Flow cytometric analysis of experimental parameters for the immunofluorescent labeling of BrdUrd in various tumour cell lines

Summary This report describes the results of the comparison of three different methods and three monoclonal antibodies to stain cells in suspension for incorporated bromodeoxyuridine and total DNA content. The procedures were tested in three different experimental tumour cell lines. The sensitivity of the different procedures was expressed as the ratio of the anti-BrdUrd fluorescence intensities of the S and G1 phase cells (FS/FG1 ratio). There were remarkable differences in sensitivity between the different procedures. With the heat denaturation the most favourable FS/FG1 ratio's were obtained but substantial cell loss occurred during this procedure which is a disadvantage for clinical application. With the pepsin digestion + acid denaturation procedure cell loss was negligible. The standard acid denaturation procedure was inferior to the other two methods. Using the pepsin digestion + acid denaturation procedure we examined the variations in sensitivity for the different monoclonal antibodies and cell lines and the influence of BrdUrd concentration, labelingtime and cell concentration. The binding characteristics for the various antibodies differed considerably in our hands. Only with the IU4 antibody we obtained FS/FG1 ratio's comparable with those desenbed in the literature. No difference was observed between the cell lines. Variation in cell concentration between 1 × 10⁴ to 1 × 10⁶ ml nor BrdUrd concentration appeared to influence the sensitivity of the procedure. A labelingtime of 1 h or even 30 min seems to be more than sufficient for an optimal FS/FG1 ratio.Our results indicate that using the appropriate antibody and immunofluorescence BrdUrd can be detected by flow cytometry, after incorporation into the DNA of tumour cells under a wide range of culture conditions.For clinical application, the pepsin digestion + acid dena uration method in combination with IU4 antibody seems to be the procedure of choice due to its good reproducibility, sensitivity and its low cell loss.     

Flow cytometric analysis of experimental parameters for the immunofluorescent labeling of BrdUrd in various tumour cell lines

This report describes the results of the comparison of three different methods and three monoclonal antibodies to stain cells in suspension for incorporated bromodeoxyuridine and total DNA content. The procedures were tested in three different experimental tumour cell lines. The sensitivity of the different procedures was expressed as the ratio of the anti-BrdUrd fluorescence intensities of the S and G1 phase cells (FS/FG1 ratio). There were remarkable differences in sensitivity between the different procedures. With the heat denaturation the most favourable FS/FG1 ratio's were obtained but substantial cell loss occurred during this procedure which is a disadvantage for clinical application. With the pepsin digestion + acid denaturation procedure cell loss was negligible. The standard acid denaturation procedure was inferior to the other two methods. Using the pepsin digestion + acid denaturation procedure we examined the variations in sensitivity for the different monoclonal antibodies and cell lines and the influence of BrdUrd concentration, labelingtime and cell concentration. The binding characteristics for the various antibodies differed considerably in our hands. Only with the IU4 antibody we obtained FS/FG1 ratio's comparable with those described in the literature. No difference was observed between the cell lines. Variation in cell concentration between 1 x 10(4) to 1 x 10(6) ml nor BrdUrd concentration appeared to influence the sensitivity of the procedure. A labelingtime of 1 h or even 30 min seems to be more than sufficient for an optimal FS/FG1 ratio. Our results indicate that using the appropriate antibody and immunofluorescence BrdUrd can be detected by flow cytometry, after incorporation into the DNA of tumour cells under a wide range of culture conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow cytometric analysis of the cell cycle in chronic gastritis.

Flow cytometric cell cycle analysis was recorded in gastric biopsy specimens from patients with normal gastric mucosa (GM), superficial gastritis (SG) and chronic atrophic gastritis (CAG). Cell-cycle analysis showed significantly higher percentages of cells in S- and S+G2/M-phase in CAG than in SG and normal GM (P < 0.0001). Moreover, CAG with severe or moderate atrophy showed significantly higher percentages of cells in S-phase (P < 0.05) and S+G2/M-phase (P < 0.02) than CAG with mild atrophy in antrum. In fundus, even if this increase was observed, it did not reach statistical significance. Consideration of concomitant pathologic findings such as oesophagite, gastric or duodenal ulcer, duodenite or benign polyp allowed a better differentiation of CAG both in antrum and in fundus. Significantly higher S-phase was observed in CAG with severe or moderate atrophy than in CAG with mild atrophy (P < 0.05). No statistically significant results were observed in patients with normal gastric mucosa or chronic gastritis and a concomitant pathologic finding.     

Flow cytometric analysis of isolated liver mitochondria to detect changes relevant to cell death.

BACKGROUND: Mitochondria are key players in many forms of cell death, and mitochondrial production of reactive oxygen species (ROS), membrane depolarization, permeability changes, and release of apoptogenic proteins are involved in these processes. Flow cytometric analysis of isolated mitochondria enables parallel analysis of mitochondrial structure and function in individual mitochondria, and small mitochondrial samples are sufficient for analysis. This article describes a well-characterized protocol for flow cytometric analysis of isolated liver mitochondria that can be used to detect mitochondrial alterations relevant to cell death. METHODS: Fluorescent probes were used to selectively stain mitochondria (nonyl acridine orange), and to measure membrane potential (tetramethylrhodamine-methyl-ester, 1,1',3,3,3',3'-hexamethylindodicarbocyanine-iodide), as well as production of ROS (2',7'-dichlorodihydrofluorescein-diacetate). Calcium-induced mitochondrial swelling was detected as a decrease in SSC. To ensure optimal concentrations of all probes, the effect on mitochondrial respiration was evaluated. RESULTS: This protocol can be used to determine the purity of the mitochondrial preparation, to detect calcium-induced morphological changes, small mitochondrial de- and hyperpolarizations, as well as physiological changes in ROS generation. CONCLUSIONS: Flow cytometry is a very useful tool to simultaneously analyze several mitochondrial parameters that are important in the induction of mitochondria-mediated cell death.     

Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea

The effects of the mutagenic agent ethylnitrosourea (ENU) on spermatogenic function and sperm chromatin structure were studied by flow cytometry and the results compared with sperm head morphology measurements. Groups of mice received daily exposures ranging from 0 to 75 mg/kg body weight X 5 days and were sacrificed 28 days later. Fresh testicular cell suspensions and epididymal sperm were stained with acridine orange (AO) and measured by flow cytometry. Sperm nuclei were isolated, fixed, rehydrated, and then either subjected to thermal stress or not prior to staining with AO. Body weights were unaffected by the chemical exposure while the testicular weights were reduced by about 50%. Two-parameter (DNA, RNA) flow cytometry measurements showed a dose-response relationship in the loss of certain cell types, particularly the elongated spermatids, from the testes of treated animals. Flow cytometric analysis of both heat-stressed and non-heat-stressed nuclei showed a relationship between dosage and the coefficient of variation of alpha t [red/(red + green fluorescence)] measurements of AO stained nuclei, thereby demonstrating that alterations of chromatin structure occurred in response to ENU. Enzymatic digestions with RNAse, DNAse, and nuclease S1 suggest that the increase in red fluorescence is due to an increase of single-stranded DNA induced by heat or acid treatment of chemically altered chromatin structure. The lowest daily dosage used (5 mg/kg) caused no significant changes in ratios of testicular cell types, a questionable increase in abnormal sperm head morphology and a detectable change in chromatin structure expressed as alpha t. This report shows that our technique for assaying sperm nuclear chromatin structure appears to have the same level of sensitivity to ENU induced nuclear alterations as the sperm head morphology test.     

Dose-dependent induction of apoptosis in human tumour cell lines by widely diverging stimuli

Cell death may occur by either of two mechanisms: apoptosis or necrosis. Necrosis, the first type of cell death to be recognized, is an uncontrolled degenerative phenomenon invariably caused by noxious stimuli and is the result of irreversible failure of membrane function. Apoptosis, on the other hand, is a death process which involves a series of well-organized events which require active cell participation, and is primarily caused by physiological stimuli. In the present study we show that cell death induced by a range of varied agents may take the form of either apoptosis or necrosis. Apoptotic cell death was found to occur at low levels of these agents, while at higher levels necrosis occurred. Hence, cells which are not killed directly, but merely injured by these agents, have the capacity to activate an internally programmed suicide death mechanism, whereas cells receiving greater injuries apparently do not. In addition, the presence of extracellular calcium was found to be necessary for the induction of apoptosis with all agents tested.     

Flow cytometric assessment of cell viability: a multifaceted analysis

Flow cytometry offers the possibility to simultaneously analyze, on a cell by cell basis, different parameters related to cell viability i.e. cell size, morphology and incorporation of dyes. Different types of analysis: light absorption of unstained/stained cells, forward angle light scattering (FALS), right angle light scattering (RALS) or both, cell fluorescence based on dye retention or dye exclusion (due to erythrosin B, ethidium bromide, fluorescein diacetate, rhodamine 123) were tested and compared, with the classical Trypan blue exclusion test, for their effectiveness in the determination of cell viability. Two types of cells in monolayer cultures (L929, SIRC) and a freshly isolated suspension of mouse splenocytes were used. For each dye, the optimal dose, incubation time and conditions for analysis were determined. Viability indications by different techniques for the three type of cell line and their reliability as compared with Trypan blue were analyzed.     

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 cell cycle-dependent changes in cell thiol level by combining a new laser dye with Hoechst 33342.

By halogenation of methylfluorescein-diacetate (MFDA) or eosin-diacetate, two new dyes for cellular thiol compatible with visible laser excitation have become available. These probes circumvent the use of an ultraviolet (UV)-excitation system as required by bimane-based dyes and allow combination with probes for other cellular parameters. The thiol dyes attain maximal staining after 10 min at 37 degrees C, and fluorescence is sensitive to pretreatment with diethylmaleate but not to buthionine sulfoximine. In a dual-laser system, analysis of the cellular thiol level as a function of cell cycle distribution can be achieved in viable cells by simultaneous staining with the bisbenzimidazole dye Hoechst 33342 and one of the halogenated dyes. Using this approach, we were able to show that cells in the G2 phase of the cell cycle were more sensitive to thiol depletion with diethylmaleate than were cells in the G1 compartment. The new thiol dyes allow a more flexible selection of wavelengths of excitation and emission for assessing changes in cellular thiol (glutathione and other thiol compounds) and allow this parameter to be examined as a function of cell cycle position.     

Flow cytometric analysis of recombinant murine GM-CSF (rmuGM-CSF) induced changes in the distribution of specific cell populations in vivo

The changes in the distribution of granulocytes, monocytes, and lymphocytes in various tissue compartments following subcutaneous (SC) administration of recombinant murine GM-CSF (rmuGM-CSF) in vivo was determined by flow cytometry in time course studies. Balb/c mice were given single, daily SC injections of 1 or 4 micrograms of rmuGM-CSF for 10 days. Flow cytometric analysis was performed on bone marrow (BMC), peritoneal exudate (PEC), and peripheral blood (PBC) cell preparations from mice treated for 1, 3, and 10 days. Dual fluorescence was employed to gate on leukocytes (T200+) and analyze for Ig+, Thy 1.2+, MAC+, and 8C5+ (granulocytes) cells. The analyses indicated that SC-rmuGM-CSF increased the percentage of 8C5+ cells in PBC after 1 day of treatment. However, significant changes in the cell composition of PEC and BMC were not observed until day 10 of treatment and included increases in 8C5+ cells and the myeloid cell population, respectively. Side scatter analysis (cell density) of PBC and PEC indicated that the percentage of the granulocytic cell population increased significantly in rmuGM-CSF treated mice. The changes observed in PEC and BMC appeared to be dose-related whereas those observed in PBC were not. These data clearly demonstrate the utility of flow cytometric analyses for detecting selective effects of cytokines on cell populations that are involved in host defense mechanisms.     

Flow cytometric determination of cell cycle phase-specific changes in cellular phosphatase and glycosidase activities

The activities of two phosphatases (E.C. 3.1.3.1 and 3.1.4.1) and four glycosidases (E.C. 3.2.1.21, 3.2.1.30, 3.2.1.31 and 3.2.1.51) were measured by fluorescence spectrophotometry, and flow cytometry, in mitogen-stimulated lymphocytes, and in cultures of Molt-4-F and F-89 cell lines, synchronized by hydroxyurea or thymidine. All enzymes were active throughout the cycle but the activities of three enzymes were elevated at specific points in the cycle, alkaline phosphatase activity increased at G2 + M/G1 boundary and in early S-phase, the activity of beta-L fucosidase was elevated in G1 and late S-phase. Orthophosphate diesterase activity was elevated at the G1/S boundary, and during G2 + M. The increase in beta-L fucosidase activity was due to an increased number of cells showing activity, whilst the increase in orthophosphate diesterase activity was attributable to an increase in cellular enzyme activity. Only the activities of orthophosphate diesterase and beta-L fucosidase were measurable by flow cytometry, alkaline phosphatase activity was mainly extracellular, and therefore not detectable by flow cytometric methods employed.     

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 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 sorting and analysis of human epidermal stem cell candidates

ESC (epidermal stem cells) play a central role in the regeneration of human epidermis. These cells are also responsible for wound healing and neoplasm formation. Efficient isolation of ESC allows their use in medicine and pharmacy as well as in basic science. Cultured keratinocytes and ESC may be used as biological dressing in burn injuries, chronic wounds and hereditary disorders. Therefore, the isolation and characterization of ESC have been goals in biomedical science. Here, we present a flow cytometric method for the isolation and analysis of human ESC candidates. The strategy presented for the isolation of ESC combines previously proposed enzymatic digestion and FACS‐sorting of the obtained cell suspension that utilizes morphological features, integrin‐β1 expression and Rh123 (Rhodamine 123) accumulation of the cells. We also performed a flow cytometric analysis of sorted cells using a cell tracer.     

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.     

Modelling the flow of cytometric data obtained from unperturbed human tumour cell lines: Parameter fitting and comparison

In this paper we firstly present three alternative formulations of a mathematical model for human tumour cell lines unperturbed by cancer therapy. The model counts the number density of cells in each phase of the cell cycle over time where cells are differentiated by their DNA content. Data are available from the Auckland Cancer Society Research Centre, Auckland, New Zealand, in the form of DNA histograms or profiles from 11 different human tumour cell lines (i.e. in vitro) unperturbed by cancer therapy. We then apply one (computationally fast) formulation of the model and discover that although in general different combinations of parameter values give rise to very different DNA profiles it is possible that different combinations of parameter values give rise to virtually identical profiles. Experimental estimates of the rate of transition from the G ₁-phase (growth) to the S-phase (DNA synthesis) enable us to uniquely determine other model parameters of interest that give the least square error between the model and data. We finally apply our model to each of the 11 different cell lines and compare cell cycle phase transit times. Although the DNA histograms of each of the cell lines have similar shapes these cell lines have different combinations of transit times to each other, which could explain why they often react very differently when exposed to anti-cancer therapies during laboratory experiments. An understanding of the in vitro situation may give an insight into why some human cancer patients do not respond to cancer therapy. An erratum to this article is available at .     

Flow cytometric analysis of megakaryocyte differentiation

Megakaryocytes were isolated quantitatively from rat bone marrow by centrifugal elutriation (CE). CE-enriched megakaryocytes were stained supravitally for either DNA content with Hoechst 33342, surface membrane immunofluorescence with fluorescein isothiocyanate (FITC)-conjugated antiplatelet antibody, or both. The cells were then measured using a Becton Dickinson FACS IV flow cytometer. The following correlations were analyzed: DNA content and light scatter, light scatter and antiplatelet immunofluorescence, and DNA content and antiplatelet immunofluorescence. Although the range of light scatter increased as a function of DNA content, discrete subpopulations of megakaryocytes with different light scatter properties were detected within each of the three principal ploidy classes (8C, 16C, and 32C). Other discrete megakaryocyte subpopulations were revealed in the analysis of antiplatelet surface immunofluorescence as a function of degree of light scatter. The nonlinear relationship between the latter suggested that the degree of membrane immunofluorescence did not bear a simple relationship to cell size as reflected in light scatter. Megakaryocyte DNA content, on the other hand, varied in a linear fashion with membrane immunofluorescence, supporting the conclusion that there may be a proportional increase in the expression of platelet antigens with DNA content. The use of multiple markers, correlated multiparameter flow cytometry and multivectorial analysis to define differentiation on a single cell basis have revealed new complexities in this process. Flow cytometric analysis holds promise as a useful method for further characterization of megakaryocyte differentiation.     

Flow cytometric analysis of mature adipocytes

Flow cytometry is an excellent method for studying the physiological function in adipocytes because their response to hormones, especially insulin, varies with cell maturity and therefore size. Adipocytes present a unique technical challenge. A freshly prepared adipocyte suspension contains cells and fat droplets ranging from 10 to greater than 120 microns in diameter. Stored fat occupies 90-98% of the cell volume, making it difficult to distinguish cells from fat droplets. Other difficulties include buoyancy, large size, fragility, and tendency to aggregate and clog the sample tube and nozzle. These obstacles were overcome by 1) maintaining the sample, sample line, sheath fluid, reservoir, and nozzle assembly at 37 degrees C; 2) using a 200 microns diameter orifice; 3) using a short, 300 microns inside diameter Teflon sample delivery line; 4) injecting the sample at constant flow rate into the sheath fluid at low pressure; and 5) using the pH-sensitive vital stain, biscarboxyethylcarboxyfluorescein (BCECF) to distinguish cells from fat droplets. Stained cells are brightly fluorescent when excited at 488 nm. Because fat droplets do not fluoresce, they can be distinguished from fat cells by gating on the BCECF emission. The cytosolic pH of intact, viable, mature adipocytes was derived from the ratio of the fluorescent emission intensities at 520 and 620 nm and was estimated to be 7.2. Unlike BCECF, several other useful fluorescent probes of cell function, e.g., the intracellular calcium indicator, indo-1, label both fat cells and fat droplets.(ABSTRACT TRUNCATED AT 250 WORDS)