bluuebunny/crossref_metadata_2025_split huggingface dataset:
Light‐scattering polarization measurements as a new parameter in flow cytometry
Authors: de Grooth B. G. , Terstappen L. W. M. M. , Pupples G. J. , Greve J. 
Published: August 2005

AbstractPolarization measurement of orthogonal light scattering is introduced as a new optical parameter in flow cytometry.In the experimental setup, the electrical field of the incident laser beam is polarized in the direction of the sample flow. The intensity of the orthogonal light scattering polarized along the direction of the incoming laser beam is called depolarized orthogonal light scattering. Theoretical analysis shows that for small values of the detection aperture, the measured depolarization is caused by anisotropic cell structures and multiple scattering processes inside the cell.Measurements of the orthogonal depolarized light scattering in combination with the normal orthogonal light scattering of human leucocytes revealed two populations of granulocytes. By means of cell sorting it was shown that the granulocytes with a relatively high depolarization are eosinophilic granulocytes. Similar experiments with human lymphocytes revealed a minor subpopulation of yet‐unidentified lymphocytes with a relative large orthogonal light‐scattering depolarization. The results were obtained with an argonion laser tuned at different wavelengths as well as with a 630‐nm helium neon laser.These results show that measurement of depolarized orthogonal light scattering is a useful new parameter for flow‐cytometric cell differentiation.
Improved methodology for intracellular enzyme reaction and inhibition kinetics by flow cytometry
Authors: Dive Caroline , Workman Paul , Watson James V. 
Published: August 2005

AbstractFlow cytoenzymology is the determination of enzyme activities or concentrations in single intact cells. Using the flow cytometer built and designed in our laboratory and recent modifications to hardware and software, we have developed an improved dynamic flow cytoenzymological procedure for the assay of cellular enzyme kinetics. The reaction mixture is sampled continuously, and the computer clock incorporates time as a parameter for kinetic determinations. Conditions for cellular esterase analysis were optimized and the rates of hydrolysis of two fluorogenic substrates, fluorescein diacetate (FDA) and 4‐methylumbelliferone acetate (MUA), by esterases in EMT6 mouse mammary tumor cells were studied. Reaction kinetics were characterized, and Km values of 19 and 72 μM were obtained for the hydrolysis of FDA and MUA respectively. The kinetics of the cellular efflux of fluorescein were investigated, and a half‐life of 7.5 min obtained. Enzyme inhibition kinetics were investigated using the competitive substrates p‐nitrophenyl acetate and phenyl acetate, and the carbamoylating agents physostigmine and n‐butyl isocyanate. The latter was particularly potent with an I50 of 4.8 × 10−5 M for FDA hydrolysis compared with 6.5 × 10−3 M for physostigmine. The I50 of 8.8 × 10−5 M for n‐butyl isocyanate inhibition of MUA hydrolysis was similar to that obtained with FDA as substrate. By monitoring FDA and MUA reactions separately and simultaneously, we showed them to act as competitive substrates. A comparison of flow cytoenzymological and conventional spectrofluorimetric analysis was also made, and differences identified in some cases.
Detection of very low receptor numbers on cells by flow cytometry using a sensitive staining method
Authors: Truneh Alemseged , Machy Patrick 
Published: August 2005

AbstractWe describe a staining method for flow cytometry that resolves with a high degree of sensitivity very low numbers of cell surface molecules, which are normally too few to detect using the conventional fluorescein‐conjugated reagents. We took advantage of the fact that liposomes can be constructed to contain hundreds of thousands of fluorochrome molecules per vesicle; antigen specificity can be conferred by covalently conjugating them to antibodies or protein A. Unlike fluorochromes such as fluorescein isothiocyanate (FITC) that are directly conjugated to protein ligands with a fluorochrome to protein ratio of about 2 to 1 on the average, their large encapsulating capacity gives liposomes a tremendous potential for signal amplification. In an indirect immunofluorescence study using liposomes that contained the fluorochrome carboxyfluorescein (CF) and that were covalently conjugated to protein A, we were able to obtain up to 50 times the fluorescence signal over background that could be detected with FITC‐conjugated protein A. Scatchard analysis showed that the thymoma cell line RDM4 expresses 23,000 and 2,600 binding sites for monoclonal antibodies (mAb) against H‐2K and H‐2D, respectively. When RDM4 cells were treated with anti‐H‐2K mAb followed by FITC‐conjugated protein A, at best we were able to obtain a fluorescence signal that was only 7 times above background. However, when these cells were treated with the same antibody followed by protein A conjugated to small unilamellar liposomes or large unilamellar liposomes, the fluorescence signals were 110 and 335 times above background, respectively. Using the liposome conjugates, we were also able to detect with ease the 2,600 binding sites for the anti‐H‐2D mAb, whereas the FITC conjugate failed completely to resolve specific binding from background. We estimate conservatively that by using this methodology, it will be possible to detect as few as 800 binding sites percell.
Distinction of leucocyte classes based on chromatin‐structure‐dependent DNA‐binding of 7‐aminoactinomycin D
Authors: Stokke Trond , Steen Harald B. 
Published: August 2005

AbstractBinding of the DNA‐specific dye 7‐aminoactinomycin D (7‐AMD) in chromatin of human leucocytes was studied by flow cytometry. After formaldehyde fixation and permeabilization, monocytes bound 30–130% more 7‐AMD than lymphocytes, while binding in granulocytes was 20–60% higher than in lymphocytes. Monocytes and lymphocytes bound similar amounts of 7‐AMD when cells were permeabilized by detergent prior to fixation. Digestion of DNA in formaldehyde‐fixed chromatin by DNase1 was quantitated by measuring Hoechst 33258 (H33258) fluorescence of mononuclear cells. The monocyte/lymphocyte H33258 fluorescence ratio decreased with DNase1 digestion to an asymptotic value of 0.74, showing that DNA in chromation of monocytes was more susceptible to DNase1 digestion. 7‐AMD binding increased, reached a maximum and then decreased with extent of DNase1 digestion in both mononuclear cell types. The monocyte/lymphocyte 7‐AMD fluorescence ratio also decreased after DNase1 digestion. RNA content and RNA synthesis were higher in monocytes than in lymphocytes. The results show that 7‐AMD binding in chromatin of mononuclear leucocytes correlates with transcriptional activity as measured by DNase1 susceptibility and RNA synthesis. The staining procedure may be used for differential counting of mature myeloid cells in peripheral blood and bone marrow.
Hyperdiploidy of cultured dermal fibroblasts from patients with aerodigestive squamous carcinoma
Authors: Danes B. Shannon , Boyle Paula D. , Traganos Frank , Melamed Myron R. 
Published: August 2005

AbstractThe incidence of hyperdiploidy in monolayer cultures of dermal fibroblasts from 88 patients with squamous carcinoma of the aerodigestive tract was compared with similar cultures from 39 normal subjects using three different techniques; (1) metaphase assay (MA), (2) flow cytometry of stationary cell cultures (FCMs), and (3) flow cytometry of proliferating dividing cell cultures (FCMd). In vitro hyperdiplody was considered to be present (IVH+): (1) by MA if more than 4% of metaphases were altered, excluding tetraploidy; (2) by FCMs if more than 8% of cells in stationary cultures were hyperdiploid (i.e., DNA index > 1), and (3) by FCMd if more than 6% of cells in logarithmic cultures were hypertetraploid (i.e., DNA index > (2). There was excellent concordance between the three assays, which assigned cell cultures from 55 of the 88 patients with aerodigestive squamous carcinoma (62%) to the in vitro hyperdiploidy positive (IVH+) category. Cell cultures from all 39 normal individuals were hyperdiploidy negative (IVH−).These data suggest that some substantial proportion of patients with aerodigestive squamous carcinoma may have a genetic predisposition for the disease, which can be identified by these assays. The flow cytometry methods were easier to carry out and may be substituted for the metaphase analyses.
Calibration of flow cytometric fluorescence standards using the isoparametric analysis of ligand binding
Authors: Chatelier Ronald C. , Ashcroft Robert G. 
Published: August 2005

AbstractTo extract ligand‐cell binding parameters from flow cytometric fluorescence data, one needs a method of converting the measured cellular fluorescence to the actual number of bound molecules producing that fluorescence. Bead standards containing a known number of fluorophores do not necessarily provide the correct conversion factor. We therefore present a method for calibrating flow cytometric bead standards. The technique uses the isoparametric analysis (Chatelier et al: EMBO J 5:1181‐‐1186, 1986) to construct a plot of fluorescence per cell versus the number of bound ligands per cell, thus allowing a direct comparison of the quantum yields of the bead‐associated fluorophore with that of the cell‐bound fluorophore. The potential of this analysis is demonstrated by contrasting the fluorescent brightness of fluorescein isothiocyanate associated with thymocyte nuclei and synthetic polymer beads with that of fluoresceinated epidermal growth factor bound to A431 cells. When the standards are improperly calibrated, then the number of ligand‐binding sites is incorrectly reported, and the derived Scatchard plot may exhibit an apparent positive or negative cooperativity as well as a strong dependence on cell concentration.
DNA damage, cytotoxic effect and cell‐cycle perturbation of Hoechst 33342 on L1210 cells in vitro
Authors: Erba Eugenio , Ubezio Paolo , Broggini Massimo , Ponti Mauro , D'Incalci Maurizio 
Published: August 2005

AbstractThis study was designed to evaluate the effects of vital dye Hoechst 33342 (HO 33342), at concentrations used to obtain a good DNA histogram resolution, on DNA integrity, cell growth, and cell‐cycle phase distribution of L1210 cells. HO 33342 exposure for 2 h, at 37°C produced DNA single‐strand breaks as assessed by the method of alkaline elution. DNA single‐strand breaks were concentration dependent (in the range .5–5 μg/ml) and increased significantly when HO 33342 (0.5–1.5 μg/ml) was associated with exposure in a flow cytometer to U.V. laser beam illumination. HO 33342 produced a cytotoxic effect on cell growth even at the concentration of 0.5 μg/ml—a concentration ten‐fold smaller than those required to obtain a good DNA histogram resolution. HO 33342 produced a severe block of the cells in the G2‐M phase of the cell cycle already evident 24 h after stain exposure and continuing up to 144 h after start of recovery. A new polyploid cell population (with a 4 c DNA content) not present in the unstained cells was already evident 24 h after dye exposure. The data shown in the present paper would imply caution in using sorted cells stained with HO 33342 dye for biological, biomedical, and pharmacological studies.
Selective staining of immature hemopoietic cells with merocyanine 540 in flow cytometry
Authors: Belloc Francis , Lacombe Francis , Bernard Philippe , Dachary Dominique , Boisseau Michel R. 
Published: August 2005

AbstractMerocyanine 540 (MC540) has been reported to bind hemopoietic cells specifically. In this study, MC540 was used as a probe for the cytofluorometric discrimination of hemopoietic cells. In PHA‐stimulated lymphocytes or HL‐60 cells induced to differentiate with DMSO, MC540 binding was increased in actively proliferating cells and undifferentiated cells as compared with the more differentiated cells of the same lineage. Mononuclear bone marrow cells exhibited a discrete distribution of MC fluorescence. Sorting a population with high MC540 fluorescence (MC+ population) produced a 9–14‐fold enrichment of granulocyte‐macrophage progenitors (CFU‐GM), and a recovery of all S‐G2M phase cells (BrdUrd/DNA analysis). Cytological examination of the sorted MC+ population confirmed the enrichment in immature cells from all lineages. Double‐labeling experiments using MC540 and Hoechst 33342 on total bone marrow or peripheral blood cells confirmed that the MC+ population included all the cycling cells. The proportion of S‐G2M phase cells in this MC+ population was 29.3 ± 7.8 for 15 bone marrow samples and 16.3 ± 6.8 for 10 blood samples. MC540 could therefore be used as a marker for human hemopoietic cells, and it represents a useful tool for investigation of hemopoiesis in normal or leukemic bone marrow.
Resolution of complex fluorescence spectra recorded on single unpigmented living cells using a computerised method
Authors: Salmon Jean Marie , Vigo Jean , Viallet Pierre 
Published: August 2005

AbstractThe identification and quantification of fluorescent compounds in a complex fluorescence spectra are always difficult, especially in the case of low signal:noise ratio.We propose a computerised method that allows the resolution of low light level complex fluorescence spectra into its components.Based on a linear combination of N possible characteristic fluorescence spectra, and using N weighting functions, this method allows the integration of fluorescence intensities over the entire fluorescence spectra and the generation of n equations with N unknowns. The compounds that participate in complex fluorescence spectra are identified and quantified. Because fluorescence intensities can be integrated we can resolve complex fluorescence spectra presenting a low signal:noise ratio. The reliability and sensitivity of our method are shown through examples of resolution of complex intracellular fluorescence of single living cells pretreated with benzo(a)pyrene. Depending on the cell type and treatment, two, four, or five components can be identified in the complex fluorescence spectra.
Flow cytometric characterization of the chlorophyll contents and size distributions of plant protoplasts
Authors: Galbraith David W. , Harkins Kristi R. , Jefferson Richard A. 
Published: August 2005

AbstractWe have employed flow cytometry for the characterization of populations of protoplasts prepared from tobacco (Nicotiana tabacum) leaf tissues. We first investigated the possibility of using flow cytometric analysis of the emission of chlorophyll autofluorescence for measurement of the chlorophyll contents of leaf protoplasts. Defined numbers of leaf protoplasts were sorted according to different, nonoverlapping windows placed on the one‐dimensional histograms of chlorophyll autofluorescence emission. The amounts of cellular chlorophyll were measured in cell‐free extracts of these sorted protoplasts using fluorometry. A high degree of correlation (r2 = 0.983) was observed between these two parameters.We then examined the distribution of protoplast diameters in these protoplast populations through the use of pulse‐width time‐of‐flight (TOF) analysis. Through sorting of protoplasts using a series of narrow, nonoverlapping TOF windows, we were able to demonstrate that the TOF parameter was linearly correlated with protoplast diameter, over the range of 15–55 μm (r2 > 0.99). We also compared the use of fluorescein diacetate (FDA) fluorochromasia and chlorophyll autofluorescence as the source of fluorescent signals for TOF analysis. We found that the presence of chloroplasts introduced distortions into the measurement of apparent size afforded by TOF analysis of FDA fluorochromasia.These results are discussed in terms of the application of techniques of flow analysis and sorting for the measurement of gene expression within the various different cell types found in plant tissues and organs.
Nuclear RNA quantification in protoplast cell‐cycle phases
Authors: Bergounioux C. , Perennes C. , Gadal P. , Brown S. C. 
Published: August 2005

AbstractUsing acridine orange staining and flow cytometry the DNA and RNA levels (arbitrary units) of individual cells may be established. Here, this method has been applied to nuclei isolated from plant protoplasts during culture. The specificity of the technique has been validated for such plant material; ribonuclease markedly reduced nuclear staining without modifying the DNA histogram; ribonuclease inhibitor prevented the action of released cell nucleases; and protoplasts cultivated with actinomycin D did not synthesize RNA. First RNA synthesis was evident 18 h after Petunia hybrida protoplasts had been put into culture. An increase of RNA above a critical level was required for cells to be able to initiate DNA replication from G1, termed G1B. G2 nuclei had an RNA:DNA ratio similar to that of G1 nuclei.
Effects of denaturation with HCL on the immunological staining of bromodeoxyuridine incorporated into DNA
Authors: Sasaki Kohsuke , Adachi Shiro , Yamamoto Takeshi , Murakami Tomoyuki , Tanaka Kazunari , Takahashi Manabu 
Published: August 2005

AbstractImmunochemical procedures for detection of BrdUrd incorporated into DNA require a denaturation step of DNA. Denaturation with HCl is widely used for flow cytometric analysis of the cell cycle and for histological preparations. This brief communication describes an attempt to standardize a denaturation procedure with HCl. Various denaturation conditions at 20°C were examined for human promyelocytic leukemia cells (HL‐60 cells) fixed in ethanol. After denaturation of DNA, the cells were stained by an indirect immunofluorescence method using a commercially available monoclonal anti‐BrdUrd antibody or by propidium iodide. The relative fluorescence intensities of stained BrdUrd and double‐stranded DNA were altered reciprocally by changing HCl concentration and/or denaturation time. Treatment with 4N HCl for 10‐20 min at 20°C allowed denaturation of more than 80% of DNA and the maximum BrdUrd‐linked immunofluorescence. Under this condition, the coefficient of variation of the DNA histograms remained relatively small.
Pulse profile analyses of endocytosis in capped B lymphocytes and BCL1 cells
Authors: Yang Meng‐Chun Wang , Harvey Nancy E. , Cuchens Marvin A. , Buttke Thomas M. 
Published: August 2005

AbstractThe effect of temperature on the kinetics of endocytosis by B lymphocytes and BCL1 cells was examined by using flow cytometry. Mouse B cells were stained with FITC‐RαMIg and induced to undergo cap formation in the temperature‐controlled sample compartment of the flow cytometer. Capping and subsequent endocytosis were measured by continually monitoring the pulse profile descriptors (width and area) of the electronic signal curves generated during flow cytometric analyses. Decreases in area values which immediately followed cap formation were shown to result from a pH‐dependent quenching of fluorescence emission as the internalized FITC‐RαMIg entered acidic subcellular compartments. Similar results were obtained with BCL1 cells, but, in addition to cap formation and acidification, cytoplasmic diffusion of the fluorescent ligand could also be discerned by flow cytometry. With either cell type the rates of cap formation and endocytosis were shown to be temperature dependent with temperature coefficient (Q10) values of 2.072.7. Based upon Arrhenius plots of width and area changes, activation energies for capping and endocytosis ranged from ∼ 12‐18 kcal/mol.
DNA and S‐phase distribution and incidence of metastasis in human primary lung carcinoma
Authors: Volm Manfred , Bak Michael , Hahn Eric W. , Mattern JÜRgen , Weber Ernst 
Published: August 2005

AbstractThe aim of the study was to investigate the relationship between DNA and S‐phase distribution in primary non‐small‐cell lung carcinomas with the incidence of metastasis. Patients with non‐small‐cell lung carcinomas were divided into two groups depending on whether at time of surgery there were metastases or not, and these groups were correlated with the data obtained by flow cytometry or autoradiography.As expected from other studies, survival time was significantly longer for those patients without metastases at time of surgery (P = .0002) and the incidence of metastasis was significantly higher when the primary tumor was ≥ 70 cm3 (P = .026).In this study, a total of 185 fresh specimens of lung carcinomas were investigated by flow cytometry. Patients with aneuploid tumors had a higher tendency to have metastases (P = .016). Patients with tumors with a higher proportion of S‐phase cells measured by either flow cytometry or autoradiography demonstrated significant increase in the formation of metastases (P = .02 and P = .05). We feel that these results warrant further investigation with other primary tumors. A comparison of primary tumors that are known to rapidly metastasize vs. those that either slowly or rarely metastasize may prove to yield valuable insight into the important factors associated with metastatic potential.
Symmetry analysis of cell nuclei
Authors: Panno J. P. 
Published: August 2005

AbstractAn algorithm is described that is used to analyze the two‐dimensional spatial symmetry of cell nuclei. The method provides two symmetry features: the symmetry index (SI), which estimates the precise spatial symmetry of a given chromatin component, Cn, and the quadrant symmetry index (QSI), which estimates the number of quadrants being occupied by Cn. A previous analysis is used to show that age‐related chage in Malpighian tubule nuclei from the adult housefly is associated with significant alterations in the spatial symmetry of low‐, medium‐, and high‐density chromatin components (LDC, MDC, HDC). This included a seven‐fold increase in the spatial symmetry of HDC and a shift in the symmetry profile (from highest to lowest degree of symmetry) from LDC‐MDC‐HDC to MDC‐LDC‐HDC. The increased spatial symmetry of HDC suggests that it occurs at new nuclear sites as the fly ages and that these sites are distributed over approximately 60% of the chromosome population.
In situ flow cytometric analysis of nonyl acridine orange‐stained mitochondria from splenocytes
Authors: Ratinaud Marie Héaleègne , Leprat Patrick , Julien Raymond 
Published: August 2005

AbstractDue to its spectral characteristics, the fluorochrome nonyl acridine orange (NAO) (λabs:489 nm, λem:525 nm), which is spontaneously incorporated by mitochondria with a high relative specificity, provides a new probe for the in situ study of these organelles by flow cytometry. In 15 min at 20°C, the dye at 4.75 × 10−6 M saturates the mitochondrial binding sites present in 1.5 × 106 cells. Unlike Rh 123, the fixation of the probe is not affected by the action of uncouplers and ionophores. Unlike acridine orange, its binding is not sensitive to nucleases.By studying the mitochondrial incorporation of the fluorochrome during the cell cycle of murine splenocytes, it was possible to show that the biogenesis of NAO‐stained mitochondrial constituents mainly occurs during the G1 phase.