bluuebunny/crossref_metadata_2025 huggingface dataset:
Two‐parameter data acquisition system for rapid slit‐scan analysis of mammalian chromosomes
Authors: Weier H.‐U. L. , Eisert W. G.
Published: August 2005

A data acquisition system is described for recording two independent signals simultaneously from a laser‐based flow cytometer for rapid slit‐scan chromosome analysis. High‐aperture microscope optics allow recording of fluorescence distributions along the longest axis of metaphase chromosomes with a spatial resolution better than 1 μm. Fluorescence and small angle forward light scatter as well as dual‐wavelength fluorescence signals from Indian muntjac chromosomes stained with propidium iodide (PI) or acridine orange (AO) have been recorded simultaneously. While maintaining the multi‐user operation of the computer, photomultiplier signals are digitized at a rate of 400 signals per second, stored temporarily in high‐speed cache memories, and transferred subsequently to a minicomputer for further storage. Extensive software packages for data acquisition, analysis, and display of the results are described. Data acquisition is generally done in list mode, allowing complete reconstruction of individual signals (profiles) at any time. The distribution of stained constituents along the chromosomes can be displayed. Furthermore, histograms of various parameters of the input signals may be generated.
Fluorescence properties of allophycocyanin and a crosslinked allophycocyanin trimer
Authors: Yeh Sheila Walsh , Ong Linda J. , Glazer Alexander N. , Clark John H.
Published: August 2005

Data on the wavelength and temperature dependence of both time‐resolved and steady state fluorescence emission are presented for allophycocyanin (AP) and for a crosslinked allophycocyanin trimer (XL‐AP) (Ong LJ and Glazer AN: Physiol Veg 23:777–787, 1985). AP dissociates at high dilution and is not stable above 40°C even at moderate protein concentration. In contrast, XL‐AP does not dissociate even at very low protein concentrations and is completely stable up to 60°C in the presence of 0.75 M NaK‐phosphate, pH 7.0. The results show that XL‐AP is superior to AP for use in conjugates that abosrb and emit in the red region of the spectrum. The high stability of XL‐AP at elevated temperatures at high phosphate concentrations suggests that this derivative may be useful in conjunction with nucleic acid probes.
Coefficient of variation in flow cytometry of phagocytosis
Authors: Fujikawa‐Yamamoto Kohzaburo , Odashima Shizuo
Published: August 2005

Fluorescence histograms of V79 Chinese hamster lung cells containing phagocytized fluorescent microspheres were measured by flow cytometry. In the fluorescence histograms, the coefficient of variation (CV) of the peak for cells ingesting microspheres was not constant. Rather, it decreased with the number of microspheres ingested by the cells.
Experimental parameters and a biological standard for acridine orange detection of drug‐induced alterations in chromatin condensation
Authors: Block Adrienne L. , Bauer Kenneth D. , Williams Thomas J. , Seidenfeld Jerome
Published: August 2005

We investigated a number of sample‐preparative parameters for use of flow cytometry to detect chromatin condensation in cells stained with acridine orange after DNA in situ is partially denatured by acid treatment. Stability and data reproducibility for both control and drug‐treated ME‐180 and HT‐29 cells were assessed over: (1) a range of cell concentrations in 2.56 × 10−5 M acridine orange; (2) 15 days of storage in fixative; (3) various times between RNase digestion and staining; and (4) increasing times between staining and analysis. Listmode data for red and green fluorescence were collected and mean fluorescence intensities of G1, S, and G2 subpopulations of HT‐29 and ME‐180 cells were computed. These were normalized to data from HeLa‐S3 cells and fluorescent microspheres to control for inter‐experiment variations in staining and instrumental parameters, respectively. The normalized red and green fluorescence data were used to calculate αt for G1 cells [αt = red fluorescence/(total fluorescence)]. Exponentially growing HeLa‐S3 cells were a very consistent and reproducible biological standard to control for fixation and staining variability. Mean fluorescence intensities of control and difluoromethylornithine‐treated (i.e., polyamine depleted) cells remained stable and reproducible across all tested ranges for cell concentration, storage in fixative, and time after RNase digestion. This technique can thus be used to evaluate difluoromethylornithine‐induced changes in chromatin condensation of samples stored for as long as 2 weeks and analyzed all on 1 day.
Simultaneous increased expression of E‐rosette receptor (CD2, T11) and T cell growth factor receptor on human T lymphocytes during activation
Authors: Redelman Doug
Published: August 2005

The E‐rosette receptor (CD2, T11) is a differentiation antigen expressed on immature and mature human T lymphocytes. Activation of T cells from human peripheral blood with phytohemagglutinin (PHA) or with monoclonal antibody to the CD3‐Ti complex (anti‐Leu‐4) caused the expression of CD2 to increase 10‐ to 20‐fold. Dual parameter correlated analyses with antibody to the T cell growth factor (TCGF) receptor (anti‐Tac) and anti‐CD2 antibody demonstrated that the increase in CD2 expression occurred at the same time and on the same cells that expressed the TCGF receptor after stimulation with PHA. The increased expression of CD2 and the initial expression of Tac were totally inhibited by cycloheximide, but were not affected by sufficient actinomycin‐D to block the T cell proliferative response. The expression of CD2 was compared with the expression of CD4 and CD8, i.e., T cell differentiation antigens on cytotoxic/suppressor or helper T cell, respectively. Althouth virtually all of the small percentage of freshly isolated Tac+ peripheral blood cells belonged to the CD4+, CD8− subset, both CD4+ and CD8+ T cells were equivalently activated by PHA to express Tac. By 20–30 hr after activation, the expression of CD4 or CD8 was initially decreased 10–50%. Subsequently, the expression of CD4 and CD8 returned to the levels on resting T cells but did not increase further. Therfore, the increase in CD2 expression does not reflect a universal property of cell surface antigens on activated T lymphocytes.
On multiparameter data analysis in flow cytometry
Authors: Mann Reinhold C.
Published: August 2005

Increasing numbers of parameters that are accessible to simultaneous measurement in flow cytometric instruments, combined with the extremely large sample sizes common in flow cytometry, make it necessary to examine methods of multivariate statistics for their applicability to problems of visualization and quantitative analysis of flow cytometric data. This article describes some approaches to dimensionality reduction that appear well suited for data sets obtained by flow cytometry.
Direct hybridization to DNA from small numbers of flow‐sorted nucleated newborn cells
Authors: Bianchi Diana W. , Harris Peter , Flint Alan , Latt Samuel A.
Published: August 2005

A technique is described that allows direct hybridization to the DNA of cells flow sorted onto nitrocellulose filters, which obviates an intervening DNA isolation step. The feasibility of this technique for studying small numbers of cells is demonstrated with human cord blood, which has a high proportion of nucleated cells. The cells are stained with fluorescein‐conjugated anti‐HLe‐l, a monoclonal antibody that recognizes mature leucocytes. Anti‐HLe‐l‐positive cells are all nucleated, and a controlled, precise number of them may be sorted directly onto a nitrocellulose membrane. In cord blood, a small percentage of anti‐HLe‐l‐negative cells are nucleated erythrocytes, which may also serve as a source of DNA.

Studies were performed on male or female newborn cells flow sorted onto introcellulose membranes and hybridized with either a nonspecific human repeat DNA probe or a Y chromosome‐specific probe. Importantly, the sex of the newborn could be determined at the DNA level from as few as 50 sorted cord blood leucocytes or 5,000 HLe‐l‐negative cells. Since nucleated erythrocytes are common in fetal blood but rarely found in the peripheral circulation of adults, the method has potential application for the determination of fetal sex from analysis of flow‐sorted nucleated erythrocytes present in the maternal circulation during pregnancy.
Cytometry of breast carcinoma: Significance of ploïdy balance and proliferation index
Authors: Opfermann Martina , Brugal Gérard , Vassilakos Pierre
Published: August 2005

Image cytometry of DNA distribution in fine needle biopsies of breast carcinomas at first diagnosis was performed to see if there were significant differences in DNA histograms between patients having very different outcome but same tumor histological typing and similar therapy. Two groups of patients were considered retrospectively: the first (20 patients) with survival time shorter than 5 years and the second (20 patients) with survival time longer than 10 years. Seven benign tumors were used as controls. Ten ploïdy classes were defined. The frequencies of cells in those classes were used as independent features in a supervised multivariate analysis. The advantages of this approach was pointed out with respect to the four‐type classification of Auer. The scattering of DNA histograms within the feature space showed that a subgroup of patients with poor prognosis was clearly separated from a subgroup of patients with good prognosis but both long survival patients and short survival patients were scattered in between. In order to replace the multivariate classification of histograms by a simpler approach, two parameters were computed which explained most of the scattering in the feature space: the ploïdy balance (difference between the percentages of euploid and aneuploïd cells) and the proliferation index (percentage of cells between peaks). The scattergram of patients according to these parameters showed again that some DNA distributions were specific for either good or bad prognosis. But the separation was uncertain for seven short‐survival patients and six long‐survival patients. For six patients, the DNA distributions were very similar between long and short survival times. Those patients thus could not be separated even by means of discriminant analysis. The main conclusion of this study was that, for a significant number of patients, the objective multivariate classification of tumors DNA profiles is of little assistance to the pathologist who has to give a prognosis for the one patient under consideration.
DNA ploidy and survival in breast cancer patients
Authors: Cornelisse C. J. , van de Velde C. J. H. , Caspers R. J. C. , Moolenaar A. J. , Hermans J.
Published: August 2005

Flow cytometric DNA ploidy measurements using frozen or deparaffinized tumor specimens were performed on 565 primary breast cancers from patients treated in the period 1975–1984. Twenty‐nine percent of the cases were diploid, 61% had a single aneuploid stemline, and 10% were multiploid. Aneuploid tumors more often had negative estrogen receptor values than diploid tumors, but no significant correlation was found between ploidy class and TNM stage. Patients with more than ten positive axillary lymph nodes had predominantly aneuploid tumors. Overall and distant relapse‐free survival were higher for patients with diploid tumors and low‐aneuploid tumors. Stratification of the patients according to degree of lymph node involvement, TNM stage, and menopausal stage showed that the prognostic effect of aneuploidy was apparent predominantly in patients with locally advanced disease. Postmenopausal node‐positive patients with diploid tumors had a significantly better prognosis than those with aneuploid tumors, but this difference was not found for the comparable premenopausal group. Multivariate analysis with the Cox proportional hazards model indicated that ploidy is an additional, independent prognostic factor in postmenopausal patients.
Immunochemical analysis of the structure and function of chromosomal proteins
Authors: Bustin Michael
Published: August 2005

Immunochemical appxroaches are useful in studying the nuclear organization and cellular function of chromosomal components. Antibodies specific to histones and to defined nonhistone proteins have been used to study nucleosome heterogeneity, to visualize the presence of histone in transcriptionally active chromatin, and to isolate DNA sequences associated with specific chromosomal proteins.
Purification and analysis of rat hematopoietic stem cells by flow cytometry
Authors: McCarthy Kenneth F. , Hale Martha L. , Fehnel Paula L.
Published: August 2005

The monoclonal antibody OX7 recognizes an epitope expressed on the Thy‐1 glycoprotein, OX22 recognizes the high molecular weight forms(s) on leukocyte common antigen, and W3/13 recognized determinants found on certain sialoglycoproteins. Recently, the rat colony‐forming unit spleen (CFU‐S) was characterized as being OX7 upper 20% positive (OX7u20%), OX22 negative (OX22−), and W3/13 weakly positive (W3/13+). In the present study these observations have been extended to include the hematopoietic stem cell (HSC). Rat marow cells were incubated with allophycocyanine‐OX7 Fab' (APC‐OX7 Fab') and phycoerythrin (B‐OX22) Fab' (PhyB‐OX22 Fab'). The cells were sorted with a FACS‐ii instrument by using a Krypton laser tuned to the 530 nm spectral line for phycobiliprotein excitation. It was found that marrow cells capable of protecting lethally irradiated Lewis rats (9.5 Gy total body radiation, 0.4 Gy/min Co60) had the phenotype OX7u20%, OX22−. The percentage of cells in the marrow with this phenotype was found to be 0.34 ± 0.01 (mean ± S.E.). Three thousand of these cells were required to rescue 50% of lethally irradiated recipients (30‐d survival), while the number of unsorted bone marrow cells required was 1.05 × 106. Thus, a 350‐fold purification of the HSC was realized. Although CFU‐S copurified with HSC, purification of only 105‐fold was obtained. This might indicate that purified HSC have a reduced capacity to generate splenic hematopoietic colonies. The OX7u20%, OX22−‐enriched HSC population could be further divided into W3/13 dimand W3/13+ subpopulations by three‐parameter immunofluorescence analysis with the use of a new optical bench arrangement.
Quantitation of the binding, uptake, and degradation of fluoresceinylated neoglycoproteins by flow cytometry
Authors: Midoux Patrick , Roche Annie‐Claude , Monsigny Michel
Published: August 2005

The fluorescence properties of the fluorescein residues bound to a protein are used to analyze by flow cytometry the neoglycoproteins' endocytosis mediated by membrane lectins of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein bound to a protein is dependent on the number of fluorophore molecules bound to a protein molecule and the pH of the environmental medium. The mean fluorescence intensity of a fluorescein molecule bound to a protein decreases when the number of fluorescein residues per protein molecule increases. However, after proteolytic digestion, the mean fluorescence intensity of a fluorescein molecule is constant and equal to that of free fluorescein. The binding of fluorescein‐labeled α‐glucosylated serum albumin to 3LL cells at 4°C can easily be determined by flow cytometry because under these conditions the environmental pH is neutral, and the neoglycoprotein is not degraded. When the cells are incubated at 37°C in the presence of a fluorescein‐labeled neoglycoprotein, the fluorescence intensity of a cell is low because of the low pH of endosomes and lysosomes but is increased upon a postincubation at 4°C in the presence of monensin, a proton/sodium ionophore. The extent of the proteolytic digestion of an endocytosed neoglycoprotein can be assessed by comparing, upon a monensin postincubation at 4°C, the high cell‐associated fluorescence of cells incubated in the absence of leupeptin (an inhibitor of lysosomal proteases) and the relatively low fluorescence intensity of cells incubated in the presence of leupeptin.
Influence of systematic errors on the evaluation of the S phase portions from DNA distributions of solid tumors as shown for 328 breast carcinomas
Authors: Haag Dietrich , Feichter George , Goerttler Klaus , Kaufmann Manfred
Published: August 2005

The portion of cells in S phase has proved to be a valuable prognostic indicator of early relapse and life expectancy, particularly in breast carcinoma. Comparisons of published data on samples of primary breast carcinoma biopsies showed that the values obtained by analyses of flow cytometric DNA distributions were generally higher than those of determinations based on the tritiated thymidine (3H‐ThdR) labeling index (LI).

Flow cytometric DNA analyses of 328 biopsy samples of primary breast carcinomas revealed that these differences could be explained by varying contributions of debris background. Since this influence is inversely proportional to the cell counts in each channel, it may cause considerable errors, particularly in the S phase channels, which normally contain the lowest counts of the DNA distributions. Two different mathematical rationales were tested in order to separate DNA distributions from the debris superimposition. No appreciable differences were found with respect to the essential results. After appropriate subtraction of the background levels, the previously reported discrepancies between cytometrically determined S phase portions and 3H‐ThdR LI values disappeared, and good agreement was achieved for the comparable tumor samples of the present study.

In conclusion, debris background subtractions should generally precede the DNA histogram analyses, particularly of solid tumors, in order to obtain reliable S phase values.
Cell cycle perturbation of cultured C6 glioma cells following short‐term contact with a low dose of ACNU
Authors: Genka Shigeru , Shitara Nobuyuki , Tsujita Yoshihiko , Kosugi Yukio , Wu Yaochen , Takakura Kintomo
Published: August 2005

The purpose of this study was to investigate the cell cycle perturbation of cultured C6 rat glioma cells induced by 1‐(4‐amino‐2‐methyl‐5‐pyrimidil) methyl‐3‐ (2‐chlorethyl) 3‐nitrosourea hydrochloride (ACNU) using simultaneous flow cytometric measurements of DNA and bromodeoxyuridine (BrdU) content. A new graphic computer program permitted the quantification of cell density in hexagonal subareas and allowed the fraction of BrdU‐labeled cells with mid‐S phase DNA content (FLS) to be defined in a narrow window. The cell kinetic parameters such as cell cycle time (Tc) and S phase time (Ts) were estimated from a manually plotted FLS curve at 18 and 6 hr, respectively.

The major effect of ACNU on the cell cycle was an accumulation of the cells in the G2M phase 12 to 24 hr posttreatment when compared to G2M traverse of untreated cells. For the two‐dimensional analysis, cells were labeled with BrdU and then treated with ACNU, or treated with ACNU and then labeled with BrdU. It was concluded that the cells in the S and G2M phases at the time of ACNU administration progressed to mitosis but that the G1 phase cells accumulated in the subsequent G2M phase. Two‐dimensional FCM analysis using BrdU provided a useful tool in studying cell cycle perturbation.
Flow cytometric evaluation of anti‐herpes drugs
Authors: Rosenthal Ken S. , Hodnichak Cheryl M. , Summers Jack L.
Published: August 2005

A rapid means of screening drugs for toxicity and anti‐herpes simplex virus activity was developed based on the flow cytometric detection of HSV induced changes in cellular DNA content. Subconfluent monolayers of human diploid fibroblasts (HEL 299) were assayed for DNA content with propidium iodide 24 h after infection with HSV‐1 (multiplicity of infection 1–10) and treatment with the drug to be tested. Infection was detected by a broadening of the normal diploid and tetraploid peaks and presence of greater than 4‐n DNA staining. Inhibition of viral DNA synthesis and maintenance of the normal growth pattern of control cells was indication of antiviral activity. Toxicity of the compound was indicated by the loss of S phase and tetraploid cell populations. Using this assay, we evaluated the activities of one experimental and two established antiviral agents.
A rapid analytical technique for flow cytometric analysis of cell viability using calcofluor white M2R
Authors: Berglund Deborah L. , Taffs Rolf E. , Robertson Nancy P.
Published: August 2005

Analysis of dead versus live cells is shown to be possible using Calcofluor White M2R (CFW), a fluorescent brightener. Comparison of CFW with both propidium iodide (PI) and fluorescein diacetate (FDA) was performed on a FACS 440 dual laser flow cytometer on several populations of cultured rat and mouse cell lines, peripheral leukocytes, splenocytes, diatoms, and plant protoplasts. As a measure of cell viability, staining results with CFW were strongly associated with PI (correlation coefficient of 0.9886) and FDA (inverse correlation coefficient of 0.9647). With plant and algal cells, controls are necessary as CFW does stain live cells to some extent. CFW (excitation: UV, emission max: 435 nm) can be used in conjunction with two‐color immunofluorescence analysis using fluorochromes excited at 488 nm with no interference.
Solid tumor preparation for flow cytometry using a standard murine model
Authors: Ensley J. F. , Maciorowski Z. , Pietraszkiewicz H. , Klemic G. , Kukuruga M. , Sapareto S. , Corbett T. , Crissman J.
Published: August 2005

The application of flow cytometry (FCM) to solid human tumors has been hindered by the difficulty in producing high yield, viable, unaltered single cell suspensions. Carcinomas containing a high desmosomal content, such as well‐differentiated squamous cell (SCC) cancers of the head and neck (H&N) region, are particularly difficult to prepare. The desire to employ FCM to study cellular DNA parameters of these tumors led to the use of a 3‐methylcholanthrene induced murine SCC for the comparative testing of preparative techniques. Dissociation techniques, including mechanical, enucleation, chemical, single and combination enzymes methods, were comparatively tested. Of these, the combination enzyme treatment employing trypsin and collagenase produced the highest cell yields in the shortest time with the highest dye exclusion viability and the least expense. Several fixation systems including glutaraldehyde, paraformaldehyde, acetic acid, and ethanol were comparatively tested using percent of cell loss and quality of the DNA histograms produced as end points. Ethanol‐water systems with added fetal calf serum provided minimal cell loss and high quality histograms which were stable for extended periods of time. A murine tumor, closely mimicking the histology of the human tumor of interest, may be used as a model for the determination of optimum techniques of solid tumor preparation for flow cytometric analysis.
Solid tumor preparation for clinical application of flow cytometry
Authors: Ensley J. F. , Maciorowski Z. , Pietraszkiewicz H. , Hassan M. , Kish J. , Al‐Sarraf M. , Jacobs J. , Weaver A. , Atkinson D. , Crissman J.
Published: August 2005

Intense interest in advanced squamous cell cancers of the head and neck (SCC of H&N) has resulted from the recent progress made in tumor responses with chemotherapy and radiotherapy. Unfortunately, the response patterns and clinical outcome of such patients are not adequately predicted on an individual patient basis using clinical parameters or conventional morphology. The study of flow cytometrically determined cellular parameters in such tumors is therefore of interest, but is hindered by inadequate tumor preparative technology. The previous article (10) in this journal describes the use of a murine SCC tumor, LC12, which was employed for comparative testing and determination of optimum techniques of preparation for this tumor. This report describes the application of these techniques to 144 specimens of human SCC of H&N. The mean total yield for these specimens is 7.4 × 107 cells/g of tissue. The mean viable enzymatic yield (3.3 × 107 cells/g) was higher than the mean viable mechanical yield (2.0 × 107 cells/g) except when lymph nodes were the source of the specimen (5.4 × 107 cells/g). The mean dye exclusion viability from enzymatically dissociated specimens were above 90%. Significant aneuploidal subpopulation losses were evident in mechanically dissociated and enuclated specimens. 65% of the enzymatically dissociated specimens were successfully cultured with a mean cloning efficiency of 2.1 × 10−3. Preparative techniques derived from comparative testing with a murine standard tumor have been successfully applied to 144 specimens of SCC of H&N with resultant high yields and excellent viability. Technical problems detected during the preliminary testing with LC12 were confirmed in the human tumors.
Flow cytometric analysis and sorting of plant chromosomes from Petunia hybrida protoplasts
Authors: Conia J. , Bergounioux C. , Perennes C. , Muller P. , Brown S. , Gadal P.
Published: August 2005

A method to obtain a high metaphase index and therafter a plant chromosome suspension is described for Petunia hybrida (2n = 14). Mesophyll protoplast cultures have been used, giving easily disrupted cell walls and a high percentage of dividing cells after 42 h. On 2.5 mM colchicine‐treated cells, metaphase indexes reaching 10% were routinely obtained. The lysis medium in which the protoplast‐derived cells were disrupted was a simplified culture medium. After chromosome release, samples were stained with Hoechst 33342 dye and analysed by flow cytofluorometry. The histogram of fluorescence intensities included three peaks of metaphase chromosomes and a duplication of this flow karyotype provoked by “monochromatid chromosome.” This interpretation was established after flow sorting; micronuclei could also be observed and sorted. Of the 7 chromosomes, only the largest formed a distinct peak while the others were incompletely resolved, due to the similar DNA content of various chromosomes. Model distributions of Petunia hybrida chromosomes have been computed according to the relative chromosome length. The theoretical histograms indicated that low variability is indispensable for resolving distinctive chromosome peaks. The experimental flow karyotype was consistent with one of the models having CV of 2.5%.
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

Polarization 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

Flow 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

We 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

Binding 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

The 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

To 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

This 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

Merocyanine 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

The 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

We 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

Using 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

Immunochemical 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

The 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

The 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

An 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.