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PubMed1993

Increased sensitivity of damaged DNA to digestion with nuclease S1 as assessed in single cells by flow cytometry.

DNA sensitivity to digestion with nuclease S1 was investigated in cells irradiated with gamma rays, or treated with the antitumor drug adriamycin (Adr). The nuclease-resistant DNA fraction was determined by propidium iodide staining. Treated cells were found to be more sensitive to nuclease digestion than the undamaged controls. Gamma ray-induced strand breaks were detectable at doses up to 10 Gy; an increase in the reaction temperature, from 37 degrees to 63 degrees C, was necessary in order to detect higher levels of damage. Nuclease S1 sensitivity in Adr-treated cells showed a single-peak, concentration-dependent relationship, in agreement with the known self-inhibitory effect exerted by high drug doses. Determination of DNA digestion could be performed in combination with other cellular parameters (e.g., protein content). Detection of drug-resistant cells in a heterogeneous population of small-cell lung carcinoma was achieved on the basis of the different sensitivity of the cells to enzymatic digestion. These results indicate that nuclease S1 may be a useful probe for studying in single cells DNA alterations induced by drugs or radiation.

Cell Separation↗
PubMed1993

Flow cytometric analysis of DNA and nuclear protein in paraffin-embedded tissue.

Previously we described the simultaneous quantification of DNA and nuclear protein in unfixed tissue from solid tumors. The resultant 2 parameter flow cytometric analysis has several advantages over that of DNA alone. In this report, we describe a modification of the technique for the analysis of formalin-fixed paraffin-embedded tissue. Paraffin-embedded material was prepared by hydrating sections, incubating in 0.5% pepsin solution, washing, and resuspending in buffer containing nonionic detergent. The nuclei were then stained with fluorescein isothiocyanate and propidium iodide in the presence of ribonuclease. Several solid tumor tissue types have been analyzed, including breast, colon, kidney, and thymus. The best results were obtained when the initial pepsin treatment was for 1.5 h, instead of 0.5 h. Pepsin treatment for 1.5 h improved the CVs of both the DNA and nuclear protein parameters, and did not appear to reduce nuclear protein levels or to cause significant disintegration of nuclei. The DNA/nuclear protein histograms of unfixed and fixed, paraffin-embedded tissue were similar. Since tumor nuclei typically have higher protein levels than DNA-diploid nuclei, the technique reduces population overlapping and permits less subjective identification of DNA aneuploidy.

Aneuploidy↗
PubMed1993

Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase-sensitive flow cytometry.

A flow cytometric method has been developed that uses phase-sensitive detection to separate signals from simultaneous fluorescence emissions in cells labeled with fluorochromes having different fluorescence decay lifetimes. By CHO cells were stained with propidium iodide (PI) and fluorescein isothiocyanate (FITC). These dyes bind to DNA and protein and the fluorescence lifetimes of the bound dyes are 15.0 and 3.6 ns, respectively. Cells were analyzed as they passed through a modulated (sinusoidal) laser excitation beam. Fluorescence was measured using only a long-pass filter to block scattered laser excitation light and a single photomultiplier tube detector. The fluorescence detector output signals were processed by dual-channel phase-sensitive detection electronics and the phase-resolved PI and FITC signals were displayed as frequency distribution histograms and bivariate plots. By shifting the phase of one detector channel reference signal by pi/2 + phi 1 degrees and the phase of the other detector channel reference signal by - pi/2 + phi 2 degrees, where phi 1 and phi 2 are the phase shifts associated with the PI and FITC lifetimes, the PI and FITC signals were separately resolved at their respective phase-sensitive detector outputs. This technology is also applicable to suppressing background interferences caused by cellular autofluorescence, unbound/free dye, nonspecific dye binding, and Raman and Rayleigh scattering.

Animals↗
PubMed1993

Characterization of hypoxia-dependent peroxide production in cultures of Saccharomyces cerevisiae using flow cytometry: a model for ischemic tissue destruction.

Peroxide production in cultures of Saccharomyces cerevisiae was measured using the H2O2-sensitive fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) and flow cytometry. Aeration of cultures of S. cerevisiae exposed to a period of hypoxia was found to induce elevated levels of peroxide that were 100-fold higher than the levels observed in cultures maintained under exclusively aerated or hypoxic conditions. Simultaneous viability analysis, using the fluorescent DNA-intercalating dye propidium iodide, indicated that the increase in peroxide generation preceded cell damage and death. Various agents were found to influence the effect of peroxides on cell viability. The addition of ethanol to hypoxic stationary cultures dramatically increased the rate of cell death without further increasing the amount of peroxide produced, while glucose inhibited peroxide production and decreased the rate of cell death. Surprisingly, elevated peroxide levels of hypoxic/reaerated cultures were maintained upon addition of KH2PO4, although the cells remained viable for extended periods of time when compared to control and other test cultures. Similarities between our observations and those of other investigators using anoxic/reperfused organs suggest that hypoxic/reaerated yeast cultures may be a useful model system to study ischemia-dependent tissue destruction of mammals.

Cell Death↗
PubMed1993

Two-color flow cytometric analysis of intraerythrocytic malaria parasite DNA and surface membrane-associated antigen in erythrocytes infected with Plasmodium falciparum.

A method for fixation of Plasmodium falciparum infected erythrocytes and solubilization of the erythrocyte membrane with detergent was developed. This method was applied to two-color flow cytometric analysis of both intraerythrocytic (IE) malaria DNA and parasite-derived antigen on the erythrocyte surface membrane. Infected erythrocytes were fixed with 0.025% glutaraldehyde followed by treatment with 1% saponin to gain access to intramembranous components and allow antibody to interact with antigen. DNA of IE parasite was subsequently stained with propidium iodide. Using this procedure cell morphology was well preserved with excellent parasite DNA staining. Using anti-malaria antibodies which recognize ring-infected erythrocyte surface antigen (Pf155/RESA), we observed that glutaraldehyde-fixed saponin treated infected erythrocytes exhibited a variable immunofluorescence intensity as assessed by both flow cytometry and fluorescence microscopy. Ring-infected cells displayed strong immunofluorescence staining, whereas a weak signal was detected on cells containing schizonts. Simultaneous measurement of parasite DNA and antigen in the infected erythrocyte membrane can facilitate the study of antigen expression in the cell membrane in association with development of IE parasites.

Animals↗
PubMed1993

Recognition and reduction of artifacts from autolysis in paraffin-embedded tissue using DNA/nuclear protein flow cytometry.

Artifacts from autolysis can be a problem in retrospective flow-cytometric analyses of DNA content in paraffin-embedded tissues. Autolyzed tissue from rat liver, human liver, and rat spleen were stained for DNA and nuclear protein to determine if this technique would be useful in identifying partially degraded cells. After the tissue was deparaffinized and rehydrated, the nuclei were isolated using 0.5% pepsin. Propidium iodide (PI) and fluorescein isothiocyanate (FITC) were used to stain DNA and nuclear protein. When unfixed rat liver tissue was allowed to undergo autolysis at 4 degrees C for 24-48 h before fixation, there was a progressive broadening of the G1 and G2M DNA peaks and a slight increase in the average DNA contents of these peaks. Nuclei that stained more intensely with PI also stained more intensely with FITC. Similar results were obtained using human liver and rat spleen. Sometimes the increased PI staining resulted in a false aneuploid peak. The distinctive skewing of the DNA/nuclear protein histograms from autolysis was reduced by increasing the incubation of the tissue in 0.5% pepsin from 0.5 h to 1.5 h during the nuclei-isolation step. The DNA/nuclear protein method provides a means for identifying artifacts from autolysis, whereas the extended pepsin treatment provides a means for reducing these artifacts.

Animals↗
PubMed1993

Analysis of lymphocyte activation and proliferation by video microscopy and digital imaging.

Video microscopy and digital imaging were used as a noninvasive method to quantitatively analyze lymphocyte activation and proliferation. This method takes advantage of the fact that upon activation lymphocytes blast and become significantly larger before proliferating. The mean cell sizes of T lymphocytes in an activation kinetics assay were measured by digital image analysis and compared to [3H]-thymidine incorporation of cells under the same treatment. An increase in cell size was observed before [3H]-thymidine incorporation; therefore the digital imaging assay is more sensitive in determining the earliest time-point of activation. Also, the digital imaging assay was comparable to the [3H]-thymidine incorporation assay in providing information about the extent and rates of T lymphocyte proliferation. Cellular DNA was stained with propidium iodide to show that the larger blasting cells in the population of activated T lymphocytes were indeed the cells that accounted for the increase in DNA synthesis and thus an increase in cell size can be correlated with activation.

Analog-Digital Conversion↗
PubMed1994

TOTO and YOYO: new very bright fluorochromes for DNA content analyses by flow cytometry.

Flow cytometric (FCM) studies were performed on nuclei, ethanol-fixed CHO cells, and isolated human GM130 chromosomes stained with two new cyanine dyes, TOTO and YOYO. These fluorochromes, which are dimers of thiazole orange and oxazole yellow, respectively, have high quantum efficiencies and exhibit specificities for both DNA and RNA. Bound to dsDNA in solution, TOTO and YOYO emit at 530 and 510 nm, respectively, when excited at 488 nm and 457 nm, wavelengths available from most lasers employed in FCM. RNase-treated CHO nuclei, stained with either TOTO or YOYO, provided DNA histograms, with low coefficients of variation, that were as good as or better than those obtained with nuclei stained with propidium iodide (PI) or mithramycin (MI). In addition, by comparison on an equimolar basis, nuclei stained with YOYO fluoresced over 1,000 times more intensely than nuclei stained with MI. Fluorescence ratio analyses of nuclei stained with both YOYO and Hoechst 33258 showed that the ratio of YOYO to Hoechst fluorescence remained relatively constant for G1 and S phase cells, but decreased significantly for cells in G2/M. These results indicate that the cyanine dyes may be useful in examining specific changes in chromatin structure during G2/M phases of the cell cycle. Ethanol-fixed CHO cells stained with TOTO or YOYO did not yield reproducible DNA histograms of good quality, presumably because of the poor accessibility of DNA to these large fluorochromes. However, bivariate analyses of human GM130 chromosomes stained with TOTO or YOYO alone and excited sequentially with uv and visible wave-lengths showed resolution of many individual chromosome peaks similar to results obtained for chromosomes stained with HO and chromomycin A3. Collectively, these studies show potential advantages for the use of these new cyanine dyes in FCM studies that require the sensitive detection of DNA.

Animals↗
PubMed1994

Further flow cytometric studies of the effects of triethylenemelamine on somatic and testicular tissues of the rat.

Exposure to the mutagen triethylenemelamine on rat bone marrow, blood, and testis was studied using flow cytometry of DAPI-stained nuclei. Increased coefficients of variation (CVs) of the G1 peaks were observed in bone marrow and blood after both 1 d and 5 d exposures. After 5 d exposure and 7 d recovery both tissues had recovered, in some cases to significantly lower CVs. Increased CVs of the 1C peak of testis were observed only after 5 d exposure to the high dose with no subsequently observed recovery. Bone marrow cells also were stained with Hoechst 33258 and Propidium Iodide. No differences among dyes were observed indicating that increased CVs likely are due to DNA damage resulting from interactions with the mutagen rather than differences in how the dyes bind to DNA relative to mutagen binding. This study demonstrates that differences occur among tissues in how quickly they respond and recover from mutagen exposure. Increased CVs, cell cycle alterations, and decreased CVs after recovery are all potentially useful biomarkers of effect for laboratory and field studies in environmental toxicology.

Animals↗
PubMed1994

Flow cytometric detection of apoptosis: comparison of the assays of in situ DNA degradation and chromatin changes.

The aim of this study was to compare three methods of detection of apoptotic cells: (1) the method based on elution of low molecular weight DNA from the ethanol fixed cells followed by cell staining with DAPI (diamidino-2-phenylindole) or propidium iodide as the DNA fluorochromes, (2) the method of in situ labeling of DNA strand breaks with biotinylated dUTP, utilizing exogenous terminal deoxyribonucleotide transferase, and (3) the method of analysis of DNA denaturation in situ using acridine orange to differentially stain denatured and double-stranded DNA sections following cell exposure to 0.1 M HCl. Cells of the human promyelocytic HL-60 line, treated in vitro with the DNA topoisomerase I inhibitor camptothecin, which selectively triggers apoptosis of S-phase cells, were chosen as a model. The method based on analysis of changes in DNA denaturability was the most sensitive in terms of detection of the earliest changes in chromatin of cells undergoing apoptosis; the increased sensitivity of DNA to denaturation in S-phase cells was measured as early as 100 min after addition of camptothecin. DNA cleavage, assayed either by the univariate measurement of DNA content following extraction of low molecular weight DNA, or by labeling DNA strand breaks with biotinylated dUTP, was detected in S-phase cells after 120 min incubation with camptothecin. The percentage of apoptotic cells at the late stage of apoptosis, the kinetics of cell transition to apoptosis, and kinetics of the loss of S phase cells were all essentially similar when measured by any method.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoptosis↗
PubMed1994

Rapid quantification of lymphocyte subsets in heterogeneous cell populations by flow cytometry.

Determination of the number of viable cells or quantification of lymphocyte subsets in heterogeneous cell populations is critically important for cytotoxicity assays, apoptosis assays, or the analysis of differential activation of T-cell subsets by distinct stimuli. In this report, we describe a rapid flow cytometry method termed Standard Cell Dilution Analysis (SCDA) specifically to quantify any subset of phenotypically definable, viable cells in heterogeneous populations using a FACScan flow cytometer. This method combines: (1) specific detection of lymphocyte subsets by phycoerythrin-conjugated monoclonal antibodies, (2) electronic exclusion of dead cells or cell debris by propidium-iodide staining and gating on forward vs. sidescatter, respectively, and (3) admixture of a known amount of fixed, fluorescein isothiocyanate stained cells immediately before analysis as a constant parameter to allow for calculation of cell quantity. We have used SCDA to analyze the in vitro growth characteristics of various human T-lymphocyte subpopulations in response to different activation stimuli.

Antibodies, Monoclonal↗
PubMed1994

Evaluation of interspecific DNA content variations and sex identification in Falconiformes and Strigiformes by flow cytometric analysis.

A high interspecific karyotype variability has been evidenced in birds especially in Falconiformes and Strigiformes. Avian cytogenetic analysis, conventionally used for this study, presents several difficulties. We used flow cytometric analysis in order to obtain further information on the DNA patterns of different species of birds belonging to the above-mentioned orders. Our study was performed on blood samples while chicken erythrocytes and human lymphocytes, with known cytometric DNA content, were used as reference cells. The blood samples of the birds under study were stained, simultaneously to the reference cell, with a lysis-staining buffer containing propidium iodide. The nuclear DNA content of the bird samples was calculated as DNA index in relation to reference cells, and was expressed as nuclear DNA mass in picograms (pg) with respect to the standard value of 7.0 pg per human lymphocyte nucleus. The results obtained showed an interspecific variability of DNA content and evidenced the usefulness of FCM analysis as a rapid and easy tool for studying the DNA pattern of different species of birds. Moreover, our results have confirmed and extended the possibility of sex identification in species of birds characterized by sexual monomorphism by evaluating the small DNA content difference which exists between males and females.

Animals↗
PubMed1994

Flow cytometric detection and quantitation of the epidermal growth factor receptor in comparison to Scatchard analysis in human bladder carcinoma cell lines.

The epidermal growth factor receptor (EGFR) is considered a tumor-related marker with potential diagnostic and prognostic value. In order to assess the sensitivity of flow cytometry to detect EGFR and to quantify receptors objectively, two human bladder carcinoma cell lines with different urothelial differentiation, RT4 and J82, were grown in vitro, and their membrane EGFR content was measured by flow cytometry. Exponential monolayers showed decrease of EGFR content after 20 min pulses with 10 ng/ml EGF in medium, as detected with the antibody EGFR1 in a double staining technique with propidium iodide for DNA evaluation. Further decrease of green fluorescence intensity was seen in cells constantly exposed to EGF. Absolute receptor numbers were determined by Scatchard analysis with radioactive EGF and resulted in relatively low receptor numbers for both cell lines (approximately 3-4 x 10(4) EGFR/cell), as well as one affinity class. These findings could be matched by absolute receptor quantification by flow cytometry, adding beads with defined antigenic sites (Quantum Simply Cellular, Microbead Corporation) to the cell suspension for staining. Our data suggest that flow cytometric EGFR detection and quantitation may be supplied to in vivo tumor samples and that measurements by multiparameter analysis may define subpopulations valuable for tumor diagnosis and judgment on tumor progression.

Biomarkers, Tumor↗
PubMed1994

Detection of bromodeoxyuridine incorporation by alteration of the fluorescence emission from nucleic acid binding dyes using only an argon ion laser.

A method was developed that uses paired DNA dyes to detect the incorporation of bromodeoxyuridine (BrdUrd) into cellular DNA and requires only 488 nm excitation. The fluorescence of thiazole blue, TO-PRO-3, and LDS-751 was found to be enhanced by the presence of BrdUrd in DNA. Pairing LDS-751, thiazole blue, or TO-PRO-3 with propidium iodide (PI) for flow cytometry allowed the differentiation of cells containing BrdUrd from BrdUrd unlabeled cells. LDS-751 can be excited directly at 488 nm, and TO-PRO-3 or thiazole blue can be excited indirectly by resonance energy transfer from PI. The enhancement of fluorescence from these dyes is correlated with a decrease in PI fluorescence, suggesting an increased energy transfer from PI to the red emitting dye. Fluorescence from other dyes, including thiazole orange, TO-PRO-1, rhodamine 800, oxazine 750, and 7-aminoactinomycin D, was not altered by the presence of BrdUrd in DNA. Results also were obtained showing that BrdUrd detection using PI and TO-PRO-3 is compatible with immunofluorescence staining with FITC-labeled antibodies.

Bromodeoxyuridine↗
PubMed1995

Cell membrane-dependent chromatin condensation.

It is shown by means of flow cytometry that during several seconds after cell membrane damage by a non-ionic detergent in physiologically relevant buffer solution, the chromatin of mouse thymocyte nuclei undergoes a drastic decondensation, which is revealed by a sharp increase of binding of DNA-specific fluorochromes (olivomycin or propidium iodide) and of DNA accessibility to DNAse I digestion. A similar change is observed in dead cells. Roughly half of this decondensation can be prevented by lowering the pH of the outside medium to the level known to be inside the cells; the other half remains thus far unexplained (divalent cations and the difference between small anion species seem not to be involved). The approach is based on a novel observation that fixation by formaldehyde conserves chromatin structure before the action of detergent. Flow cytometric assay is proposed for monitoring these condensation/decondensation events in media of different composition. In addition, a new approach to viable/dead cell determination, which has the advantage of immediately fixing the cell state and preserving it for a reasonably long time, is proposed.

Animals↗
PubMed1995

Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry.

Dead cells represent a significant source of interference in the flow cytometric analysis of viable cells primarily due to nonspecific uptake of probes, increased autofluorescence, and altered antigen expression and DNA content. Traditional methods of dead cell exclusion, based on light scatter or uptake of dyes such as propidium iodide (PI) or fluorescein diacetate (FDA), are appropriate for the analysis of fresh, relatively homogeneous samples. However, they are incompatible with the development in this laboratory of a solid tumor monoclonal antibody panel incorporating combined surface and intracellular staining: Light scatter is unreliable in heterogeneous samples such as solid tumors, and most of the widely used viability probes are incompatible, due to weak or reversible binding, with the use of permeabilizing agents for intracellular staining. To determine the best viability marker for inclusion in the solid tumor panel, we compared cultured cells held under hypoxic conditions for up to 15 days after harvest, stained with eight viability probes, and processed according to the solid tumor panel procedure (unprocessed cells from each day, stained with PI, were used as standards). The viability probes included PI (in processed and unprocessed samples); 7-aminoactinomycin D (7-AAD); TO-PRO-3; laser dye styryl (LDS)-751; ethidium monoazide (EMA); and actin, cytokeratin, and tubulin indirectly labelled with sheep-alpha-mouse-FITC (SAM-FITC). The selection criteria for the best viability probe included broad cell type specificity: low nonspecific staining of live cells, specific staining of dead cells strong enough to withstand the permeabilization procedure, high signal-to-noise ratio throughout the time course, and compatibility with the four other fluorescent probes making up the tumor antibody panel. TO-PRO-3, LDS-751, and PI (in processed cells) stained both live and dead cells indiscriminately. Actin-SAM-FITC, EMA, and 7-AAD did not display sufficiently high signal-to-noise ratios over the entire time course. Cytokeratin-SAM-FITC was acceptable in every respect other than its specificity only for cells of epithelial origin. Tubulin-SAM-FITC alone satisfied all the criteria and was selected for inclusion in the monoclonal antibody panel as a viability probe.

Azides↗
PubMed1995

Mouse testis cell sorting according to DNA and mitochondrial changes during spermatogenesis.

Flow cytometry can measure variations in DNA content and chromatin structure as well as dramatic changes in the mitochondria of germ cells during maturation from spermatogonia to elongated spermatids. Using 10-N nonyl acridine orange (NAO), an inner mitochondrial membrane dye, it is easy to follow mitochondria rearrangements. Mouse testis cells stained with the DNA fluorescent probe propidium iodide (PI) and analyzed by flow cytometry can be discriminated on the basis of their ploidy levels into five main regions corresponding to elongated spermatids, round spermatids, diploid, S-phase, and tetraploid cells. The simultaneous use of PI and NAO demonstrated the presence of cells having low and high mitochondrial content in the haploid, diploid, and tetraploid compartments. Eleven sorting windows were selected from the bivariate analysis (PI/NAO) and the corresponding cells were identified by microscopic observation. Cells were also discriminated by two parameter analysis of DNA content vs. cell diameter. The definition of seven different regions allowed us to determine NAO or rhodamine 123 (Rh 123) uptakes in each compartment. We observed that the ratio (Rh 123/NAO) dramatically changed according to the progression of cell differentiation which occurs during spermatogenesis.

Acridine Orange↗
PubMed1995

A new flow cytometric method for discrimination of apoptotic cells and detection of their cell cycle specificity through staining of F-actin and DNA.

Drug-initiated apoptosis of human leukemia HL-60, THP-1, and U-937 cells was studied via multiparameter flow cytometry and cell sorting. A new flow cytometric method that allows both identification and quantitation of apoptotic cells and estimation of their cell cycle specificity is presented. The method is based on paraformaldehyde fixation followed by staining of F-actin and DNA with fluorescein isothiocyanate (FITC)-phalloidin and propidium iodide (PI), respectively. Bivariate green fluorescence (F-actin) vs. side scatterplots of HL-60 cells treated with 10 microM etoposide for 4 h showed two cell populations, one with high green fluorescence and low side scatter and one with low green fluorescence and high side scatter. Sorting revealed cells with intact nuclei in the high green fluorescence/low side scatter population and cells with fragmented nuclei in the low green fluorescence/high side scatter population, demonstrating that the cells in the latter population were apoptotic. Exposure of HL-60 cells to 10 microM etoposide for 4 h resulted in S-phase selective apoptosis, whereas 5 micrograms/ml cycloheximide initiated apoptosis mainly in G0/G1-phase and S-phase cells. The apoptotic response of HL-60 cells to 20 GY gamma-irradiation was selective for S-phase and G2 + M-phase cells. The present method offers the opportunity to estimate the cell cycle distributions of both the apoptotic and the nonapoptotic cell populations, which is especially valuable when apoptosis occurs in association with cell cycle perturbations. A similar shift from one to two cell populations in green fluorescence vs. side scatter-plots, similar to that observed for HL-60 cells, was observed in the THP-1 and U-937 cell lines secondary to etoposide treatment.

Actins↗