bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of membrane fluidity and lipidic metabolism in transformed rat fibroblasts induced by the sesquiterpenic hormone farnesylacetone
Authors: Rodes Jean‐François , Berreur‐Bonnenfant Josette , Trémolières Antoine , Brown Spencer C.
Published: June 2005

Farnesylacetone is a natural terpene extracted from androgenic glands of the crustacean Carcinus maenas and is capable of inhibiting proliferation, notably in transformed mammalian cells. Flow cytometry with three lipophilic probes, diphenylhexatriene, trimethylammonium‐diphenylhexatriene, and Nile red, has revealed modifications of the lipidic metabolism in transformed FR3T3‐mTT4 rat fibroblasts treated by farnesylacetone, including changes in membrane fluidity. Farnesylacetone strongly increased the number of neutral lipidic droplets in the cytoplasm. Moreover, after prolonged terpene treatment, the membrane fraction of cells contained a substantial level of triglycerides. Farnesylacetone provoked an immediate but transitory increase in membrane fluidity of the cell membrane. The change in measured lipid fluidity appears to be due to these triglycerides rather than to the phospholipids. © 1995 Wiley‐Liss, Inc.
Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry
Authors: O'Brien Melissa C. , Bolton Wade E.
Published: June 2005

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. © 1995 Wiley‐Liss, Inc.

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‐α‐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. © 1995 Wiley‐Liss, Inc.
Limited loss of nine tumor‐associated surface antigenic determinants after tryptic cell dissociation
Authors: Corver Willem E. , Cornelisse Cees J. , Hermans Jo , Fleuren Gert Jan
Published: June 2005

Disodium ethylenediaminetetraacetic acid (EDTA) or trypsin/EDTA are frequently used for the dispersion of monolayer cells into single cell suspensions allowing flow cytometric analysis of surface antigenic determinants. A disadvantage of EDTA is the slow action of this agent, whereas trypsin might affect the antigenic determinants under focus. We studied the possible deleterious effect of trypsin on three different ovarian carcinoma cell lines, COV413b, COV362.c14, and NIH:OVCAR‐3, on cell surface antigenic determinants by flow cytometry. Either EDTA or trypsin/EDTA was used for detachment and dissociation of monolayer ovarian cancer cell lines, followed by indirect immunofluorescence with a panel of monoclonal antibodies directed against nine different surface antigenic determinants, including six markers directed against widely distributed antigens. Compared to EDTA, trypsin/EDTA resulted in higher total cell yields and rapid detachment and dissociation into single cell suspensions with significantly lower amounts of dead cells detected by both trypan blue and propidium iodide (PI). Large differences in antigen expression were observed for the different cell lines. However, all antigenic determinants tested could still be detected after tryptic proteolysis. Three antigenic determinants were significantly decreased after trypsin/EDTA compared to EDTA detachment. CA 125 was decreased on COV362.c14 and NIH:OVCAR‐3 cells, respectively. BMA 180 and ICAM‐1 were decreased on COV413b cells. This cell line‐dependent decrease might be caused by differences in glycosylation. We conclude that trypsin/EDTA can be used for rapid monolayer cell detachment with high cell yields and limited loss of antigenic determinants tested. © 1995 Wiley‐Liss, Inc.
Computer software for testing drug susceptibility of malaria parasites
Authors: Reinders Paul P. , van Vianen Philip H. , van der Keur Maarten , van Engen Anneloes , Janse Chris J. , Tanke Hans J.
Published: June 2005

A computer program is described for the automated analysis of data obtained by flow cytometry for in vitro antimalarial drug susceptibility testing. Samples of malaria‐infected red blood cells (RBC), which were cultured in the presence of different concentrations of antimalarial drugs, were stained with Hoechst. The Hoechst fluorescence intensity of infected RBC corresponds to DNA content of the parasites and to their stage of development. After measurement of the samples by a FACStar flow cytometer equipped with a UV laser and an autosampler, FCS 1.0 data files were generated. The HP PASCAL program developed for these files identifies five different populations—uninfected RBC, infected RBC, free parasites, leukocytes, and debris—on the basis of their light scatter and fluorescence characteristics. The program calculates the percentage of infected cells, the total number of parasite nuclei, and the average number of nuclei per parasite. The results of each culture are presented as a drug dose‐response curve. During data analysis, user interaction is limited to selecting the first file of the first culture. The algorithm then processes each culture automatically. Potential problems or difficulties in analysis are flagged. To date, a total of 862 drug tests have been evaluated and fall into two classes, an extended microtest and the World Health Organization standardized microtest. These tests gave satisfactory results in more than 99% of the cases. © 1995 Wiley‐Liss, Inc.
Mouse testis cell sorting according to DNA and mitochondrial changes during spermatogenesis
Authors: Petit J. M. , Ratinaud M. H. , Cordelli E. , Spanò M. , Julien R.
Published: June 2005

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. © 1995 Wiley‐Liss, Inc.
Topical application of 12‐O‐tetradecanoylphorbol‐13‐acetate induces dyssynchronous expression of keratins K1 and K10 in mouse epidermis
Authors: Heyden Anders , Lützow‐Holm Claus , Clausen Ole Petter F. , Brandtzaeg Per , Huitfeldt Henrik S.
Published: June 2005

12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) is a potent tumor promoter that causes severe alterations in the biosynthesis of epidermal keratins. This study shows that TPA induces a dyssynchrnous effect on keratin expression in stratified squamous epithelium. The effect of TPA on the separate expression of the maturation‐associated keratins K1 and K10 was studied by immunohistochemistry in an unperturbed replicative keratinocyte population of hairless mice epidermis in relation to changes in the cell cycle time during regeneration. Keratinocytes in DNA synthesis were pulse‐labeled by intraperitoneal injection of the thymidine analogue 5‐bromodeoxyuridine (BrdUrd) 1 h before a single topical application of TPA. The BrdUrd‐labeled cell cohort, representing an originally unperturbed replicative keratinocyte population exposed to TPA mainly in the postreplicative period, was followed for up to 97 h. The results suggested unaltered timing of the onset of K1 and K10 expression comparared with normal epidermis (18 and 24 h, respectively, following DNA synthesis). This indicates that the synthesis of both keratins was programmed before the keratinocytes entered their last DNA synthesis. A reduction in K10 expression from about 30 h compared with that of K1 expression was observed. Mathematical modeling suggested a delay in K10 expression related to the second and third rounds of cell divisions after pulse‐labeling. How TPA induces such dyssynchrony in K1 and K10 regulation remains unknown. © 1995 Wiley‐Liss, Inc.
S‐phase arrest of nutrient deprived lung cancer cells
Authors: Tinnemans Monique M. F. J. , Lenders Marie‐Hélène J. H. , ten Velde Guul P. M. , Blijham Geert H. , Ramaekers Frans C. S. , Schutte Bert
Published: June 2005

The human small cell lung cancer cell line NCI‐H82 was used to study the effect of nutritional status on cell proliferative parameters. Incorporation of bromodeoxyuridine (BrdUrd) was used to characterize actively proliferating cells and to obtain information on cell cycle dynamics. During several days, in which the culture medium was not changed, a gradual decrease in overall cell growth, labeling index, and vitality was observed. Simultaneously, an increase in the number of S‐phase cells that did not incorporate BrdUrd was noticed. From a more detailed kinetic study on d 6 of nutrient depletion, it appeared that, although the cells incorporated BrdUrd, they stopped cycling. When the same cells were regrown in fresh culture medium, a delay of 10 h in G1‐phase entry and exit was measured. After this delay the cells resumed the cell cycle at normal phase transit rates. In addition, BrdUrd unlabeled S‐phase cells were gradually lost from the culture. Bivariate flow cytometric DNA/ proliferating cell nuclear antigen (PCNA) and DNA/ Ki67‐antigen analyses confirmed a delay in G1 phase entry and exit. In this paper we show that nutrient depletion can cause cell cycle arrest as indicated by the occurrence of BrdUrd unlabeled S‐phase cells. This arrest could lead to overestimation of kinetic parameters such as S‐phase transit time (Ts) and potential time (Tpot) as determined after in vivo labeling of tumors. © 1995 Wiley‐Liss, Inc.
Flow cytometric monitoring of allergen induced basophil activation
Authors: Gane Pierre , Pecquet Catherine , Crespeau Hervé , Lambin Patrick , Leynadier Francisque , Rouger Philippe
Published: June 2005

Human basophils express many clustered differentiation antigens (CD), including CD45; however, none are specific for these cells. In a previous study, we described a two‐color immunofluorescence procedure, employing antibodies to CD45 and IgE for the cytometric evaluation of basophils. In the present work, we show that when sensitized basophils are stimulated by allergenic preparations, they demonstrate an upregulation of CD45 as well as a decrease in anti‐IgE binding. Since CD45 antigen modulation was observed with all aeroallergens tested and the decrease in IgE expression varied with allergenic preparations, the measurement of CD45 upregulation was used to evaluate basophil activation. Using this approach, reproducible results were observed when atopic patients were tested at different time intervals. In addition, we show that the upregulation of CD45 on allergen stimulated basophils is a very rapid phenomenon that is observed after a few minutes and that this rapid flow cytometric method can also be used for investigations on nonallergenic mediators of basophil stimulation such as calcium ionophores. © 1995 Wiley‐Liss, Inc.
Differential effects of two fluorescent probes on macrophage migration as assessed by manual and automated methods
Authors: Denholm Elizabeth M. , Stankus Gerald P.
Published: June 2005

Fluorescent probes have been utilized to label leukocytes for both in vivo and in vitro studies of cell migration; however, the effects of such probes on migration have not been determined. The aim of this study was to examine the effects of two commonly used fluorescent probes on leukocyte chemotaxis. J774 macrophages were labeled with either calcein‐acetoxymethyl ester (calcein‐AM) or 2′,7′‐bis‐(2‐carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein, acetomethyl ester (BCECF‐AM), then assayed for their ability to migrate to zymosan‐activated serum (ZAS). Cell migration was quantified by two methods: visual counting of cells and measuring cell fluorescence. Using the cell counts, commison of unlabeled and fluorescently labeled macrophages demonstrated that BCECF‐AM decreased the number of cells responding to ZAS, while cal‐n‐AM had essentially no effect. Neither probe significantly affected the number of cells migrating to medium alone. The inhibitory effects of BCECF‐AM on cell migration increased with probe concentration (0.1–1.0 μM) and cell fluorescence. Cell viability was unaffected by either probe. In contrast to the results obtained by visual counting, measuring fluorescence of migrated cells did not reveal a significant difference between the chemotactic response of macrophages labeled with BCECF‐AM and those labeled with calcein‐AM. These experiments indicated that fluorescent probes can affect the chemotactic response and that inhibitory activity of these probes may not be detected when chemotaxis is quantified solely by automated methods. © 1995 Wiley‐Liss, Inc.
Enumeration of viable Candida albicans blastospores using tetrabromofluorescein (Eosin Y) and flow cytometry
Authors: Costantino Paul J. , Budd Dianne E. , Gare Norman F.
Published: June 2005

A rapid assay was developed for determination of the viability of blastospores of Candida albicans utilizing flow cytometry to detect the accumulation of tetrabromofluorescein in non‐viable yeast cells. Eosin Y was shown to stain non‐viable C. albicans blastospores selectively without affecting the cellular viability of competent yeast cells or resulting in non‐specific staining of viable cells. Results of these studies show that this flow cytometric method of determining cell viability of C. albicans is more accurate and more precise than the more common method of enumerating colony forming units. © 1995 Wiley‐Liss, Inc.
Organic material concentration in auditory outer hair cells measured by laser interferometry
Authors: Laffon E. , Dulon D. , Aurousseau C. , Dilhaire S. , Claeys W.
Published: June 2005

Outer hair cells (OHC) of the mammalian cochlea are quasicylindrical cells of different length, which play a major role in hearing at threshold. Their particular shape allows the use of a noninvasive laser interferometric technique of isolated cells in vitro in order to measure the organic material concentration (OMC), hence the density of each cell body. In most (95 %) of the OHCs isolated from the same guinea pig, when the cell diameter is normalized, the results show that the cell body OMC does not vary with cell length. In different animals, the respective normalized OMC mean values can vary between 70 kg/m3 and 103 kg/m3. A few OHCs with morphological particularities often possess cell body OMCs > 103 kg/m3. The results of the interferometric measurements in isolated OHCs confirm that density variations in the cell bodies are not involved in a sound frequency coding. The in vitro OMC variations of the OHCs could be related to the isolation procedure; however, they could also correlate with actual in vivo OMC variations. © 1995 Wiley‐Liss, Inc.
Flow cytometric assay of lung macrophage uptake of environmental particulates
Authors: Stringer Bradley , Imrich Amy , Kobzik Lester
Published: June 2005

We sought to establish a quantitative method using flow cytometry to study uptake of environmental particulates by alveolar macrophages (AMs). We used right angle light scatter (RAS) to measure uptake of titanium dioxide, quartz, and diesel particulates. After incubation with TiO2 in vitro, AMs showed dose‐dependent increases in both cell‐associated particles visualized by microscopy and RAS measured by flow cytometry (e.g., fold increase RAS at 4, 8, 16, 32, and 80 μ/ml, respectively, = 2 ± 0.1, 4.0 ± 0.5, 5.5 ± 0.5, 9.1 ± 2.5, 14.3 ± 0.9; mean ± SEM). Similar results were obtained with quartz and diesel particles. A strong correlation was observed between particle load per cell and AM RAS after uptake of fluorescent latex beads or fluorescent TiO2 (coated with BODIPY‐BSA) (R2 = 0.984, 0.997, respectively). Using this technique, we found AM uptake of environmental particulates to be substantially greater than that of a panel of myelomonocytic and epithelial cell lines, consistent with their physiologic role in pulmonary defenses. RAS measurements have also identified both calcium‐dependent and calcium‐independent components in AM interactions with inert particles. Although this technique does not allow precise quantitation of particle number or mass per cell, flow cytometric analysis of relative increases in RAS is a useful tool to study AM interactions with a variety of environmental particulates. © 1995 Wiley‐Liss, Inc.
Karyotypic heterogeneity and its relation to labeling index in interphase breast tumor cells
Authors: Balazs Margit , Matsumura Kouji , Moore Dan , Pinkel Daniel , Gray Joe W. , Waldman Frederic M.
Published: June 2005

We have used fluorescence in situ hybridization (FISH) with chromosome‐specific probes and immunofluorescent detection of in vivo bromodeoxyuridine (BrdUrd) incorporation to evaluate simultaneously numerical chromosome aberrations and proliferative activity of breast cancers. The number of distinct hybridization domains specific for repetitive pericentromeric sequences on chromosomes 1, 7, 11, 15, 17, and X was used as an indicator of copy number of these chromosomes in interphase tumor cells from 23 human breast cancers. Every tumor analyzed showed a heterogencous distribution of copy number for at least one chromosome type. The copy number distribution for different chromosomes within a tumor frequently showed differing patterns. Major cell populations showing monosomy were relatively rare, occurring only in five cases for chromosome 17, once for chromosome 1, and once for chromosome 15. Flow cytometric analysis of DNA ploidy correlated well with FISH analysis, although flow cytometry failed to detect aneuploidy when only a few chromosomes were affected. To determine whether cell populations with different chromosomal copy numbers have identical proliferation characteristics in vivo, BrdUrd incorporation and centromeric copy number were detected simultaneously. Comparison of the chromosome copy number distribution in BrdUrd‐positive cells vs. the distribution of the entire cell population showed different distributions in seven of the 20 cases analyzed. This study demonstrates the common occurrence of chromosome copy number heterogeneity and suggests that a cell phenotype (proliferation) may be associated with genotypic subpopulations. © 1995 Wiley‐Liss, Inc.
Flow cytometric analysis using digital signal processing
Authors: Zilmer Nick A. , Godavarti Mahesh , Rodriguez Jeffrey J. , Yopp Timothy A. , Lambert Georgina M. , Galbraith David W.
Published: June 2005

Current commercial flow cytometers employ analog circuits to produce the feature values of the pulse waveforms that result from particle analysis. The use of analog pulse processing limits the features that can be measured to pulse integral, pulse height, and pulse width, and a large amount of potentially relevant information about the shape of the pulse waveform is lost. Direct digitizing of the waveform provides a means for the extraction of additional features, for example, pulse skewness and kurtosis, as well as the Fourier properties of the pulse. Here we describe a digital pulse waveform processing system that is compatible both with a commercial flow cytometer, and with a readily available computational platform. The performance of the digital and analog systems were compared through analysis of synthetic waveforms, and the waveforms produced by standard fluorescence microspheres and biological particles. The digital waveform processing system was found to be accurate and flexible, and the value of several of its unique attributes was demonstrated using biological cells. A protocol was designed in which digital pulse processing provided a means for the quantitative monitoring of the optical alignment of the flow cytometer. It was shown that digital pulse processing could be used to discriminate between particle classes which produce feature values indistinguishable through analog pulse processing, and to discriminate accurately single cells from doublets and larger aggregates. © 1995 Wiley‐Liss, Inc.
Rapid flow cytometric method for measuring lymphocyte subset activation
Authors: Maino Vernon C. , Suni Maria A. , Ruitenberg Joyce J.
Published: June 2005

Standard in vitro methods for assessing T‐cell activation have typically measured either the proliferative responses of peripheral blood mononuclear cell (PBMC) cultures to various provocative stimuli employing tritiated thymidine incorporation or the secretion of specific cytokines from activated cells. These bulk assay methods suffer the drawback of being lengthy assays, and, in addition, they do not provide information about functional responses of individual lymphocyte subsets. This report describes a three‐color flow cytometric method for the rapid analysis (4 hours) of individual T‐cell subsets in whole blood responding to various provocative stimuli, including pokeweed mitogen, the comitogenic monoclonal antibodies (mAbs) CD2/CD2R, the superantigen staphylococcal enterotoxin B (SEB), and the specific antigen Candida albicans. After 4 hours, CD69 expression in response to CD2/CD2R paralleled thymidine incorporation measured after 72 hours. Variations in the proportions of CD4+ and CD4− T cells expressing CD69 were observed with different stimuli. These observations demonstrate the potential of multiparameter flow cytometry for the investigation of functional responses of individual T‐cell subsets to a variety of stimuli in whole blood. © 1995 Wiley‐Liss, Inc.
Cell cycle progression and phenotypic modification of Ki67 antigen‐negative G1‐ and G2‐phase cells in phorbol ester‐treated molt‐4 human leukemia cells
Authors: Tsurusawa Masahito , Fujimoto Takeo
Published: June 2005

To elucidate the relationship between the level of cellular Ki67‐reactive antigen and cell proliferation, the effects of 12‐O‐tetra‐decanoylphorbol 13‐acetate (TPA) on Ki67 expression, cell cycle progression, and surface phenotypes of human T‐lymphoblastic leukemia Molt‐4 cells were investigated by multiparameter flow cytometry. The Ki67 antigen is constitutionally expressed in almost all untreated exponentially proliferating Molt‐4 cells. Treatment with 10 nM TPA prolonged the duration of the cell cycle time and resulted in a progression arrest of cells in G1‐ and G2‐phases, during which Ki67 expression was decreased to an undetectable level. However, in TPA‐treated cultures, the Ki67‐positive fraction was invariably smaller than the growth fraction as estimated from continuous 5‐bromodeoxyuridine (BrdUrd) labeling curves. This discrepancy could be explained by the finding that some Ki67‐negative G1 cells do not enter the resting state but instead remain in the cycling compartment. These results show that Ki67 expression of tumor cells with relatively long G1 duration is downregulated to undetectable levels in late G1‐phase and the difference in the level of Ki67 expression between late G1 cells and resting G1 cells is undetectable by conventional immunological methods. Although TPA induced differentiation of Molt‐4 cells into mature suppressor T cells, the phenotypic modification was not correlated with cell cycle position and Ki67 reactivity of the cells. These results suggest that growth arrest and phenotypic differentiation of Molt‐4 cells are independent effects of TPA. © 1995 Wiley‐Liss, Inc.
A new flow cytometric method for discrimination of apoptotic cells and detection of their cell cycle specificity through staining of F‐actin and DNA
Authors: Endresen Petter C. , Prytz Per S. , Aarbakke Jarle
Published: June 2005

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 μM 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 μJM etoposide for 4 h resulted in S‐phase selective apoptosis, whereas 5 μg/ml cycloheximide initiated apoptosis mainly in G0/G1‐phase and S‐phase cells. The apoptotic response of HL‐60 cells to 20 GY γ‐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 scatterplots, similar to that observed for HL‐60 cells, was observed in the THP‐1 and U‐937 cell lines secondary to etoposide treatment. © 1995 Wiley‐Liss, Inc.
Single‐step procedure for labeling DNA strand breaks with fluorescein‐ or Bodipy‐conjugated deoxynucleotides: Detection of apoptosis and bromodeoxyuridine incorporation
Authors: Li Xun , Traganos Frank , Melamed Myron R. , Darzynkiewicz Zbigniew
Published: June 2005

The methods of in situ labeling of DNA strand breaks have been used to identify apoptotic cells and/or DNA replicating cells. While discrimination of apoptotic cells is based on the inherent presence of numerous DNA strand breaks in their chromatin, DNA proliferating cells can be discriminated by the selective DNA strand break induction by photolysis (SBIP) methodology at the sites that contain incorporated bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd). In both instances, DNA strand breaks are labeled with biotin‐ or digoxygenin‐conjugated deoxynucleotides, preferably in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase; fluorescein tagged avidin (streptavidin) or digoxygenin antibody is used in the second step of the reaction. In the present study, DNA strand break labeling was simplified by using directly labeled deoxynucleotides, in a single‐step reaction. Cell fluorescence was measured by flow cytometry as well as by a microscope‐based laser scanning multiparameter cytometer. Apoptotic cells in HL‐60 cultures treated with camptothecin or in primary cultures of non‐Hodgkin's lymphoma cells treated with prednisolone were easily identified utilizing BODIPY‐conjugated dUTP (B‐dUTP). Apoptotic cells were also recognized using fluorescein‐conjugated dUTP or dATP, although the discrimination was more pronounced with B‐dUTP The single‐step procedure, requiring fewer centrifugation. steps, resulted in less cell loss compared to the two‐step cell labeling technique. The cells, labeled with BrdUrd for 30 min or 1 h, could also be identified in a single‐step reaction; under the present conditions, better discrimination (signal‐to‐noise ratio) was obtained measuring cell fluorescence with the microscope cytometer compared to cell analysis by flow cytometery. Preincubation of cells with Hoechst 33258 prior to ultraviolet (UV) illumination, to enhance the photolysis, improved detection of cells which incorporated BrdUrd. The morphology of cells subjected to SBIP was excellent, allowing visualization of distinct DNA replication points. Becaus, unlike the immunocytochemical methods used to detect BrdUrd incorporation, the SBIP methodology does not require DNA denaturation by heat or acid, nuclear proteins are expected to remain undenatured in situ, allowing one to study colocalization of various constituents, detected immunocytochemically, at the DNA replication points. © 1995 Wiley‐Liss, Inc.
Binding diversity of antibodies against external and internal epitopes of the multidrug resistance gene product P‐glycoprotein
Authors: Lehne G. , De Angelis P. , Clausen O. P. F. , Egeland T. , Tsuruo T. , Rugstad H. E.
Published: June 2005

P‐glycoprotein (Pgp) is a trans‐membraneous protein that is associated with multidrug resistance (MDR) in human cancer, including hepatocellular carcinomas and leukemias. There is no consensus regarding methods of choice for analysis of Pgp expression, and development of reliable analytical methods is now essential. We have studied the Pgp expression in human hepatoma and leukemia cell lines using flow cytometry. The aim of the study was to compare binding properties of anti‐Pgp antibodies reacting with surface (MRK16, UIC2) and cytoplasmic (C219, JSB‐1) epitopes to assess which antibody performed best with respect to fluorescence discrimination. By histogram subtraction the fractions of resistant human hepatoma cells positive for Pgp were 99% (MRK16), 97% (UIC2), 77% (JSB‐1), and 51% (C219), demonstrating variations in antibody reactivity. The resolution in detecting decreasing levels of Pgp in hepatoma cells was superior for the externally binding antibodies, showing that there is a correlation between antibody reactivity and fluorescence discrimination. Similar results were obtained for parental and resistant KG1a human leukemia cell lines. The Pgp epitopes remained reactive to the anti‐Pgp MAbs after methanol fixation and cryopreservation. By dual parameter flow cytometry it was shown that Pgp expression in viable cells may be assessed together with uptake of epirubicin, which was low in cells expressing high levels of Pgp and vice versa. In conclusion, all tested antibodies proved useful for flow cytometric detection of high levels of Pgp, but the externally binding ones were superior in detection of low and variable levels of Pgp. © 1995 Wiley‐Liss, Inc.
Scatchard analysis of fluorescent concanavalin a binding to lymphocytes
Authors: Gordon Ian L.
Published: June 2005

Standard Scatchard analysis of ligand binding to cell receptors requires the use of isotopes and is imprecise at low ligand concentrations. To evaluate the feasibility of Scatchard analysis via fluorescence flow cytometry, the binding of fluorescein isothiocyanate‐derivatized concanavalin A (FITC‐ConA) to murine lymphocytes at 4°C was compared to 125I‐ConA binding. A FACS IV flow cytometer (Becton‐Dickinson, Mountain View, CA) was used for analysis of cells after fluorescent ligand binding. A simple spectrophotometric technique was used to calibrate the relation between cytometer‐determined fluorescence and ligand binding per cell. As FITC‐ConA binding showed a quasi‐Gaussian distribution, the mean number of molecules bound per cell was easily calculated. Scatchard analysis of FITC‐ConA binding yielded results (1.9 × 106 receptors/cell, K = 3.6 × 10−15) similar to those obtained with 125I‐ConA (1.4 × 106 receptors/cell, K = 5.2 × 10−15). Cytometric Scatchard plots showed less scatter and seemed more precise, suggesting superiority to radioactive ligand measurements, particularly at low ligand concentrations. © 1995 Wiley‐Liss, Inc.
Adaptation of the TdT assay for semi‐quantitative flow cytometric detection of DNA strand breaks
Authors: Bromidge Teresa J. , Howe Denise J. , Johnson Stephen A. , Phillips Malcolm J.
Published: June 2005

The enzyme Terminal Deoxynucleotidyl Transferase (TdT) is a DNA polymerase which can be used to label DNA strand breaks by the incorporation of a labelled nucleotide followed by a fluorescent detection step. The amount of label incorporated can then be assessed by flow cytometry. The mechanism of action of TdT, however, will allow the addition of varying numbers of nucleotides to the free 3' termini produced by DNA strand breaks. The substitution of Digoxigenin (DIG)TM labelled dideoxynucleotides for labelled deoxy‐nucleotides in the TdT assay will limit the addition of label to a DNA break to a single nucleotide, thus ensuring a direct relationship between an increase in DNA strand breaks and an increase in fluorescence. We have used this adaptation of the TdT Assay to evaluate DNA damage incurred in lymphocytes, from patients with Chronic Lymphocytic Leukaemia (CLL), on exposure to UV irradiation and apoptosis‐inducing drugs, fludarabine and 2‐Chloro‐2′‐deoxyadenosine (2‐CdA). This technique may give a good indication of the susceptibility of CLL patients to apoptosis inducing drugs, and hence an indication of the likely response to these therapies. © 1995 Wiley‐Liss, Inc.
Photomultiplier voltage setting: Possible important source of variability in molecular equivalents of soluble fluorochrome (MESF) calculation?
Authors: Rigolin Gian Matteo , Lanza Francesco , Castoldi Gianluigi
Published: June 2005

We evaluated the effect of flow cytometer photomultiplier (PMT) voltage setting on molecular equivalents of soluble fluorochrome (MESF) calculation by using two different quantitative microbead calibrating kits as calibrating standards and a third one as an unknown testing sample of stable intensity of fluorescence. Based on the analysis of residual values derived from linear regression and on the determinations of the resolution index, we demonstrated that, on a FACScan instrument, the window of photomultiplier setting for fluorescein isothiocyanate (FITC) fluorescence determinations is 590–670 V. However, the best region was in the range of 610–650 V. Within the 590–670 PMT voltage region, the evaluation of the same testing sample at different PMT voltages, at 590 V, gave MESF percentage differences of 15% and 13% on the two lower unknown standards of intensity compared to the values obtained at the nearby 630 V setting. In the analyzed PMT region, logarithmic amplifier response was globally maintained, whereas evaluation of the same unknown testing sample in both linear (Lin) and logarithmic (Log) amplification demonstrated (at the higher intensities of fluorescence) Log‐Lin minimal differences within the 610–650 PMT voltage range. According to our data, it can be stated that PMT voltages between 610 and 650 provide the best instrument performances and that PMT setting may be proposed as a source of variability in MESF calculation that deserves a careful evaluation in quality‐control trials. © 1995 Wiley‐Liss, Inc.
Nucleic acid dyes for detection of apoptosis in live cells
Authors: Frey Tom
Published: June 2005

Apoptotic thymocytes were found to be much dimmer than normal thymocytes when stained with several nucleic acid dyes. These dyes provide a quick and simple assay for apoptosis which works for live cells and does not require a UV laser. The collection of dyes giving this staining pattern includes reagents suitable for use in either the FL1, FL2, or FL3 channel of a standard FACScan. Cells identified by these reagents were identical to apoptotic thymocytes defined by several widely used criteria: (i) rapid uptake of Hoechst 33342 but exclusion of propidium iodide, (ii) merocyanin 540 bright, and (iii) sub‐G1 DNA content when permeabilized in a buffer that elutes fragmented DNA. In addition, L3T4/Thy‐1 dim thymocytes were included in the dye dim population. The standard Hoechst 33342 and merocyanin 540 assays were not able to separate the normal and apoptotic populations in HL‐60 cells treated with camptothecin. However, the dyes SYTO‐16 and LDS‐751 both gave adequate differentiation of apoptotic from nonapoptotic cells in this model system. Some of these dyes also emit very little in other fluorescence channels of the flow cytometer and can be used in multicolor assays on cytometers equipped with only a single argon‐ion laser. © 1995 Wiley‐Liss, Inc.
Kinetics of plasma membrane and mitochondrial alterations in cells undergoing apoptosis
Authors: Lizard Gérard , Fournel Sylvie , Genestier Laurent , Dhedin Nathalie , Chaput Christophe , Flacher Monique , Mutin Mireille , Panaye Geneviève , Revillard Jean‐Pierre
Published: June 2005

Programmed cell death or apoptosis is characterized by typical morphological alterations. By transmission electron microscopy, apoptotic cells are identified by condensation of the chromatin in tight apposition to the nuclear envelope, alteration of the nuclear envelope and fragmentation of the nucleus, whereas integrity of the plasma membrane and organelles is preserved. Conversely cells undergoing necrosis display an early desintegration of cytoplasmic membrane and swelling of mitochondria. In this study we assessed by flow cytometry the sequential alterations of forward angle light scatter, 90° light scatter, and fluorescence associated with fluorescein diacetate, rhodamine 123, and propidium iodide in two human B cell lines undergoing apoptosis induced by the topoisomerase II inhibitor VP‐16. The kinetics of these modifications were compared to those of cells undergoing necrosis induced by sodium azide. At the same time intervals, cells were examined by transmission electron microscopy and by UV microscopy after staining with Hoechst 33342. We report that sequential changes in light scatters and fluorescein diacetate are similar in cells undergoing apoptosis or necrosis, whereas apoptosis is characterized by a slightly delayed decrease of mitochondrial activity as assessed by rhodamine 123 staining. Surprisingly a part of cells undergoing apoptosis displayed an early uptake of propidium iodide followed by a condensation and then a fragmentation of their nuclei. It is concluded that uptake of propidium iodide is a very early marker of cell death which does not discriminate between necrosis and apoptosis. Along with biochemical criteria, nuclear morphology revealed by staining with Hoechst 33342 would seem to be of the most simple and most discriminative assay of apoptosis. © 1995 Wiley‐Liss, Inc.
Rhodamine 123: A useful probe for monitoring T cell activation
Authors: Ferlini Cristiano , Biselli Roberto , Nisini Roberto , Fattorossi Andrea
Published: June 2005

The T cell activation pathway involves an increase in mitochondrial activity. This can be evaluated in individual cells using the fluorescent probe rhodamine 123 (Rh123) and flow cytometry. Peripheral blood mononuclear cells (PBMC) were stimulated with optimal concentrations of phytohaemagglutinin(PHA), superantigens (Sag) SEA and SEC2, and allogeneic cells. Activation kinetics were followed at days 1, 2, 4 and 7. In all activation conditions, Rh123 uptake was augmented with the CD25 expression, cell size, and DNA synthesis. Rh123 uptake reflected an increase in mitochondrial activity and mass, as assessed by experiments in which Rh123 was substituted for by the 10‐nonyl acridine orange, which stains mitochondria in an energy‐independent manner. The spectral characteristics of Rh123 allowed us to double stain cells with Rh123 and phycoerythrin‐conjugated monoclonal antibodies. In PHA‐activated cultures, CD4+ and CD8+ cells incorporated essentially the same amount of Rh123 at all time points, suggesting that the two subsets did not differ in their activation kinetics. Accordingly, after 1 weekof culture, no significant modification in the Cd4/Cd8 ratio was observed. Sag‐activated CD4+ cells incorporated a higher amount of Rh123 than did CD8+ cells and preferentially expanded after 1 week of culture as indicated by the increase in the CD4/CD8 ratio. The different behavior of the CD4 and CD8 subsets observed by dual color flow cytometry in the PHA and Sag models was confirmed using purified CD4+ and CD8+ cell preparations obtained by immunomagnetic sorting. CD4+ cells were also the preferential target in the allogeneic model, although the magnitude of the phenomenon was lower than in the Sag model. Present data indicate that Rh123 is a reliable marker for monitoring the mitochondrial compartment during T cell activation. The possibility of phenotyping Rh123‐stained cells adds to the applicability of the probe. © 1995 Wiley‐Liss, Inc.
Use of light scatter when recording a DNA histogram from paraffin‐embedded tissue
Authors: Ormerod M. G. , Titley J. C. , Imrie P. R.
Published: June 2005

The quality of a DNA histogram as recorded in a flow cytometer can often be improved by gating on forward and orthogonal light scatter. This may be helpful when measuring DNA histograms from formalin‐fixed, paraffin‐embedded material. We report that nuclei from many human tumours scatter more light orthogonally than those from normal stroma. Gating on light scatter enabled the DNA histogram from the tumour to be recorded with reduced contamination from normal cells. In studies of human mammary carcinomas, we have found that this method has the potential to improve the estimation of S phase and the measurement of DNA index (DI) when the DI is close to 1 (near diploid). © 1995 Wiley‐Liss, Inc.
Simultaneous dual‐frequency phase‐sensitive flow cytometric measurements for rapid identification of heterogeneous fluorescence decays in fluorochrome‐labeled cells and particles
Authors: Deka Chiranjit , Cram L. Scott , Habbersett Robert , Martin John C. , Sklar Larry A. , Steinkamp John A.
Published: June 2005

In frequency‐domain lifetime spectroscopy, the apparent fluorescence lifetimes obtained from phase‐shift measurements are independent of modulation frequency only in the special case of a single exponential fluorescence decay. For heterogeneous fluorescence decay, the apparent fluorescence lifetimes measured by the phase‐shift methods are functions of the modulation frequency. This modulation‐frequency dependent property of apparent fluorescence lifetimes may be used to identify heterogeneous fluorescence decays by measuring lifetimes at multiple frequencies. In this article we explore the requirements and experimental design considerations for making such measurements in flow. We report a phase‐sensitive flow cytometric method that allows one to probe the excited statelifetimes of labeled cells by using multiple simultaneous modulation frequencies. Application of this method is demonstrated by measuring fluorescence lifetimes of labeled cells at two frequencies simultaneously, using a continuous‐wave, dual‐frequency modulated excitation in flow. The dual‐frequency method presented herein can be used to rapidly identify heterogeneity in the fluorescence decay on a cell‐by‐cell basis in real time. Information on the nature of the fluorescence decay is important in biological measurements because it can provide insight into intermolecular interactions at the subcellular level.
Changes in free cholesterol content, measured by filipin fluorescence and flow cytometry, correlate with changes in cholesterol biosynthesis in THP‐1 macrophages
Authors: Hassall David G. , Graham Annette
Published: June 2005

The free cholesterol content of cells can be monitored by the intensity of fluorescence emissions from the polyene antibiotic filipin. In a previous study (Hassall: Cytometry 13:381–388, 1992) using THP‐1 macrophages, a decrease in filipin fluorescence in response to increasing concentrations of modified lipoprotein was observed, suggesting a reduction in the free cholesterol content of the cells. In this study, THP‐1 macrophages were treated with a number of agents known to modulate cholesterol biosynthesis and cholesterol esterification. Changes in filipin fluorescence emissions were measured by flow cytometry, and correlated with changes in cholesterol biosynthesis measured by incorporation of [14C]acetate into cholesterol. A correlation between decreases in filipin fluorescence and reductions in cholesterol biosynthesis was apparent, even when cholesterol esterification was inhibited. These results suggest that the decreases in filipin fluorescence observed may be due, at least in part, to reductions in cholesterol biosynthesis. © 1995 Wiley‐Liss, Inc.
Flow sorting of the Y sex chromosome in the dioecious plant Melandrium album
Authors: Veuskens Jacky , Marie Dominique , Brown Spencer C. , Jacobs Michel , Negrutiu Ioan
Published: June 2005

The preparation of stable chromosome suspensions and flow cytometric sorting of both the Y sex chromosome of the white campion, Melandrium album, and the deleted Y chromosome of an asexual mutant, 5K63, is described. The principle has been to maintain transformed roots in vitro, synchronise and block mitosis, reduce cells to protoplasts, and lyse these to release chromosomes. Such in vitro material, unlike many cell suspensions, showed a stable karyotype. Factors critical to producing high‐quality chromosome suspensions from protoplasts include osmolality of isolation solutions and choice of spindle toxin and of lysis buffer. Agrobacterium rbizogenes transformed young growing root cultures were synchronised at G1/S with 50 μM aphidicolin for 24 h and released to a mitotic block with 30 μM oryzalin for 11 h. Protoplast preparations from such tissue routinely had metaphase indices reaching 15%. Suspensions of intact metaphase chromosomes, with few chromatids, were obtained by lysing swollen mitotic protoplasts in a citric acid/disodium phosphate buffer. Except for the presence of clumps of autosomal chromosomes near the X and Y chromosome zones, monoparametric histograms of fluorescence intensities of suspensions stained with 4′,6‐diamidino‐2‐phenylindole showed profiles similar to theoretical flow karyotypes. Two types of Y chromosomes, one full‐length and one partially deleted (from the asexual mutant), could be sorted at 90% purity (21‐fold enrichment of Y). These results are discussed in the context of sex determination and differentiation in higher plants. © 1995 Wiley‐Liss, Inc.