and analysed by flow cytofluorometry. The histogram of fluorescence intensities included three peaks of metaphase chromosomes and a duplication of this flow karyotype provoked by "monochromatid chromosome." This interpretation was established after flow sorting; micronuclei could also be observed and sorted. Of the 7 chromosomes, only the largest formed a distinct peak while the others were incompletely resolved, due to the similar DNA content of various chromosomes. Model distributions of Petunia hybrida chromosomes have been computed according to the relative chromosome length. The theoretical histograms indicated that low variability is indispensable for resolving distinctive chromosome peaks. The experimental flow karyotype was consistent with one of the models having CV of 2.5%.     

Cell-by-cell flow cytometric analysis of chromosomes

The authors discuss various aspects of a recently developed method permitting a detailed flow cytometric analysis of the individual cell karyotypes such as instrumentation, histochemistry, data proceeding algorithms. Possible drawbacks of the method and the ways of their overcoming are considered. Results of analysis of the Chinese hamster cells are presented that illustrate the possibilities of the method, including the metaphase chromosome distribution according to their fluorescence intensity, the analysed cell distribution according to their chromosomes number, the table in which the individual cell karyotypes are distributed according to their fluorescence. The results obtained show that the developed method may be successfully used for investigating chromosomal iNstability and heterogeneity of the mammalian cells.     

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.     

Identification of inverted duplicated #15 chromosomes using bivariate flow cytometric analysis

A dual laser FACS IV cell sorter has been used to obtain bivariate flow histograms of human metaphase chromosomes stained with the DNA-specific dyes, 33258 Hoechst and chromomycin A3. Approximately twenty distinct chromosomal fluorescence populations can be resolved using this double staining technique and the flow cytometer which has been modified only by the substitution of a specially designed air-spaced achromat for the standard focusing lens. Metaphase chromosomes from two different cell lines bearing inverted duplicated #15 autosomes have been subjected to bivariate chromosome analysis. In both cases, the inverted duplicated #15 chromosomes have been identified in the bivariate flow histogram. This identification was supported by experiments in which doubly stained chromosomes were counterstained with either netropsin or distamycin A, resulting in a relative increase in the 33258 Hoechst fluorescence intensity of the structurally abnormal #15 chromosomes, compared with the other chromosomes, as predicted by cytological studies. The possibility of identifying and separating small abnormal autosomes using commercially available instrumentation should facilitate the use of recombinant DNA techniques for the construction of libraries which are highly enriched for DNA sequences from limited autosomal subregions important in the study of chromosomal abnormalities such as deletions, translocations and inversion duplications.     

Flow cytometric analysis of proliferation associated nuclear antigens using washless staining of unfixed cells.

The expression of different proliferation associated nuclear antigens was analyzed using a washless double-staining method and flow cytometry. It is a simple and rapid two-step procedure which can be performed on low cell numbers. A series of hematopoietic cell lines and fresh lymphoma cells were tested and the methodology was found to be applicable to a number of nuclear antigens (PCNA, Ki-67, p105, MPM-2, fibrillarin). For PCNA, the detectability was dependent on the type of antibody used. The immunofluorescence pattern observed by microscopy was altered for antigens stained by the washless technique in comparison with the pattern obtained with fixed cells. With the washless method, detailed cell cycle analysis could be obtained by dual parameter analysis of PCNA and Ki-67.     

Flow cytometric analysis of Chlamydia trachomatis interaction with L cells

Immunofluorescent staining and flow cytometric analysis have been investigated as means of studying the early stages of in vitro infection of Chlamydia trachomatis. The lymphogranuloma venereum strain of C. trachomatis was grown in vitro in L cells, fixed in p-formaldehyde, stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody to the chlamydial major outer membrane protein, and analyzed flow cytometrically. Infected cells stained 50-100 times more intensely than uninfected cells, and they could easily be discriminated by flow analysis. The number of infected cells and the fluorescence intensity of individual cells were proportional to the multiplicity of infection. The attachment of purified elementary bodies to L cells could be analyzed by immunofluorescence and flow cytometry. Cells exposed to 0.26 inclusion-forming units/cell could be discriminated from an unexposed population. Flow analysis of purified elementary bodies was possible after fluorescent staining with the aid of a laser-based cytometer and gating on low volume.     

Flow cytometric analysis of microorganisms

The application of flow cytometry to microorganisms is as old as the technique itself, but it has historically been underexploited for microbial applications. This is now being reversed and microbiologists are ideally placed to benefit from recent technological advances. While earlier papers demonstrated the use of flow cytometry for studies of viability and taxonomy, recent developments in bioinformatics and reporter gene technologies are leading to novel applications in microbiology. Variants of green fluorescent protein have been used for the study of conditional microbial gene regulation in medically important host-pathogen interactions and fluorescence-activated cell sorting is being applied to the isolation of novel mutants in directed evolution studies. This paper reviews the reasons for the delay in the application of flow cytometry to microbial problems, the range of applications, and their limitations and considers the progress made in developing new strategies for use in microbiological investigations.     

Flow cytometric analysis of tetanus toxin binding to neuroblastoma cells

A neuroblastoma cell line was assessed for its capacity to bind tetanus toxin (TT) by using immunofluorescence and flow cytometry to analyze cells on a single cell basis. A clone of Neuro 2a, N2AB-1, was shown to bind variable amounts of TT per cell and this binding could be saturated by increasing doses of the toxin. Toxin binding was specific for neuronal cells, as the non-neuronal cell line, C6 glioma, bound negligible amounts of toxin. Variability of immunofluorescence staining was due in part to the increase in size of N2AB-1 cells as they progress through the cell cycle as measured by cell surface densities of toxin binding and DNA levels by propidium iodide (PI) staining. When N2AB-1 cells were treated with exogenous gangliosides for 24 h, cells were induced to sprout neurites and cell growth was inhibited. Analysis of DNA histograms indicated that ganglioside treatment caused more cells to appear in G0G1 of the cell cycle than that seen for untreated controls. Upon cytometric analysis of TT binding to ganglioside treated cells, it was apparent that treatment stimulated all cells to bind TT in larger amounts per cell than that seen with untreated N2AB-1 cells. These data suggest that TT binding and, therefore, toxin receptors are constant in density throughout the cell cycle of these neuroblastoma cells and that exogenous gangliosides can cause differentiation followed by increased toxin binding.     

Flow cytometric analysis of micronuclei found in cells after irradiation

Exposure of mammalian cells to either ionizing radiation or mutagenic and carcinogenic substances can induce chromosome aberrations. These aberrations in turn may give rise to micronuclei which can be found in cells during the interphase after division. A two-step method is presented that allows separation of micronuclei from cell nuclei. They can then be measured and analysed according to their DNA content in a flow cytometer. The method involves an initial detergent treatment of cells followed by a second treatment with sucrose and citric acid. Micronuclei with DNA content larger than 2% of the G1-nuclei can be measured. The method is tested and compared with microscopic observations of micronucleated cells in irradiated, asynchronous, and synchronized Ehrlich ascites tumour cells growing in vitro. The agreement between the flow cytometric technique and microscopic observations is excellent when the dose-dependent number of micronuclei per cell is taken into consideration.     

Flow cytometric analysis of a marine LAS-degrading consortia

The specific nucleic acid fluorochrome SYTO-13 was used in flow cytometric analysis to assess changes in the density and heterogeneity of marine bacterial populations which biodegrade linear alkylbenzene sulphonate (LAS). Seawater samples with LAS and incubated in the laboratory (20 degrees C, 100 rpm, 30 days) were used to monitor LAS-degrading consortia. Flow cytometric studies and culture methods were used to characterize the LAS degrading bacterioplankton consortia. Fluorescence and scatter signals enabled us to define three regions (R1, R2 and R3) in the dual parameter cytograms. The distribution of the bacterial counts in these regions allowed us to monitor the formation and evolution of the consortia.     

Flow cytometric method for analysis of cytokine expression in clinical samples

Evaluation of cytokine expression is becoming an increasingly useful approach for defining cell-mediated immunity. The interest in characterizing the role of these important molecules in a wide range of clinical conditions has provided impetus to develop new methods to assess cytokine expression in clinical samples. The whole blood flow cytometric procedure described in this report is compatible with both qualitative and quantitative analysis of cytokine expression in clinical samples and is potentially a useful method for evaluating the role of cytokines in disease pathogenesis.     

Flow cytometric analysis of hairy cell leukemia using right-angle light scatter

We report on an easy and reliable method for the enumeration of the typical cells present in the blood, bone marrow, and spleen of patients with hairy cell leukemia. Samples of five patients were analyzed. In three patients, the hairy cells were very accurately followed during treatment with alpha-interferon, using right-angle light scatter to differentiate them from other leukocytes present in total blood. Excellent correlation was obtained with the microscopic hairy cell enumeration. Additional verification by cell sorting and immunofluorescence studies confirmed these findings.     

Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells

Normal development of nuclear transfer embryos is thought to be dependent on transferral of nuclei in G0 or G1 phases of the cell cycle. Therefore, we investigated the cell cycle characteristics of porcine fetal fibroblast cells cultured under a variety of cell cycle-arresting treatments. This was achieved by using flow cytometry to simultaneously measure cellular DNA and protein content, enabling the calculation of percentages of cells in G0, G1, S, and G2+M phases of the cell cycle. Cultures that were serum starved for 5 days contained higher (p < 0.05) percentages of G0+G1 (87.5 +/- 0. 7) and G0 cells alone (48.3 +/- 9.7) compared with rapidly cycling cultures (G0+G1: 74.1 +/- 3.0; G0: 2.8 +/- 1.2). Growth to confluency increased (p < 0.05) G0+G1 percentages (85.1 +/- 2.8) but did not increase G0 percentages (6.0 +/- 5.3) compared to those in cycling cultures. Separate assessment of small-, medium-, and large-sized cells showed that as the cell size decreased from large to small, percentages of cells in G0+G1 and G0 alone increased (p < 0.05). We found 95.2 +/- 0.3% and 72.2 +/- 12.0% of small serum-starved cells in G0+G1 and G0 alone, respectively. Cultures were also treated with cell cycle inhibitors. Treatment with dimethyl sulfoxide (1%) or colchicine (0.5 microM) increased percentages of cells in G0 (24.8 +/- 20.0) or G2+M (37.4 +/- 4.6), respectively. However, cells were only slightly responsive to mimosine treatment. A more complete understanding of the cell cycle of donor cells should lead to improvements in the efficiency of nuclear transfer procedures.     

Flow cytometric analysis of granulosa cells from developing rat follicles.

Immature rats were treated with diethylstilboestrol (DES) or pregnant mares' serum gonadotropin (PMSG) and forward angle light-scatter (FALS) and 90 degrees light-scatter (90 degrees LS) signals were used to measure the size and the granularity (internal organization) of the granulosa cells, respectively. The results confirmed the presence of two major populations of granulosa cells in the ovaries of both groups of rats, with the same percentage of larger cells in both treatments (52.3% in DES, 49.5% in PMSG). Since DES treatment brings about granulosa cell growth while PMSG treatment causes growth and differentiation, it is evident that there is heterogeneity in granulosa cell sizes during different states of growth and differentiation. There was also heterogeneity in sizes of granulosa cells harvested from follicles of small (less than 210 microns), medium (210-420 microns) and large (greater than 420 microns) diameter. Quadrant analysis of granulosa cells in various fractions collected from Percoll gradients suggested an increase in granularity in the small and large granulosa cell populations. Cell cycle analysis of small and large granulosa cell populations collected from large follicles of rats treated with PMSG indicated that each population was distributed in G0/G1, S and G2/M phases. These results demonstrate that populations of small and large granulosa cells exist in rat ovarian follicles during various stages of growth and differentiation.     

Flow cytometric cell cycle analysis of cultured brown bear fibroblast cells

The aim of this study was to assess by flow cytometry the cell cycle of brown bear fibroblast cells cultured under different growth conditions. Skin biopsies were taken in Cantabria (Spain) from a live, anaesthetized brown bear. DNA analysis was performed by flow cytometry following cell DNA staining with propidium iodide. Serum starvation increased (P<0.01) the percentage of G0/G1 phase cells (92.7+/-0.86) as compared to cycling cells (39.7+/-0.86) or cells cultured to confluency (87.3+/-0.86). DMSO included for 48h in the culture significantly increased (P<0.01) the percentage of G0/G1 phase of the cell cycle at all concentrations used and decreased percentages of S phase in a dose-dependent fashion. Roscovitine increased the G0/G1 phase of the cell cycle (P<0.01) at 15microM concentration. Interestingly, the G2/M stage significantly increased at 30 and 50microM compared to the control and 15microM (P<0.02). The cell cycle of brown bear adult fibroblast cells can be successfully synchronized under a variety of culture conditions.     

Flow cytometric analysis of steroidogenic organelles in differentiating granulosa cells.

Functional and structural changes accompany the differentiation of granulosa cells during follicular development. We used flow cytometry and fluorescent dyes to characterize two organelles important to the steroidogenic process. Mitochondria, which contain the rate-limiting enzyme responsible for cholesterol conversion to pregnenolone, and lipid droplets, which store cholesterol substrate, were probed in viable hen granulosa cells during differentiation. The fluorescent dye Dio3-C5 (DiO) was used to probe mitochondrial membrane potential, indicative of mitochondrial activity and/or number, during rapid granulosa cell differentiation in a hierarchy of individual developing hen preovulatory follicles (F6, smallest, to F1, largest). Cellular DiO fluorescence, granularity, and cell size were significantly elevated with increasing maturation state. Treatment with LH significantly increased DiO fluorescence in granulosa cells from F1 but not F3. The increased mitochondrial activity/number in granulosa cells that accompanies follicular maturation and is influenced by LH may reflect, at least in part, increased activity or amount of hormone-regulated mitochondrial enzymes controlling steroidogenesis. Flow spectrofluorometry and the metachromatic lipid dye, nile red, were used to probe lipid droplets in differentiating granulosa cells from F6 to F1. There was a dramatic increase in the fluorescence component related to lipid droplets with increasing stages of follicular maturation, suggesting recruitment of lipids into droplets during the differentiation of granulosa cells into hormone-responsive steroidogenic cells. The results demonstrate the dynamic nature of the granulosa cell morphology involved in steroidogenesis during follicular development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow cytometric analysis of bromodeoxyuridine-substituted cells stained with 33258 Hoechst.

This paper describes a flow-cytometric application of the quenching of fluorescence from 33258 Hoechst stained Chinese hamster ovary-line cells due to the incorporation of 5-bromo-deoxyuridine (BrdU) into the cellular deoxyribonucleic acid. Cells were grown for 24 hr in medium containing BrdU in concentrations ranging from 1 x 10(-8) to 1 x 10(-4) M. For each concentration we measured the average fluorescence as determined by flow cytometry, the extent of BrdU substitution and the effect of the BrdU on cell growth. We determined that a BrdU concentration of 1 x 10(-5) M resulted in sufficient substitution to quench the fluorescence from 33258 Hoechst by a factor of 4, allowing discrimination between cycling and noncycling cells. The extent of BrdU substitution after growth for 24 hr in this concentration of BrdU was 64%. These data indicate the feasibility of detecting deoxyribonucleic acid synthesis in whole cells using the 33258 Hoechst-BrdU methodology.