Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90 degrees and forward scatter measurements

Mitotic cells could be well discriminated from the cells in the G1-, S- and G2-phases of the cell cycle using pulse labeling of S-phase cells with bromodeoxy-uridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2- and M-phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M-phase cells showed lower propidium iodide fluorescence emission compared to G2-phase cells. The fluorescence difference of M- and G2-phase cells was caused by the different thermal denaturation of their DNA. Best separation of M- and G2-phase cells was obtained after 30-50 min heat treatment at 95 degrees C. Mitotic index could be measured if no unlabeled S-phase cells were present in the cell culture. With additional measurements of 90 degree scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2- and S-phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulse-labeled cells progressed through the cell cycle. We conclude that this modified BrdUrd/DNA technique using prolonged thermal denaturation and the simultaneous measurement of scatter signals may offer additional information especially in the presence of BrdUrd-unlabeled S-phase cells.     

Flow cytometric discrimination of mitotic nuclei by right-angle light scatter

Flow cytometry has been used to demonstrate alterations in protein, RNA, and DNA content of cells as they traverse the cell cycle. Employing fluorescein isothiocyanate (FITC) to stain protein and propidium iodide (PI) to stain nucleic acids, multiple regions within the G1 and G2 phases of the cell cycle, in addition to the M phase, can be distinguished. In this study, cytograms of the 90 degree light scatter signal vs. PI fluorescence were remarkably similar to those of FITC fluorescence vs. PI fluorescence, suggesting a relationship between 90 degree light scatter and protein content. M-phase nuclei can be distinguished from G2-phase nuclei on cytograms of 90 degree light scatter vs. PI fluorescence. However, the percentage of mitotic nuclei obtained by this technique is less than that found by light microscopic analysis. Flow cytometric parameters of nuclei prepared by nonionic detergent (NP40) lysis in Dulbecco's PBS, Vindelov's buffer, or Pollack's hypotonic EDTA/Tris buffer were compared. The best resolution of mitotic nuclei was obtained in Pollack's buffer. However, the stainability of the M-phase nuclei is reduced, and the nuclei are located in the late S/G2 region of the single-parameter histogram.     

A new method to discriminate G1, S, G2, M, and G1 postmitotic cells

A new flow cytometric method combining light scattering measurements, detection of bromodeoxyuridine (BrdU) incorporation via fluorescent antibody, and quantitation of cellular DNA content by propidium iodide (PI) allows identification of additional compartments in the cell cycle. Thus, while cell staining with BrdU-antibodies and PI reveals the G1, S, and G2 + M phases of the cell cycle, differences in light scattering allow separation of G2 phase cells from M phase cells and subdivision of G1 phase into two compartments, i.e., G1A representing postmitotic cells which mature to G1B cells ready to initiate DNA synthesis. The method involves fixation of cells in 70% ethanol, extraction of histones with HC1, and thermal denaturation of DNA. This treatment appears to enhance the differences in chromatin structure of cells in the various phases of the cell cycle to the extent that cells could be separated on the basis of the 90 degrees scatter. Mitotic cells show much lower scatter than G2 phase cells, and G1 postmitotic cells (G1A) show lower scatter than G1 cells about to enter the S phase (G1B). Light scattering is correlated with chromatin condensation, as judged by microscopic evaluation of cells sorted on the basis of light scatter. The method has the advantage over the parental BrdU/DNA bivariate analysis in allowing the G2 and M phases of the cell cycle to be separated and the G1 phase to be analyzed in more detail. The method may also allow separation of unlabeled S phase cells from mitotic cells and distinguish between labeled and unlabeled mitotic cells.     

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.     

The optimal application of forward and ninety-degree light scatter in flow cytometry for the gating of mononuclear cells

Peripheral blood mononuclear cells from ten normal donors were labeled with a monoclonal antibody specific for monocytes and analyzed using a fluorescence activated cell sorter (FACS). Forward and 90 degrees light scatter parameters were studied in order to apply optimal computerized gating to identify and exclude monocytes from lymphocyte populations. An average of 9.45% versus 1.22% of cells, within chosen lymphocyte gates established by forward angle and 90 degrees scatter, respectively, were identified as monocytes. In samples from ten donors, the exclusion of monocytes from the lymphocyte population was more efficient using 90 degrees scatter than forward scatter. Simultaneous use of forward and 90 degrees scatter did not significantly improve the ability to accurately exclude monocytes, but did result in a significant increase in the improper exclusion of lymphocytes. Use of 90 degrees scatter alone, forward scatter alone, and forward and 90 degrees scatter simultaneously to identify lymphoid cells resulted in the exclusion of 12, 17, and 23% of lymphocytes from further analysis. The 90 degrees scatter alone appears to be the optimal method to eliminate monocytes electronically from mononuclear cell populations in which lymphocytes are being studied.     

Human bone marrow CFU-GM and BFU-E localized by light scatter cell sorting

Flow cytometric separation was performed on the normal human bone marrow (BM) by using the low-angle (0 degrees) or high-angle (90 degrees) light scatter. Four distinct subpopulations of cells can be enriched from normal human BM and these fractions were subsequently evaluated for their morphological properties as well as their clonogenic capacity in various progenitor cell assays. Our results indicate that human erythroid and granulocyte-macrophage progenitor cells can be separated from BM low-density cells by cell sorting, and these cells show similar 0 degrees and 90 degrees light scatter properties to those observed with murine bone marrow studies. Flow cytometric analysis also suggests that the majority of sorted BFU-E and CFU-GM resides in the blast cell subset of human BM mononuclear cells.     

A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification.

A method was developed for gentle fixation of mammalian cells and permeabilization of their membranes. The method is useful for staining of intracellular antigens or quantification of DNA content simultaneously with cell surface staining. Cells are treated for 1 h at 4 degrees C with 0.25% buffered paraformaldehyde then for 15 min at 37 degrees C with 0.2% Tween 20 detergent in PBS. The procedure permits excellent staining of intracellular proteins, very low coefficients of variation (CV) on the G0G1-peak of DNA distributions, and preservation of the integrity of cell surface antigens. The low vs. 90 degrees angle light scatter profile of cell clusters is maintained thereby allowing discrimination of different cell populations including human peripheral blood lymphocytes and monocytes for gating and analytic purposes. The method was successfully used on a variety of other cell types, including human thymocytes, murine thymocytes and spleen cells, and several leukemic cell lines. Dual-color surface antigen staining combined with DNA staining with 7-amino-actinomycin D (7-AAD) on peripheral blood mononuclear cells (PBMC) cultured with tetanus toxoid allowed the determination of the cell subset that was preferentially stimulated. Staining for internal antigens was done on CCRF-CEM for expression of CD3 epsilon and on NALM-6 for expression of mu. The technique we developed gave bright and specific staining of internal antigens in the examples presented here. It is particularly suited for correlations of internal antigen staining with DNA staining and/or surface immunofluorescence.     

X-irradiation--induced changes in the progression of type B spermatogonia and preleptotene spermatocytes

In response to induced DNA damage, proliferating cells arrest in their cell cycle or go into apoptosis. Ionizing radiation is known to induce degeneration of mammalian male germ cells. The effects on cell-cycle progression, however, have not been thoroughly studied due to lack of methods for identifying effects on a particular cell-cycle phase of a specific germ cell type. In this study, we have utilized the technique for isolation of defined segments of seminiferous tubules to examine the cell-cycle progression of irradiated rat mitotic (type B spermatogonia) and meiotic (preleptotene spermatocytes) G1/S cells. Cells irradiated as type B spermatogonia in mitotic S phase showed a small delay in progression through meiosis. Thus, it seems that transient arrest in the progression can occur in the otherwise strictly regulated progression of germ cells in the seminiferous epithelium. Contrary to the arrest observed in type B spermatogonia and in previous studies on somatic cells, X-irradiation did not result in a G1 delay in meiotic cells. This lack of arrest occurred despite the presence of unrepaired DNA damage that was measured when the cells had progressed through the two meiotic divisions.     

X-ray-induced G2 arrest in ataxia telangiectasia lymphoblastoid cells

Sensitivity to X-ray-induced G2 arrest was compared between ataxia telangiectasia (AT) lymphoblastoid cells and normal human cells. Flow cytometrical analysis of cells following X-ray irradiation revealed that the fraction of cells with 4n DNA content was greater in AT cells than in normal cells as previously reported by other investigators. However, the other parameters for cell-cycle progression kinetics including mitotic indices, cumulative mitotic indices and cumulative labelled mitotic indices indicated that X-ray-induced G2 arrest as a function of dose in AT cells was indistinguishable from that in normal cells. Moreover, no significant difference in cell viability was noted between AT and normal cells until 48 h following X-irradiation up to 2.6 Gy, although X-irradiated AT cells, compared to normal cells, showed a significantly decreased survival in terms of cell multiplication in growth medium and colony formation in soft agar. These data collectively suggest that the greater accumulation of AT cells with 4n DNA content in flow cytometry cannot be attributed to more stringent irreversible blockage of cell-cycle progression at the G2 phase and eventual cell death there. The possible reasons for this greater accumulation are discussed.     

Automatic cell identification and enrichment in lung cancer. I. Light scatter and fluorescence parameters.

Two physical parameters were investigated to automatically recognize cells in sputum from human squamous cell carcinoma of the lung and to separate them for preparation by the Papanicolaou methods, for human interactive identification and for automated high resolution image analysis. The two parameters, 0.5-15.0 degrees forward argon-ion laser light scatter to estimate total cell size and 546 nm Acridine orange fluorescence to approximate total cell DNA content, were measured in a flow-through fluorescence activated cell sorting system. Enrichment for neoplastic cells in three cases of squamous cell carcinoma of the lung averaged 7.8-fold over the original sputum when only green fluorescence was used and 10.5-fold using green fluorescence and forward light scatter. The average enrichment for neoplastic cells was 65.6-fold relative to polymorphonuclear deenrichment.     

Subpopulations of slowly cycling cells in S and G2 phase in mouse epidermis

Evidence has been presented supporting the existence of heterogeneity in cell-cycle progression in mouse epidermis, The present study was undertaken to characterize this heterogeneity in more detail. Hairless mice were continuously labelled with tritiated thymidine every 4 hr for 4 days. Basal cell suspensions were prepared from slices of mouse skin at intervals during the experiment and subjected to DNA flow cytometry. Cell-cycle analysis was combined with sorting of cells from windows in G1, S and G2 phase, and the proportion of labelled cells within each window was determined in autoradiographs. Reanalysis and resorting to control the purity of of sorted fractions were performed. Computer simulations of the data were made using a mathematical model assuming different S and G2 phase characteristics. A good fit to the data was only obtained when heterogeneity in mouse epidermal cell-cycle progression was assumed, indicating the existence of slowly traversing, distinct subpopulations of cells in G2 and S phase. These cells are assumed to contribute to about 40% of all cells in S phase and to about 70% of all in G2 phase. The estimated residence times in the resting states were 38 and 32 hr in S and G2 phase, respectively. Two-parameter sorting based on DNA and light scatter indicated that slowly cycling cells were larger than the average. There is no evidence of significant subpopulations of permanently non-proliferating keratinocytes in any of the cell-cycle phases.     

Doublet discrimination in DNA cell-cycle analysis.

Differences in doublet analysis have the potential to alter DNA cell-cycle measurements. The techniques for doublet determination are often used interchangeably without regard for the complexity in cell shapes and sizes of biological specimens. G(0/1) doublets were identified and quantitated using fluorescence height versus area and fluorescence width versus area pulse measurements, by enumerating the proportion of G(2) + M cells that lack cyclin B1 immunoreactivity, and modeled in the DNA histograms by software algorithms. These techniques were tested on propidium iodide-stained whole epithelial cells or nuclei from asynchronous cultures, or after exposure to chemotherapeutic agents that induced cell-cycle arrest and were extended to human breast tumor specimens having DNA diploid patterns. G(0/1) doublets were easily discernible from G(2) + M singlets in cells or nuclei that are generally homogenous and spherical in shape. Doublet discrimination based on pulse processing or cyclin B1 measurements was nonconcordant in some nonspherical cell types and in cells following cell cycle arrest. Significant differences in G(0/1) doublet estimates were observed in breast tumor specimens (n = 50), with estimates based on pulse width twice those of pulse height and nearly five times greater than computer estimates. Differences between techniques are attributed to difficulties in the separation of the boundaries between G(0/1) doublets and G(2) + M singlet populations in biologically heterogeneous specimens. To improve reproducibility and enhance standardization among laboratories performing cell cycle analysis in experimental cell systems and in human breast tumors, doublet discrimination analysis should best be accomplished by computer modeling. Shape and size heterogeneity of tumor and arrested cells using pulse-processing can lead to errors and make interlaboratory comparison difficult.     

The Bfa1/Bub2 GAP complex comprises a universal checkpoint required to prevent mitotic exit

At the end of the cell cycle, cyclin-dependent kinase (CDK) activity is inactivated to allow mitotic exit [1]. A protein phosphatase, Cdc14, plays a key role during mitotic exit in budding yeast by activating the Cdh1 component of the anaphase-promoting complex to degrade cyclin B (Clb) and inducing the CDK inhibitor Sic1 to inactivate Cdk1 [2]. To prevent mitotic exit when the cell cycle is arrested at G2/M, cells must prevent CDK inactivation. In the spindle checkpoint pathway, this is accomplished through Bfa1/Bub2, a heteromeric GTPase-activating protein (GAP) that inhibits Clb degradation by keeping the G protein Tem1 inactive [3-5]. Tem1 is required for Cdc14 activation. Here we show that in budding yeast, BUB2 and BFA1 are also required for the maintenance of G2/M arrest in response to DNA damage and to spindle misorientation. cdc13-1 bub2 and cdc13-1 bfa1 but not cdc13-1 mad2 double mutants rebud and reduplicate their DNA at the restrictive temperature. We also found that the delay in mitotic exit in mutants with misoriented spindles depended on BUB2 and BFA1, but not on MAD2. We propose that Bfa1/Bub2 checkpoint pathway functions as a universal checkpoint in G2/M that prevents CDK inactivation in response to cell-cycle delay in G2/M.     

Differential effect of D-penicillamine on the cell kinetic parameters of various normal and transformed cellular types

The cell-growth-inhibitory and phase-specific effects of D-penicillamine on cell-cycle progression were investigated using cell-proliferation patterns, quantitative cell-cycle analysis by flow cytometry, and determination of the mitotic index and binucleate cell fraction of normal (rabbit articular chondrocytes, L 809, rabbit fibroblasts) and transformed (HeLa, L 929) cells. D-penicillamine treatment resulted in an inhibition of growth within a dose range of 5 × 10^?4 M to 7.5 × 10^?3 M. Examination of DNA by flow cytometric analysis revealed that rabbit articular chondrocytes were preferentially arrested in the G0/1 phase of the cell cycle, whereas the other cell lines were blocked in the G2 + M phase; the increase in the proportion of cells with G2 + M DNA content was partially due to an enhancement of binucleate cells, resulting in a cytokinesis perturbation for HeLa and L 929 cells. These results showed that D-penicillamine affects cell proliferation through different events according to cell type.     

A unified procedure for conservative (morphology) and integral (DNA and immunophenotype) cell staining for flow cytometry

BACKGROUND: Current methods for multiparameter DNA flow cytometry suffer from several limitations. These include significant modifications of cell morphological parameters, the impossibility to counterstain cells with certain fluorochromes, and laborious tuning of the instrument that, for some procedures, must be equipped with an ultraviolet (UV) laser. To overcome these problems, we developed a novel method for the simultaneous analysis of morphological parameters, four-color immunophenotyping, and stoichiometric DNA labeling using a bench-top flow cytometer. METHODS: The method consists of a mild permeabilization/fixation treatment at room temperature, followed by labeling with fluorochrome-conjugated monoclonal antibodies (mAbs) and with the DNA dye 7-aminoactinomycin D (7-AAD) at 56 degrees C. RESULTS: Using this method, we analyzed resting peripheral blood mononucleated cells (PBMC), proliferating T cells cultured in the presence of interleukin-2 (IL-2), and lymphoblastoid B cells. Lymphocytes, monocytes, and lymphoblasts treated by this procedure retained differential light scattering (DLS) characteristics virtually identical to those of untreated cells. This allowed regions to be drawn on forward scatter (FSC) and side scatter (SSC) cytograms resolving different cell populations. DLS were preserved well enough to distinguish large lymphoblasts in the S or G2/M phases from small G0/G1 cells. Also, stainability with fluorescein-isothiocyanate (FITC), R-phycoerythrin (PE), allophycocyanin (APC)-conjugated mAbs was generally preserved. DNA labeling with 7-AAD was of quality good enough to permit accurate cell cycle analysis. CONCLUSIONS: The method described here, which we called integral hot staining (IHS), represents a very simple, reproducible, and conservative assay for multiparameter DNA analysis using a bench-top flow cytometer. Last but not least, the cytometer tuning for multiparameter acquisition is straightforward.     

Correlated analysis of cellular DNA, membrane antigens and light scatter of human lymphoid cells

Flow cytometric correlated analysis of membrane antigens, DNA, and light scatter was performed on human lymphoid cells using fluorescein (FITC)-conjugated antibodies to label B- and T-cell antigens and propidium iodide (PI) to stain DNA after ethanol fixation and RNase treatment. A FACS II flow cytometer was modified to obtain digitized measurements of two color fluorescence and light scatter emissions, simultaneously. Software was written to allow single parameter analysis or correlated analysis of any two of the three parameters acquired. Ethanol fixation preserved FITC surface labeling for at least 15 weeks, but produced marked changes in light scatter. No changes in FITC distributions were observed after RNase treatment and PI staining, and the presence of FITC labeling did not affect DNA distributions. Within heterogeneous cell populations, the DNA distribution of cell subpopulations identified by a membrane antigen was clearly demonstrated.     

Cellular heterogeneity in normal human urothelium: an analysis of optical properties and lectin binding

Flow cytometry was used to study the optical properties of normal urothelial cells in suspension. Narrow-angle light scatter, which is a function of cell size, defined one major and one minor cell population, and 90 degrees light scatter, a function of intracellular structure, showed three distinct cell populations. These properties were displayed as a 2-dimensional dot plot or "fingerprint" which proved to be characteristic and reproducible from one specimen of urothelium to the next. Cell sorting on the basis of these two parameters demonstrated that the small cells of the basal layer occupy the low narrow angle, low 90 degrees light-scatter region; the giant cells of the superficial layer lie in the high narrow angle, high 90 degrees scatter region; and the pyramidal cells of the intermediate layer lie in an intermediate zone. Studies of tissue sections using the galactose-specific, FITC-conjugated Maclura Pomifera lectin (MPA) demonstrated preferential binding to the superficial layers of intact urothelium. In order to quantify the apparent differences in lectin binding between the superficial and basal layers, urothelial cell suspensions were labeled with FITC-conjugated MPA and studied by flow cytometry. The resolution obtained on the basis of light scatter made it possible to quantify the difference in lectin binding to the three morphologically recognized cell types present in normal urothelium.     

A temperature-sensitive mutation affecting S-phase progression can lead to accumulation of cells with a G2 DNA content

Cultures of ts BN75, a temperature-sensitive mutant of BHK 21 cells, show a gradual biphasic drop in [3H]thymidine incorporation together with an accumulation of cells having a G2 DNA content when incubated at 39.5 degrees. However, when higher (41 degrees - 42 degrees) nonpermissive temperatures were used, the major block was in S-phase DNA synthesis. The cultures of ts BN75 shifted to 42 degrees at the start of the S phase, cell-cycle progress was arrested in the middle of S, while under these conditions wild-type BHK cells underwent at least one cycle of DNA synthesis. When ts BN75 cells growth-arrested at high temperature with a G2 DNA content were shifted to the permissive temperature (33.5 degrees C), the restart of DNA synthesis preceded the appearance of mitotic cells. These data suggest that the ts defect of ts BN75 cells might affect primarily the S phase of the cycle rather than the G2 phase.     

Time-temperature analyses of cell killing of synchronous G1 and S phase Chinese hamster cells in vitro

Time-temperature analyses of durations of heating required to achieve isosurvival were used to compare hyperthermic cell killing of synchronous Chinese hamster ovary (CHO) cells heated in G1 or S at temperatures of 42 to 45.5 degrees C. G1 populations were obtained by incubation of mitotic cells for 90 min at 37 degrees C. S phase populations were obtained by incubation of mitotic cells for 12 h at 37 degrees C in medium supplemented with 2 micrograms/ml aphidicolin, a reversible inhibitor of DNA alpha polymerase; S phase survival was also determined in an aphidicolin-free system by using high specific activity [3H]thymidine. In both systems, the thermosensitivity was similar and decreased as the cells progressed from early S phase, in agreement with earlier studies (R. A. Read, M. H. Fox, and J. S. Bedford. Radiat. Res. 98, 491-505 (1984]. A comparison of Arrhenius plots of the inverse of durations of heating required to achieve isosurvival for cells heated in G1 or S phase showed similar temperature dependence above 43.5 degrees C, yet the plots for heat-sensitive S phase cells were offset from those for heat-resistant G1 cells by about 1.5 degrees C, i.e., S phase cells respond to 43 degrees C with a rate similar to that observed in G1 cells heated at 44.5 degrees C. Using least-squares regression of the semilog plots, the curves were analyzed either as continually bending curves or as two straight lines with a break at 43.5 degrees C. When the data were analyzed using two straight lines, no significant differences in the slopes of the time-temperature plots of G1 or S phase cells were observed. A quantitative comparison between the two methods of data analysis demonstrated that in both phases the data were better fit with a continuously curving line, rather than two straight lines.