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PubMed1996

[Establishment of a new human ovarian serous cystadenocarcinoma cell line (IM), and influence on cell proliferations by cisplatin with or without hyperthermia].

A new cell line, designated IM has been established from operation material derived from a woman with ovarian serous cystadenocarcinoma. The population doubling time of the 35th passage IM cell was 28.8 hours. And it was successively subcultured 165 times in over 7 years, moreover still kept CA125 production. The nuclear DNA and cell surface CA125 antigen were double stained by propidium iodide and anti CA125 monoclonal antibody-FITC. Then the two color cytogram obtained by flow cytometry was drawn up. For the most part of CA125 positive cell retained G0 + G1 of cell cycle, the lesser part was in G2 + M phase. The S phase rate of IM cell incubated with cisplatin at 37 degrees C or 41 degrees C for 1 hour that estimated by BrdU-propidium iodide double stain method of flow cytometry, it was suggested that the inhibition of DNA synthesis by cisplatin was increased with 41 degrees C low hyperthermia.

CA-125 Antigen↗
PubMed1997

Sequence specificity, enantiospecificity and polyelectrolyte effect in the binding to DNA of a 6-(2-pyridyl)phenanthridine chiral photonuclease.

In order to establish the basis for the rational design of a novel family of intercalating chiral photonuclease drugs aimed at photochemotherapy, namely N, N'-dialkylated 6-(2-pyridinium)phenanthridinium (pyp) dications, a detailed investigation of the DNA binding of the dq2pyp member (where dq2 stands for -CH2CH2-), was conducted. The study addresses the sequence- and enantiospecificity, as well as polyelectrolyte effects in the drug-DNA interaction. Binding isotherms with synthetic polynucleotides, forcefield calculations, affinity chromatography in a DNA-cellulose stationary phase and salt-dependent equilibrium and kinetic studies with DNA were used. dq2pyp shows a strong preference for alternating GC over AT base pairs; binding to homopolymeric DNA is weak (< 3 x 10(4) M-1). Affinity chromatography shows enantiospecific binding of dq2pyp to DNA. The polyelectrolyte contribution to the binding free energy are shown to be relatively important (-4.8 kcal/nmol out of an overall value of -7.2 kcal/mmol at 10.2 mM Na+). The slope of the logkd (dissociation rate constant) vs. log[Na+] plot (0.7) agrees with the values predicted from counterion condensation theory for a dicationic intercalator, giving further support to such a DNA binding mode for dq2pyp. The relatively high kinetic dissociation constants (logkd = 0.70log[Na+] + 3.79) in comparison with those of propidium (two orders of magnitude larger at any Na+ concentration) seems to originate from the absence of amino groups in dq2pyp. The kinetic association constants (logka = -1.06log[Na+] + 5.53) are twice these of propidium, probably due to the less restrictive positioning of dq2pyp at the intercalation site. The kinetic studies support a mechanism of intercalation in which the drug forms a pre-equilibrium outside the complex followed by the intercalation of the drug. Molecular modelling is used throughout to rationalize all the experimental data, as well as to propose new candidates with improved DNA affinity and residence time.

Base Sequence↗
PubMed1998

Chronically HIV-1-infected monocytic cells induce apoptosis in cocultured T cells.

We have previously developed a human macrophage hybridoma model system to study the effect of HIV-1 infection on monocytic function. Upon coculture of one chronically (35 days postinfection) HIV-1-infected human macrophage hybridoma cell line, 43HIV, there was a dose-dependent decrease in the viability of cocultured Ag-stimulated T cells associated with an increase in DNA strand breaks. Enhanced apoptosis was determined by labeling with biotinylated dUTP and propidium iodide, increased staining with annexin V, increased side light scatter and expression of CD95, and decreased forward light scatter and expression of Bcl-2. There was also increased DNA strand breaks as determined by propidium iodide staining in unstimulated T cells cocultured with 43HIV and in T cells stimulated with anti-CD3 mAb and PHA. Pretreatment with 5145, a human polyclonal anti-gp120 Ab that recognizes the CD4 binding region, as well as with an anti-Fas ligand mAb blocked apoptosis in CD4+ T cells but not in CD8+ T cells. A soluble factor with a Mr below 10,000 Da was defined that induced apoptosis in CD4+ and CD8+ T cells and B cells. SDS-PAGE analysis of the active fractions revealed a band of 6000 Da that, after electroelution, had proapoptotic activity. The pI of the activity was estimated to be between 6.5 and 7.0. In conclusion, chronically HIV-1-infected monocytic cells induce apoptosis in bystander-, Ag-, anti-CD3-, and mitogen-stimulated T cells by multiple factors, which may contribute to the depletion of lymphocytes induced by HIV-1.

Apoptosis↗
PubMed1997

Staining of micronuclei in squamous epithelial cells of human oral mucosa.

OBJECTIVE: To evaluate the influence of methodologic variables, staining method and sampling, on the frequency of micronuclei scored in squamous epithelial cells of oral mucosa. Micronuclei were used as biomarkers of structural and numerical chromosome damage. STUDY DESIGN: Feulgen and Giemsa stain and fluorescent dyes Hoechst 33258 and propidium iodide were used for micronucleus staining. Cells were collected by either light or vigorous scraping of oral mucosa. RESULTS: Staining of micronuclei by Hoechst 33258 proved more handly, less time consuming and at least equally reliable to the more widely used Feulgen staining. Results with Giemsa stain and propidium iodide were unsatisfactory. Micronuclei were less frequent in superficial layers of the oral mucosa. CONCLUSION: Hoechst 33258 can be recommended for micronucleus staining in squamous epithelial cells of oral mucosa. Differences in scraping may affect the measurement of micronucleus frequency; scraping should therefore be carefully standardized in each laboratory.

Epithelial Cells↗
PubMed1999

CeReS-18 inhibits growth and induces apoptosis in human prostatic cancer cells.

BACKGROUND: Polypeptide growth factors are positive and negative regulators of prostatic growth and function, and many positive regulators of growth in the prostate have been extensively studied. However, very few inhibitors of prostate cell proliferation have been identified. We have isolated a unique 18-kDa sialoglycopeptide (CeReS-18) which inhibits cell proliferation of three separate lines of human prostate cancer cells, as well as inducing cellular cytotoxicity via an apoptotic pathway unrelated to the Bcl-2 family of proteins. METHODS: Cell cycle inhibition was analyzed by direct cell counts with a Coulter (Miami, FL) cell counter. Apoptotic cells were analyzed by electron microscopy, annexin V-fluorescein isothiocyanate (FITC) staining, fluorescence microscopy, and propidium iodide uptake measured with a fluorescence-activated cell sorter. Expression of the proteins of the Bcl-2 family was detected by Western blot analysis. RESULTS: We found that CeReS-18 inhibits cell proliferation of androgen-responsive, LNCaP.FGC human prostate cancer cells, as well as of androgen-nonresponsive DU-145 and PC3 human prostate cancer cells. Furthermore a, fivefold increase over the inhibitory concentration of CeReS-18 elicited a cytotoxic response by all three cell lines. We thus characterized the cytotoxic mechanism as apoptotic in nature, and we measured the expression of several members of the Bcl-2 family in PC3 cells upon treatment with CeReS-18. CONCLUSIONS: The data indicate that CeReS-18 is a potent inhibitor of cellular progression through the cell cycle by both androgen-responsive and androgen-nonresponsive human prostate cancer cells. In addition, treatment of both types of cells with increased concentrations of CeReS-18 induces cellular cytotoxicity, characterized as apoptosis.

Annexin A5↗
PubMed1998

Flow cytometry and cell sorting for yeast viability assessment and cell selection.

Yeast suspensions were analysed by flow cytometry after dye staining for determination of total and viable cell densities. Results were comparable to traditional colony counting and, in addition, provided further information on the percentage of total cells that were viable. The flow cytometric methods provided results within 20 min whereas colony counts were not available until 36 h. We evaluated a number of fluorescent dyes: ChemChrome Y (CY), oxonol (Ox), propidium iodide (PI), Fungolight and rhodamine 123, for accurate determination of viability of industrial yeast cultures and freshly re-hydrated high activity dried yeast (HADY). PI, Ox and CY gave the most conclusive live/dead discrimination and were the simplest to use. Culturing after dye staining and cell sorting demonstrated that the yeast remained viable after cell sorting and incubation with PI, CY or Ox. The methods, therefore, permit physical selection of individual yeast cells from populations of mixed viability. Sorting demonstrated that PI stained non-culturable cells whilst CY stained culturable cells. Analysis of yeast stained simultaneously with CY and PI or with Ox and PI demonstrated that PI and CY assays were in mutual agreement with respect to viability assessments. The Ox assay was in agreement with CY and PI for live/heat-killed mixtures. However, for re-hydrated HADY, Ox stained a significantly (P < or = 0.05) higher proportion of cells than did PI.

Colony Count, Microbial↗
PubMed1997

Progesterone induces apoptosis and up-regulation of p53 expression in human ovarian carcinoma cell lines.

BACKGROUND: Progesterone (PROG) has been shown to reduce the risk of developing ovarian carcinoma in postmenopausal women who have undergone estrogen and progestogen replacement therapy, and it has been clinically used to treat some types of ovarian tumors. It is not yet clear whether or not the antitumor activity of progestogen is due to its ability to induce apoptosis in precarcinomatous and carcinomatous ovarian cells. The apoptosis-related genes p53, bcl-2, and c-myc have important roles in the regulation of programmed cell death, and thus may be involved in the process of the suspected PROG-induced apoptosis. METHODS: Antiproliferation effects of PROG on 3AO and AO ovarian carcinoma cells were determined by 3H-thymidine incorporation. Apoptosis of the PROG-treated cells was determined by DNA laddering analysis and was quantitated by both nuclear condensation and flow cytometry after cells were stained with propidium iodide. Cell cycle analysis was also performed by flow cytometry. The expression of p53, bcl-2, and c-myc after 72 hours of PROG treatment was detected by Northern blot analysis. RESULTS: In both 3AO and AO cell lines, cells proliferation was maximally inhibited by PROG after 72 hours of treatment at 10 microM concentration. Under the same conditions, more than 50% of PROG-treated cells had undergone apoptosis, whereas less than 3% of the cells were apoptotic in untreated cell cultures. After exposure to PROG for 72 hours, cells were arrested in the G1 phase of the cell cycle, and the levels of p53 mRNA were remarkably increased in both cell lines. No changes in expression of bcl-2 or c-myc were detected. CONCLUSIONS: PROG significantly inhibited cell proliferation and induced apoptosis in both of the ovarian carcinoma cell lines tested in this study. PROG treatment markedly up-regulated p53 expression in these cells, indicating involvement of p53 in PROG-induced apoptosis.

Antineoplastic Agents, Hormonal↗
PubMed2000

Thiol-mediated apoptosis in prostate carcinoma cells.

BACKGROUND: Glutathione (GSH) maintains an optimum cellular redox potential. Chemical depletion, physical efflux from the cell, or intracellular redistribution of this thiol antioxidant is associated with the onset of apoptosis. The aim of this study was to determine the effects of a thiol-depleting agent, diethylmaleate (DEM), on androgen sensitive and insensitive prostate carcinoma cells. METHODS: LNCaP and PC-3 cell lines were induced to undergo apoptosis by DEM and diamide. Apoptosis was quantified by annexin V binding and propidium iodide incorporation using flow cytometry and was confirmed by DNA gel electrophoresis. Intracellular GSH was quantified using a thiol quantitation kit and the generation of reactive oxygen intermediates was measured using dihydrorhodamine 123. Western blot assessed caspase-3, caspase-8, Bcl-2, and Bcl-XL protein expression. Mitochondrial permeability was measured using DiOC6 and stabilized using bongkrekic acid. RESULTS: DEM and diamide induced apoptosis in both androgen sensitive and insensitive cells. Apoptosis was also induced in an LNCaP transfectant cell line overexpressing Bcl-2. Apoptosis was caspase-3 dependent and caspase-8 independent. Bongkrekic acid partially prevented the effects of DEM on mitochondrial permeability but was unable to prevent the induction of apoptosis. Decreased Bcl-2 and Bci-XL protein expression was observed at the time of initial caspase-3 activation. CONCLUSIONS: This study demonstrates that thiol depletion can be used as an effective means of activating caspase-3 in both androgen sensitive and insensitive prostate carcinoma cells. Direct activation of this effector caspase may serve as a useful strategy for inducing apoptosis in prostate carcinoma cells.

Annexin A5↗
PubMed1996

Intracellular distribution of type II collagen mRNA and prolyl 4-hydroxylase in embryonic avian corneal epithelia.

BACKGROUND: The distribution of endoplasmic reticulum (ER) proteins and type II collagen mRNA in whole mount preparations of embryonic corneal epithelia was investigated. We asked, do transmission electron microscopy (TEM) and confocal microscopy of chick prolyl 4-hydroxylase (CPH) show similar ER distributions as CPH is necessary for collagen biosynthesis? METHODS: The overall distribution of ER was analyzed by TEM. Collagen secreting ER was localized with antibodies to chick prolyl 4-hydroxylase (CPH, CPH-alpha, or CPH-beta subunits). Type II collagen mRNA distribution was determined by in situ hybridization. RESULTS: The ER localized with TEM, and immunohistochemistry using anti-CPH antibodies, showed similar distribution patterns. Immunostaining for CPH and CPH-beta was prominent in both periderm and basal cells and also appeared to stain the periderm apical surface. CPH-alpha was less intense, only localizing to ER regions. The basal cell CPH distribution appeared perinuclear in optical sections that contained nuclei, but occupied nearly all the cytoplasm in a reticular pattern above and below nuclei, similar to that seen with TEM cross-sections. Epithelia double labeled with propidium iodide and CPH showed overlapping cytoplasmic staining. The distribution of type II collagen mRNA was similar to the ER staining pattern, appearing to represent a subset of total ER. CONCLUSIONS: This study demonstrates that ER markers have a similar distribution as type II collagen mRNA in embryonic avian corneal epithelia. In addition the CPH subunits had distinct, but overlapping distributions, suggesting that they may act independently.

Animals↗
PubMed1996

Down-regulation of tumour gelatinase/inhibitor balance and preservation of tumour endothelium by an anti-metastatic ruthenium complex.

The anti-metastatic ruthenium complex Na[trans-RuCl4(DMSO)Im] was given i.p. at 22 and 44 mg/kg/day, on days 8-13 after tumour implantation, to mice carrying s.c. implants of MCa mammary carcinoma. The aim of the study was to compare the effects on lung metastasis formation with those on primary tumour cells. This investigation was based on flow cytometry analysis after propidium iodide and acridine orange staining, histology of tumour parenchyma and RT-PCR analysis for the type-IV collagenases MMP-9 and MMP-2 and their respective inhibitors TIMP-1 and TIMP-2 mRNAs. Na[trans-RuCl4(DMSO)Im] is not cytotoxic for tumour cells but has the capacity of interacting with nucleic acids, giving a general reduction of nucleic acid content as shown by a marked reduction of acridine orange staining and a tendency to a reduction of DNA polyploidy with marked reduction of 8n and 4n cell populations. Na[trans-RuCl4(DMSO)Im] also influences a proteolytic system which has the potential of degrading the basement membrane and has been related to metastatic aggressiveness: it markedly reduces, in a dose-dependent manner, MMP-2/TIMP-2 balance, but not that of MMP-9/TIMP-1. The different enzyme/inhibitor mRNA levels between untreated and treated tumours seem to be unaffected by tumour-infiltrating lymphocytes and are paralleled by the maintenance of connective tissue around blood vessels in the tumour mass. Correspondingly, lung metastasis formation is markedly reduced, to less than 10% of that seen in controls.

Acridine Orange↗
PubMed1996

Reliable flow cytometric estimation of nuclear DNA content in coffee trees.

Flow cytometry gave high resolution of genome size in two coffee taxa (Coffea liberica dewevrei and C. pseudozanguebariae). Propidium iodide (PI) and Petunia hybrida were used as dye and internal standard, respectively. Proportionally between the DNA content and the digitized fluorescence signal was checked. Five main results were evident. First, optimal experimental conditions were established for peak location estimation (mean or mode), staining time (at least 2 minutes), high voltage (557 V) for the photomultiplier tube (PMT), and PI concentration (333 micrograms/ml). Second, a parameterization of the effects of high voltage and PI concentration were deduced from curve fitting. Third, two biases in DNA content estimation were recorded for high voltage and PI concentration, and were minimized. Fourth, the genome sizes of C. liberica dewevrei and C. pseudozanguebariae were estimated with accuracy 2C = 1.421 +/- 0.005 pg and 2C = 1.129 +/- 0.005 pg, respectively. Fifth, between-genotype variance was emphasized within each taxon.

Cell Nucleus↗
PubMed1996

Flow cytometric analysis of the early phases of apoptosis by cellular and nuclear techniques.

The early events occurring during apoptosis at the plasma membrane, chromatin, and mitochondrial levels were investigated in freshly isolated irradiated human lymphocytes, growth factor-deprived cultured human lymphocytes, and dexamethasone (DEX)-treated murine thymocytes. In intact, unfixed cells, evaluation of the light scatter properties and of DNA stainability with ethidium bromide (EB) allowed a cell subset suggestive for initial apoptosis to be identified. The apoptotic nature of these cells was confirmed by cell sorting in irradiated human lymphocyte model. EB could not be substituted for by propidium iodide, indicating that the nature of DNA probe used is of major importance for detecting initial apoptotic changes. Because mitochondria are thought to represent a primary target during apoptosis, we measured the uptake of mitochondria transmembrane potential sensitive (Rhodamine 123) and nonsensitive (10-nonyl-acridine-orange) probes concomitantly with EB uptake. Cells starting apoptosis had an enhanced incorporation of both mitochondria dyes, which in combination with EB identified several cell subsets. This suggests that complex alterations in mitochondrial structure and functioning occur in the early stages of apoptosis. To investigate phenomena occurring at the chromatin level in similar phases of apoptosis, irradiated human lymphocytes and DEX-treated murine thymocytes were disrupted and DNA stainability assessed in nuclear suspensions. A transient increase in DNA stainability, i.e., the appearance of distinct hyperdiploid peaks in the human model and a generalised upward shift of the G0/1 peak in the murine model, was observed in the early phases of apoptosis concomitantly with specific alterations in light scattering properties. These findings suggest that chromatin texture is altered in early apoptosis and affects DNA stainability.

Acridine Orange↗
PubMed1996

Interactions of intercalating fluorochromes with DNA analyzed by conventional and fluorescence lifetime flow cytometry utilizing deuterium oxide.

Deuterium oxide (D2O) has been shown in previous studies to increase both the fluorescence lifetime and fluorescence intensity of propidium iodide (PI) and ethidium bromide (EB) when bound to nucleic acid structures. We have used spectroscopic analysis and conventional and phase-sensitive flow cytometry to compare changes in PI and EB fluorescence intensity and lifetime bound to DNA and fixed Chinese hamster ovary (CHO) cells in the presence of D2O vs. phosphate-buffered saline (PBS). Spectroscopic and flow cytometric studies showed a twofold enhancement of fluorescence intensity of PI and EB bound to fixed CHO cells in D2O and a 5 ns increase in PI and EB fluorescence lifetimes in D2O. The fluorescence lifetime of HL-60 cells stained with PI or EB was found to be 1-2 ns different from that of CHO cells, indicating that the lifetime of these fluorochromes is sensitive to chromatin configuration in different cells types. Apoptotic subpopulations of HL-60 cells had a significantly reduced fluorescence lifetime compared to nonapoptotic subpopulations. Results indicate that different chromatin states, or differences in the structures of PI and EB, lead to alterations in the fluorescence intensity and fluorescence lifetime of these intercalating probes.

Animals↗
PubMed1996

Adaptation of a simple flow cytometric assay to identify different stages during apoptosis.

Apoptosis occurs under many different physiological and pathological conditions, and it reflects a genetically encoded suicide program that can be triggered by different stimuli in susceptible cells. We adapted a flow cytometric assay for the detection of apoptosis based on differential staining of viable cells with two different DNA binding dyes, propidium iodide (PI) and Hoechst 33342 (Ho342). Apoptosis was induced in different cell lines by gamma irradiation, an anti-FAS monoclonal antibody, or ganciclovir in Herpes simplex virus-thymidine kinase-expressing cells. We could identify three different populations that appeared sequentially after the induction of apoptosis. Cells corresponding to these populations were sorted and assessed for evidence of apoptosis as determined by alterations of nuclear morphology and detection of endonucleolytic activity. This analysis revealed a PI- population with subtle apoptotic changes and increased Ho342 fluorescence compared with untreated cells. Extensive apoptotic alterations were observed in a PI+ population that increased over time following the induction of apoptosis. A third population was characterized by an intermediate intensity of PI fluorescence and decreased Ho342 fluorescence compared with the other populations. This population appeared late after treatment and consisted of apoptotic bodies. Taken together, these data suggest that distinct stages of apoptosis can be identified by differential staining of cells with Ho342 and PI. This assay should be useful for the detection and further characterization of cells at different stages in the apoptotic process.

Apoptosis↗
PubMed1996

One-tube triple staining method for flow cytometric analysis of DNA ploidy and phenotypic heterogeneity of human solid tumors using single laser excitation.

We have developed a "one-tube" triple staining procedure that allows the identification of intratumor phenotypic subpopulations by FCM. Solid tumors were dissociated by a combined mechanical/ enzymatic method. Ovarian ascites tumor cell aggregates were enzymatically dissociated using trypsin. An antikeratin 8/18 MAb was used to label the epithelial fraction of these tumor samples. A second MAb directed against the leukocyte common antigen (LCA) was applied to identify nonneoplastic DNA-diploid cells. Other MAbs used as a second marker were directed against a tumor-associate surface, a cytoplasmic, or a nuclear antigen. Cells were stained using subclass-specific fluorescein-isothiocyanate (FITC) or R-phycoerythrin (PE)-conjugated antibodies. DNA was stained with propidium iodide (PI). Triply stained samples were measured on a standard bench-top flow cytometer (FACScan). Keratin 8/18-positive cells, LCA-positive cells, and DNA could be simultaneously detected in dissociated breast carcinomas, mixed Müllerian tumors, and ovarian ascites specimen for refining DNA index (DI) calculations and S phase fraction (SPF) determination. Coefficients of variation (CV) of the G0G1 peak of the DNA histograms obtained ranged from 2.55% to 4.64% and from 2.71% to 4.71% for the DNA-diploid and -aneuploid fractions, respectively. In DNA-diploid tumors, antigen expression (HER-2/Neu, proliferating cell nuclear antigen) could be analyzed without interference of fluorescence signals from nonneoplastic cells. Neoplastic tumor subpopulations were clearly identified based on both DNA-ploidy status and heterogeneity of antigen expression. The present method offers new possibilities for multiparameter DNA FCM on clinical samples and enables the identification of intratumor neoplastic subpopulations based on antigen expression and DNA-ploidy status.

Biomarkers↗
PubMed1997

Flow cytometric assessment of hydroxypyridinone iron chelators on in vitro growth of drug-resistant malaria.

The resurgence of drug-resistant malaria makes urgent the evaluation of new antimalarial agents. This study describes a flow cytometric method (FCM) for testing in vitro drug susceptibility of Plasmodium falciparum malaria to several orally active hydroxypyridinone (CP) iron chelators and to the parenteral iron chelator desferrioxamine (DF). After exposure of parasites to various concentrations of iron chelating agents, aliquots of cultures were fixed with glutaraldehyde. The fixed samples were washed and stained for parasite DNA with propidium iodide and analyzed by flow cytometry. The remaining cells were pulsed with 3H-hypoxanthine, using the microdilution radioisotope method. Both CP and DF showed dose-dependent inhibition of parasite growth. Of the compounds studied, DF exerted a stronger inhibitory effect. Fifty percent of inhibitory concentrations (IC50) of CP and DF determined by DNA fluorescence profiles in the flow cytometer were consistent with those obtained from the radioisotope method and by microscopic examination. Moreover, the minimum inhibitory concentrations (MIC) of drug required to inhibit parasite growth, as detected by the decreasing DNA fluorescence intensity of the schizont, correlated with observed abnormal microscopic morphology. The validity of the MIC, as indicated by decreased fluorescence intensity, was confirmed by subsequent parasite culture. Our FCM study demonstrated the sensitivity of both chloroquine- and pyrimethamine-resistant malaria parasites to iron chelators. Addition of equimolar concentrations of ferric ion completely abolished the inhibitory effect of iron chelators, indicating the importance of iron for parasite growth and the primary effect of the compounds as iron (III) chelating agents. These data demonstrate that FCM provides a simple and reliable means for antimalarial drug susceptibility testing, and suggest that iron chelators have potential for the treatment of drug-resistant malaria.

Animals↗
PubMed1997

Three-parameter flow cytometric analysis of rat spermatogenesis.

Mammalian spermatogenesis is a complex process which is not yet fully understood. In this paper we describe the analysis of rat spermatogenesis by means of 3-parameter flow cytometry. Since the analysis of DNA content only provides sufficient information for the identification of 4 cell populations, additional parameters were combined with propidium iodide (PI) staining. Immunostaining of the intermediate filament vimentin allowed the identification of somatic (vimentin positive) and germ (vimentin negative) cells. Utilizing the combination of DNA and vimentin staining, we have been able to quantitate the somatic cells present in a testicular cell suspension and to analyze somatic and germinal cells separately. Furthermore, the addition of mitochondrial staining with the fluorochrome nonyl acridine orange (NAO) allowed several cell subpopulations within each ploidy group to be distinguished. After 3-color staining and subsequent cell sorting, 11 testicular cell subpopulations could be identified: somatic cells, and 10 subtypes of germinal cells. The method described in this paper represents a valuable tool for the evaluation of spermatogenesis in both normal and perturbed situations.

Acridine Orange↗
PubMed1997

Sample preparation from paraffin-embedded tissue specimens for laser scanning cytometric DNA analysis.

We have developed a simple, rapid method for isolating cells from a block of formalin-fixed, paraffin-embedded tissue specimen for laser scanning cytometric (LSC) DNA analysis by using a grater. The scraping-like tissue samples were obtained by grating a paraffin-embedded tissue block. The grated samples were collected, put into a small plastic tube, and deparaffinized with xylene. Subsequently, the samples were immersed in 100% ethanol to remove the xylene. After using a syringe with a 26-gauge needle and filtering through 40-microm nylon mesh, the cells suspended in ethanol were dropped directly onto a glass slide. As a result, isolated cells adhered tightly to the glass slide. The slides mounted with isolated cells were treated with 0.1% pepsin in 0.1 N HCl for 1 h at 37 degrees C and then 0.1% RNase for 10 min at room temperature. The slides were dipped in propidium iodide (25 microg/ml) to stain DNA and sealed with nail varnish. The coefficients of variation for histograms were small enough to detect an aneuploid peak close to the diploid peak.

Aneuploidy↗