bluuebunny/crossref_metadata_2025 huggingface dataset:
Precision of DNA flow cytometry in inter‐institutional analyses
Authors: Wheeless Leon L. , Coon John S. , Cox Christopher , Deitch Arline D. , White Ralph W. Devere , Fradet Yves , Koss Leopold G. , Melamed Myron R. , O'Connell Mary J. , Reeder Jay E. , Weinstein Ronald S. , Wersto Robert P.
Published: August 2005

A Bladder Cancer Flow Cytometry Network study has been carried out to further identify and quantify sources of inter‐ and intra‐laboratory variability. Replicate samples containing four mixtures of peripheral blood lymphocytes and aneuploid cell lines were distributed together with reference standards to six laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Two of each of the four sample types and a reference standard were analyzed by each laboratory on 3 separate days to obtain cellular DNA distributions. DNA index (DI) and hyperdiploid fraction (HDF) were calculated for each histogram using an automated technique. The results showed significant inter‐ and intra‐laboratory differences. Results were evaluated by a two‐way analysis of variance to estimate components of the overall variation attributable to individual sources. Error variation was found to be the major component of random variation. Specimen means were also compared for each laboratory. No significant differences were noted in mean DI for similar specimens; however, agreement in HDF between similar specimens was lacking in most laboratories. Prediction intervals were computed to estimate the range of values expected for a single specimen based on the analysis of the previous six. Prediction intervals for DI were quite good while those for HDF were troublesome due to wide variation. The results of these studies indicate that intra‐ and inter‐laboratory variability are high enough that results for a single sample may not be sufficiently precise to allow comparison to results obtained in other laboratories. This study demonstrates the need for measurement and control of laboratory variability and for the routine use of laboratory standards and check samples if the results from one laboratory are to be compared with the results of another or if classification criteria are to be transferred between laboratories.
Calibration of fluorescence intensities to quantify antibody binding surface determinants of cell subpopulations by flow cytometry
Authors: Dux R. , Kindler‐Röhrborn A. , Lennartz K. , Rajewsky M. F.
Published: August 2005

Quantitative indirect immunofluorescence analysis by flow cytometry was used to determine the mean number of antibody binding sites per cell in a small subpopulation of rat brain cells expressing low levels of a cell surface differentiation antigen recognized by monoclonal antibody (Mab) RB13‐6 (Kindler‐Röhrborn et al.: Differentiation 30:53–60, 1985). For these non‐disjunct distributions of fluorescence intensities, the cut‐off border between antigen‐positive and antigen‐negative cells was defined by a statistical test. To eliminate the influence of accidental disturbances leading to incorrect statistical decisions, the curves for antigen‐negative cells were fitted according to cell number and shape. The flow cytometer was calibrated with the use of a clonal cell line for which the average number of Mab RB13‐6 binding sites per cell had previously been determined by radioimmunoassay and Scatchard‐plot analysis. Using this analytical procedure, both the proportion of Mab binding brain cells and the mean number of Mab binding sites per Mab binding cell could be determined as a function of developmental stage.
Flow cytometric measurement of p53 protein expression and DNA content in paraffin‐embedded tissue from bronchial carcinomas
Authors: Mørkve Odd , Laerum Ole Didrik
Published: August 2005

The nuclear protein p53 has been measured in archival lung cancer biopsies. The monoclonal antibody PAb 1801, which recognizes human p53, was used. After immunostaining, the nuclei prepared from paraffin‐embedded tissue were stained with propidium iodide for simultaneous measurement of DNA content; 17 of 24 lung cancers were p53 positive. The S‐phase fraction in positive tumors was 22.9±6.4%, as compared to 13.6±6.1% in negative tumors (P < 0,02). In ten of the positive tumors (two small cell carcinomas and eight non‐small cell carcinomas), the p53 expression varied through cell cycle, whereas in seven tumors (five small cell carcinomas and two non‐small cell carcinomas), no such variation of p53 expression was observed. Freezing the nuclear suspensions did not substantially reduce the p53 signals. Control experiments with the SV40‐transformed human foreskin fibroblast cell line HSF4‐T12 showed that the enzymatic digestion utilized to dissociate paraffin‐embedded tissue did not significantly reduce p53 fluorescence. Immunohisto‐chemical staining of biopsy specimens indicated that only cancer cells were overexpressing p53. In conclusion, using the monoclonal antibody PAb 1801, p53 is detectable in cell nuclei prepared from paraffin‐embedded bronchial carcinoma biopsies. P53 positive tumors have increased proliferative activity compared to p53 negative tumors. Furthermore, the lack of cell cycle variation of p53 in small cell carcinomas indicates that this pattern may be related to high‐grade malignancy.
Cell kinetic analysis of mixed populations using three‐color fluorescence flow cytometry
Authors: Begg A. C. , Hofland I.
Published: August 2005

The development of antibodies to DNA‐incorporated thymidine analogs has in turn led to the development of flow cytometric techniques for rapidly measuring cell kinetics parameters. More recently, these techniques have been applied to clinical tumor material. One problem with such measurements has been the difficulty of distinguishing malignant cells from coexistent normal cells in the biopsy material. In the present study, the feasibility of selecting out the desired malignant cell population for kinetic analysis from a mixture of cells was tested in vitro. An anticytokeratin antibody was used to discriminate between a mixture of tumor cells (cytokeratin positive) and normal cells (cytokeratin negative). The cell lines chosen for the study, A549 human lung carcinoma cells and Chinese hamster ovary (CHO) cells, were pulse labeled with iododeoxyuridine (IdUrd) and sampled every hour up to 16 hours.

Selecting out cells from the mixture required the application of three‐color fluorescence flow cytometry, which was carried out using the fluorochromes FITC (fluorescein isothionate, green fluorescence, IdUrd‐DNA antibody), PE (phycoerythrin, orange fluorescence, cytokeratin antibody), and PI (propidium iodide, red fluorescence, DNA). This allowed single laser excitation. The staining procedure involved incubation with the IdUrd antibodies (specific antibody plus FITC‐conjugated second antibody) followed by the cytokeratin antibodies (specific antibody plus PE‐conjugated second antibody) and lastly by the DNA stain containing RNase. Two analysis methods of the IdUrd/DNA cytograms were applied: a mid‐S window analysis and a relative movement (RM) analysis. Results of the analyses for cells selected out of mixtures were compared with results of cells stained and analyzed separately.

A clear separation of the two cell lines could be obtained on the basis of orange fluorescence (cytokeratin content) despite a large overlap of their DNA histograms. By gating on high or low orange fluorescence, almost pure populations of the individual cell types could be selected out for further kinetic analysis. Little difference was seen, with both the mid‐S and RM analyses, between cells gated from mixtures or stained separately. It is concluded that this technique is feasible for use on clinical material, provided good cell suspensions can be obtained, leading to the hope of increasing the accuracy of kinetic measurements on human tumors.
Very early detection of changes associated with cellular activation using a modified flow cytometer
Authors: Kelley Keith A.
Published: August 2005

A sample station modification previously described has been redesigned to provide greater flexibility and enhanced performance. The improved modification provides mixing and temperature regulation in a compact unit that mounts close to the nozzle holder for reduced transit times, allowing for addition of mediators to a sample in place on the flow cytometer, with observation of results in approximately 1 s. An electronic circuit activated at the time of injection generates full‐scale pulses in the forward scatter channel. This provides the data with a time stamp for direct correlation of injection and cellular response. A detailed description of the modification, performance verification data, and practical applications in the measurements associated with cellular activation are presented.
Experimental and theoretical investigations on the validity of the geometrical optics model for calculating the stability of optical traps
Authors: Schut Tom C. Bakker , Hesselink Gerlo , De Grooth Bart G. , Greve Jan
Published: August 2005

We have developed a computer program based on the geometrical optics approach proposed by Roosen to calculate the forces on dielectric spheres in focused laser beams. We have explicitly taken into account the polarization of the laser light and thd divergence of the laser beam. The model can be used to evaluate the stability of optical traps in a variety of different optical configurations.

Our calculations explain the experimental observation by Ashkin that a stable single‐beam optical trap, without the help of the gravitation force, can be obtained with a strongly divergent laser beam. Our calculations also predict a different trap stability in the directions orthogonal and parallel to the polarization direction of the incident light.

Different experimental methods were used to test the predictions of the model for the gravity trap. A new method for measuring the radiation force along the beam axis in both the stable and instable regions is presented. Measurements of the radiation force on polystyrene spheres with diameters of 7.5 and 32 μm in a TEM00‐mode laser beam showed a good qualitative correlation with the predictions and a slight quantitative difference.

The validity of the geometrical approximations involved in the model will be discussed for spheres of different sizes and refractive indices.
Application of laser optical tweezers in immunology and molecular genetics
Authors: Seeger S. , Monajembashi S. , Hutter K.‐J. , Futterman G. , Wolfrum J. , Greulich K. O.
Published: August 2005

Optical tweezers, based on a compact diode pumped Nd:YAG laser providing 350 mW at 1,064 nm coupled into a Zeiss IM 35 microscope, were used to sort CD4+ T cells into a capillary for further mechanical handling and to establish contact between single human natural killer (NK) cells and human erythroleukemia cells (K562) as targets. After contact and a lag phase of a few tens of seconds, the target cell starts to change its morphology and membrane blebbing occurs. The kinetics of the attack of the NK cell on K562 cells is not straightforward but governed by temporal oscillations in the shape of the target cell (zeosis).

In a second application, the optical tweezers are combined with a UV laser microbeam based on a pulsed UV laser and with flow cytometry and sorting. With the pulsed laser, segments of sorted chromosome 1 of the Chinese hamster karyotype (CHV 79) can be easily micro‐dissected and subsequently collected using the optical tweezers. This allows preparation of a few hundred chromosome segments per day without mechanical contact and in an absolutely sterile way and thus may provide an interesting basic technique in any type of genome sequencing project.
Laser induced cell fusion in combination with optical tweezers: The laser cell fusion trap
Authors: Steubing Rosemarie Wiegand , Cheng Steve , Wright William H. , Numajiri Yasuyuki , Berns Michael W.
Published: August 2005

A single‐beam gradient force optical trap was combined with a pulsed UV laser microbeam in order to perform laser induced cell fusion. This combination offers the possibility to selectively fuse two single cells without critical chemical or electrical treatment. The optical trap was created by directing a Nd:YAG laser, at a wavelength of 1.06 μ, into a microscope and focusing the laser beam with a high numerical aperture objective. The UV laser microbeam, produced by a nitrogen‐pumped dye laser (366 nm), was collinear with the trapping beam. Once inside the trap, two cells could be fused with several pulses of the UV laser microbeam, attenuated to an energy of ≈ 1 μJ/pulse in the object plane. This method of laser induced cell fusion should provide increased selectivity and efficiency in generating viable hybrid cells.
Three‐dimensional DNA image cytometry by confocal scanning laser microscopy in thick tissue blocks
Authors: Rigaut Jean Paul , Vassy Jany , Herlin Paulette , Duigou Françgoise , Masson Eric , Briane Dominique , Foucrier Jean , Carvajal‐Gonzalez Santos , Downs Angela M. , Mandard Anne‐Marie
Published: August 2005

A method for the quantification of nuclear DNA in thick tissue blocks by confocal scanning laser microscopy is presented. Tissues were stained en bloc for DNA by chromomycin A3. Three‐dimensional images, 60 μm deep, were obtained by stacking up confocal fluorescent images obtained with an MRC‐500 (Bio‐Rad, Richmond, CA). The effects due to bleaching and attenuation by depth of fluorescence emission were corrected mathematically. The DNA contents were estimated by summing up the detected emission intensities (discretized into pixel gray levels) from each segmented nucleus. Applications to an adult rat liver and to a human in situ carcinoma of theesophagus are shown to demonstrate, respectively, the precision of the method and its potential usefulness in histopathology. Comparisons are made with DNA histograms obtained on the same materials by image cytometry on smears and by flow cytometry. Ploidy peaks obtained with the confocal method, although wider than with other methods, are well separated. Confocal image cytometry offers the invaluable advantage of preserving the tissue architecture and therefore allowing, for instance, the selection of histological regions and the evaluation of the degree of heterogeneity of a tumor.
Quantitative flow cytometric analysis of ABO red cell antigens
Authors: Sharon Raphael , Fibach Eitan
Published: August 2005

A flow cytometry method has been employed to quantitatively compare the expression of A, B and H antigens on various red blood cells (RBC). The H substance was directly labelled by fluoroscein‐conjugated anti‐H lectin and the A and B antigens by indirect staining first with monoclonal anti‐A or anti‐B antibodies followed by fluorescently, fluorescein (FITC) or phycoerythrin (PE), labelled anti‐mouse immunoglobulin (Ig) antibodies. More than a ten‐fold difference in cellular fluorescence intensity was found within each sample. Both the percentage and the mean fluorescence of the positive subpopulation for each antigen were determined. Each RBC population was characterized with respect to the expression of A, B or H antigen by a compound mean value that was the calculated product of these two parameters. The results demonstrated a reciprocal relationship between the compound means of A or B and H. The ratio of A/H or B/H was found to be most informative. Homozygotes for A or B had ratios of >200 and >30, respectively, while heterozygotes (AO or BO) had ratios of <5. This method could also distinguish between A1 and A2; RBC carrying the A1 phenotype (as determined by agglutination with anti‐A1 lectin) showed a higher A/H ratio than those carrying A2. In contrast to the reciprocity in the expression of A (or B) and H found in RBC obtained from different individuals, a direct correlation was found in the expression of these antigens by individual cells within a given population. These results suggest that in addition to a specific gene dosage effect, other factors (e.g., aging of the RBC) may affect the expression of all these antigens in a coordinate way.
Membrane cell permeabilisation with saponin and multiparametric analysis by flow cytometry
Authors: Jacob Marie Christine , Favre Mireille , Bensa Jean‐Claude
Published: August 2005

Saponin, a detergent like molecule, can permeabilise cell membranes without destroying them, and thus can be used for the detection of intracellular antigens on intact cells with a flow cytometer.

First experiments were reported that demonstrated the detection of intracytoplasmic antigens such as intermediate filaments and CD3 in T acute lymphoblastic leukemia (ALL). Further experiments were also performed to prove that intranuclear structures were equally accessible: dyes such as propidium iodide (PI) and monoclonal antibodies (mAb) such as Ki67 could penetrate the nucleus and lead to the analysis of DNA content and to the discrimination between the different cell cycle phases (G0, G1, S, G2‐M).

This rapid and sensitive method retained sufficient integrity of cells being treated to enable differentiation of cell types on the basis of morphology. Furthermore, it did not alter membrane expression of most antigens. Therefore, it was of particular interest for multiparametric analysis, especially for simultaneous study of membrane and intracellular structures.
Bivariate flow karyotyping of human chromosomes: Evaluation of variation in Hoechst 33258 fluorescence, chromomycin A3 fluorescence, and relative chromosomal DNA content
Authors: Boschman Gert A. , Rens Wim , van Oven Carel H. , Manders Erik M. M. , Aten Jacob A.
Published: August 2005

The total variation of chromosome peak positions, in bivariate distributions of Hoechst 33258 and chromomycin A3 fluorescence of 19 healthy individuals, was compared with the experimental variation, determined from 23 bivariate distributions of chromosomes prepared separately from a single cell lineage. The experimental variation in Hoechst and chromomycin fluorescence and the relative chromosomal DNA content were determined from experiments performed over several days. The additional variance contributed by time was the same as the daily variance. The accuracy by which the relative chromosomal DNA content can be calculated from bivariate peak positions was investigated. A least squares method was used to fit the distributions of relative DNA content, obtained, respectively, from mono‐ and bivariate flow analyses of chromosomes from the same cell lineage. In general the DNA contents match quite well, but for a few chromosomes a difference was found, statistically discernible at the 5% level. The average relative chromosomal DNA content of the chromosomes from the 19 normal individuals, calculated from bivariate peak positions, showed a linear relation with the estimates published by other investigators.
CUBIC: A three‐dimensional colored projection of consort 30 generated trivariate flow cytometric data
Authors: Greimers Roland , Rongy Anne Michel , Schaaf‐Lafontaine Nicole , Boniver Jacques
Published: August 2005

The CUBIC program displays three‐dimensional colored dot plots of flow cytometric trivariate data collected by unmodified commercial instruments (FACScan flow cytometer, FACS 440 cell sorter). Assuming a bimodal distribution of the fluorescence intensity of the cells, the eight theorical subpopulations involved in a three‐color fluorescence histogram are clearly localized in the 3‐D space by colored dots that are clustered near each corner of a cubic frame. Rotation, tilting, and zoom functions are available. Table look‐up is not needed. CUBIC was illustrated by two experiments: 1) three‐color immunofluorescence of antigens on human lymphocytes using monoclonal antibodies conjugated either to fluorescein (FITC), to R‐phycoerythrin (PE), or to biotin revealed by a streptavidin coupled to a PE‐Texas red tandem conjugate (TC); 2) two‐color immunofluorescence of CD4 and CD8 antigens on thymocytes of healthy or preleukemic mice correlated to the DNA content quantified by 7‐amino‐actinomycin D (7‐AAD). The three fluorescences were excited by a single argon‐ion laser emitting at 488 nm.
Towards a quantitative grading of bladder tumors
Authors: De Meester Uneco , Young Ian T. , Lindeman Jan , van Der Linden Hans C.
Published: August 2005

The histological inspection of tumor tissue for the purpose of reporting a tumor grade is a problem of significant clinical importance. The grading by a pathologist is only partly reproducible due to vaguely defined, subjective criteria. In this article we describe and evaluate a set of measurable features that quantitate the differences in tumor tissue. Different aspects of the reproducibility of the measurements under varying conditions of image selection, focus, and noise have been investigated.

Three hundred thirty‐three images were digitized from 111 bladder tissue sections (4 μm thick, Feulgen stained), using the ICAS microscope‐camera platform. A segmentation routine was developed to segment the images into nuclei and background without any user interaction. Size, shape, optical density, and texture features were measured on and among the objects found by this segmentation routine using the image analysis package Acuity.

The results of the measurements showed that there is a significant quantitative difference between grade 1 and grade 3 tumors. Grade 2 tumors can be described as “in between grade 1 and grade 3” and falling somewhere on an increasing scale between grades 1 and 3. Grade 2 tumors do not seem to represent a statistically distinct population.

The procedure described here is shown to be quite reproducible in the presence of noise, reasonably reproducible in the event of a modest amount of defocusing (with grade 3 tumors exhibiting the most sensitivity), and less reproducible in the context of which fields‐of‐view are chosen for analysis.
Influence of tissue preparation techniques on p53 expression in bronchial and bladder carcinomas, assessed by immunofluorescence staining and flow cytometry
Authors: Mørkve Odd , Høstmark Jens
Published: August 2005

In a series of bronchial and bladder carcinomas, p53 protein expression was examined. Samples from formalin‐fixed, paraffin‐embedded tissue (routine‐treated) were compared with parallel samples of fresh tissue and tissue fixed in paraformaldehyde and ethanol. The expression of p53 was measured by immunofluorescence staining and dual parameter flow cytometry, with simultaneous monitoring of DNA content. For each tumor, p53 fluorescence with different fixatives was expressed relative to fresh tissue. The p53 fluorescence signals were on average brighter from routine‐treated tissue than from fresh tissue. The tissue fixed in paraformaldehyde showed no difference from fresh tissue. In the ethanol‐fixed tissue, however, fluorescence signals were reduced by nearly 70%, and the fraction of detectable p53 positive cells in tumor tissue was reduced by more than 50%. This loss of fluorescence was probably due to a leakage of the antigen from nucleus to cytoplasm. Pepsin treatment did not influence p53 fluorescence. Within the same tumor, the Sphase fraction in p53 positive cells was significantly higher than in p53 negative cells (13.1 ± 2.0% vs. 6.5 ± 0.8%). This pattern was not influenced by formalin fixation or pepsin treatment.

Our study demonstrates that in measuring a nuclear antigen, tissue handling may influence the results, and care should be taken to optimize the preparation procedure. Using the antibody PAb 1801, p53 expression measured in archival material is not reduced as compared to fresh tissue.
Flow cytometric double labeling technique for screening of multidrug resistance
Authors: Gheuens Eric E. O. , van Bockstaele Dirk R. , van der Keur Maarten , Tanke Hans J. , van Oosterom Allan T. , De Bruijn Ernst A.
Published: August 2005

We investigated the capabilities of flow cytometry in the analysis of a multidrug resistant (MDR) human ovarian cancer cell line 2780AD and its drug sensitive parental A2780. A functional assay using daunorubicin (DNR) as a fluorescent probe was combined with an immunofluorescence assay of P‐glycoprotein (P‐gp) using the monoclonal antibody MRK‐16. Functionally MDR could be demonstrated by the lower DNR‐content of MDR cells compared to DNR‐content of drug sensitive cells. When incubation was performed with DNR in the presence of verapamil, DNR‐content increased in the MDR cells. However the content of the A2780 cells was never attained. Differences in DNR‐content were not related to differences in DNA‐content. In experimental cell lines immunofluorescence data were inversely related with those of DNR‐content: MDR cells had high levels of P‐gp expression and low levels of DNR‐content (and vice versa in drug sensitive cells). Both assays can be easily combined in a multiparametric flow cytometric procedure to evaluate both parameters simultaneously in the same cells. Analysis of clinical samples demonstrates the existence of aberrant subpopulations which would not be detected by using a single parameter assay.
Measuring potential doubling times of murine tumors using flow cytometry
Authors: Carlton Jeffrey C. , Terry Nicholas H. A. , White R. Allen
Published: August 2005

Flow cytometric analysis of bromodeoxyuridine (BrdUrd) labeled cells has led to the description of various methods for determining population potential doubling times (Tpot) from samples obtained at a single time point. There have been several reports of results from human tumor samples using these various methods of analysis, but little documentation of the validity of these approaches in experimental tumor models. Recently, we reported results using in vitro cell cultures and determined the most reliable method for these. In this paper we report the results of this methodology when applied to two in vivo murine tumor models (MCaK and FSA). The analytic approach tested is summarized as follows: The relative movement (RMlu(t)), a measure of the mean DNA fluorescence of BrdUrd‐labeled cells that have not undergone division at the time of sampling, and a quantity ν, a function of the fractions of BrdUrd‐labeled divided and undivided cells, were measured for a series of time points following labeling. From each value of RMlu(t) an estimate of the length of S‐phase (Ts) was computed and from each ν and Ts a value for Tpot was found. These results were compared to the values of Ts and Tpot obtained by fitting all the values simultaneously. Tumor MCaK values of Ts and Tpot of 9.6 and 28.0 h were obtained from fitting all of the data. Tumor FSA gave values of 16.8 and 42.3 h for Ts and Tpot, respectively. The results of this analysis show that single time point measurements can give reliable estimates of Ts and Tpot.
Recombinant glutathione S‐transferase (GST) expressing cells purified by flow cytometry on the basis of a GST‐catalyzed intracellular conjugation of glutathione to monochlorobimane
Authors: Puchalski Ralph B. , Manoharan T. Herbert , Lathrop Angela L. , Fahl William E.
Published: August 2005

COS cells transiently expressing glutathione S‐transferase (GST) π, Ya, or Yb1 (human Pi, rat Alpha or Mu, cytosolic classes) were purified by flow cytometry and used in colony‐forming assays to show that GST confers cellular resistance to the carcinogen benzo[a]pyrene (±)‐anti‐diol epoxide (anti‐BPDE). We developed a sorting technique to viably separate recombinant GST+ cells (20%) from the nonexpressing electroporated population (80%) on the basis of a GST‐catalyzed intracellular conjugation of glutathione to the fluorescent labeling reagent monochlorobimane (mClB). The concentration of mClB, length of time cells are exposed to mClB, and activity of the expressed GST isozyme determined the degree to which recombinant GST+ cells fluoresced more intensely than controls. On‐line reagent addition ensured that all cells were exposed to 25 μM mClB for 30–35 s during transit before being analyzed for fluorescence intensity and sorted. The apparent Km for mClB of the endogenous COS cell GST‐catalyzed intracellular reaction was 88 μM. Stained GST Ya+ or Yb1+ cells catalyzed the conjugation 2 or 5 times more effectively than GST π+ cells. Enzyme activity in cytosolic fractions prepared from sorted recombinant GST+ cells was 1.8 ± 0.3‐fold greater than that of the control (80 ± 4 nmol/min/mg protein). Upon a 5‐fold purification of GST π+ cells in the electroporated population, resistance to anti‐BPDE in colony‐forming assays increased 5 times, from 1.1‐fold (unsorted) to 1.5‐fold (sorted) (P <0.001).
Simultaneous flow cytometric method to measure phagocytosis and oxidative products by neutrophils
Authors: Perticarari S. , Presani G. , Mangiarotti M. A. , Banfi E.
Published: August 2005

We developed a rapid and sensitive two‐color flow cytomotric method which allows the simultaneous quantification of both the phagocytosis rate and the oxidative burst activation of polymorphonuclear leukocytes (PMNLs). The oxidation of hydroethidine (HE) to ethidium bromide (EB) was performed by the oxidative neutrophil products within the cells during the respiratory burst, which was stimulated by phagocytized fluorescein‐labeled Staphylococcus aureus. By means of flow cytometry we measured red EB fluorescence emission together with green fluorescence, which was emitted by the ingested fluoresceinated bacteria. The fluorescence intensity was proportional to the number of bacteria ingested. Adherent bacteria were distinguished from the ingested ones. This two‐color cellular staining permits measurement of two different functions of neutrophils in one step. This method could be of interest for the determination of the interactions between neutrophils and bacteria and for the investigations on infectious diseases in chronic granulomatous disease patients.
New chamber for flow cytometric analysis over an extended range of stream velocity and application to cell adhesion measurements
Authors: Amblard François , Cantin Claude , Durand Jocelyn , Fischer Alain , Sékaly Rafick , Auffray Charles
Published: August 2005

When analyzed in a flow cytometer, particles are suddenly accelerated to high velocities (1–10 m.s−1) over very short distances. This feature is essential to obtain high analysis rates and low coincidence levels, but translates into very strong velocity gradients (> 105 s−1): particles experience strong hydrodynamic stresses that elongate them acid tend to dissociate weakly associated complexes.

In order to analyze fragile conjugates formed by heterotypic adhesion between two cell types, a flow cytometer was modified to make hydrodynamic stress not only much weaker but also adjustable. A new and easily adaptable flow cell was designed for the instruments of the FACS™ series; it provided satisfactory hydrodynamic conditions on a wide continous range of flow rates. Accompanying electronic adaptations permitted standard analysis between 0.01 and 10 m.s−1.

At 0.01 m.s−1, the velocity gradient roughly amounts to 50 s−1. Conjugates formed by the adhesion between human B and resting T lymphocytes, disrupted in conventional flow cytometers, could be detected and precisely quantified provided analysis velocity was kept below 0.1 m.s−1. We conclude that low velocity flow cytometry makes possible the quantification of weak intercellular adhesion phenomena, and is potentially useful for the future development of new biomechamical techniques and other applications.
Gene transfer by electroporation, lipofection, and DEAE‐dextran transfection: Compatibility with cell‐sorting by flow cytometry,
Authors: Puchalski Ralph B. , Fahl William E.
Published: August 2005

The aim of this work was to define a transfection procedure that is compatible with the sorting and propagation of cells that transiently express a heterologous gene. Three requirements were established for the procedure and were meet with COS monkey kidney cells that express a recombinant glutathione Stransferase (GST) gene. The transfection procedure used had to generate (i) populations in which at least 10% of the cells expressed recombinant GST, (ii) cellular morphological homogeneity throughout the population, and (iii) viable cells with at least a 5% colony‐forming ability. Of the transfection techniques tested, only electroporation satisfied all three requirements. Usually 20–22% of the cells that survived electroporation expressed recombinant GST 3 days after electroporation as measured by flow cytometry, and 25% of the cells that survived electroporation formed colonies in cloning assays. Transfection. with DEAE‐dextran and chloroquine did enable 40% of the surviving cells to express GST, but only 0.01% of the cells that survived transfection formed colonies in cloning assays. Finally, with lipofection, only 1% of the surviving cells expressed recombinant GST, although 25–40% of the cells that survived transfection formed colonies. These studies define the merits and limitations of transfection techniques relative to the analysis and sorting of transfected cells by flow cytometry.
Flow cytometric analysis of whole blood lysis, three anticoagulants, and five cell preparations
Authors: Carter Patricia H. , Resto‐Ruiz Sandra , Washington Glennelle C. , Ethridge Steve , Palini Alessio , Vogt Robert , Waxdal Myron , Fleisher Thomas , Noguchi Philip D. , Marti Gerald. E.
Published: August 2005

We studied the effects of anticoagulants and cell preparation methods on lymphocyte forward‐angle s catter (FSC), autofluorescence, and immunofluorescent staining for CD45, CD14, and CD13. Blood samples collected in ethylenediaminetetracetic acid (EDTA), heparin, and acid citrate dextrose (ACD) were processed by using conventional Hypaque‐Ficoll (HF) separation and four whole blood (WR) lysis techniques: Immuno‐lyse, Q‐Prep, FACS Lyse, and Gen Trak Lysis. Lymphocytes prepared by using three of the four whole blood methods gave FCS values comparable to those isolated by HF, while one method (FACS Lyse) gave consistently lover values. Autofluorescence values were comparable by all methods except Immuno‐lyse, which showed consistently higher values in blood stored for 24 h with any anticoagulant. Immunofluorescent values for CD45‐stained cells were quite consistent across all methods, and among the whole blood methods, FACS Lyse and Q‐Prep uniformly gave the highest purity of CD45‐positive cells in the lymphocyte light scatter gates. Additionally, propidium iodide (PI) analyses of CD45‐stained whole blood, and analyzed without lysis, confirmed that ACD and heparin were superior to EDTA for maintaining viable leucocytes overnight. Future studies should focus on other commonly used reagents, a wide variety of abnormal samples, and cell viability.
Rapid multivariate analysis and display of cross‐reacting antibodies on human leukocytes
Authors: Robinson J. Paul , Ragheb Kathy , Lawler Gretchen , Kelley Steve , Durack Gary
Published: August 2005

We present an application which can rapidly determine the binding patterns of monoclonal antibodies on mixed populations of cells simultaneously in a single rapid analysis. It is an application of the tube identifier parameter (TIP) system which can provide fully correlated listmode data of the entire patient phenotype in a single file. Using the phenogram analytical display, we are able to determine the cross‐reacting antibodies for an entire antibody panel for each cell type. This information can be displayed in a single plot. Using light scatter gating to select different populations of lymphocytes, monocytes, and neutrophils, phenograms can be simultaneously generated. This provides a directly comparable means of displaying the positive and negative binding characteristics of each antibody on each cell population. Any marker combination that is abnormal will be identifiable in the phenogram. Additionally, by plotting the fluorescence distributions of each marker beside one another (termed overview), quantifiable differences in intensity can be determined. There are 3 major benefits of the proposed analysis. By using the TIP concept, several sets of antibodies can be compared simultaneously. Any light scatter gate can be used and this gate can be changed on one histogram or plot, yet apply to the total analysis. Data analysis is particularly rapid since the entire phenotype of a patient can be evaluated by performing a single rapid analysis.
Detection of granulocyte reactive oxygen species formation in whole blood using flow cytometry
Authors: Himmelfarb Jonathan , Hakim Raymond M. , Holbrook Diane G. , Leeber Donald A. , Ault Kenneth A.
Published: August 2005

We have developed a technique for analysis of granulocyte reactive oxygen species formation in whole blood using flow cytometry and two color immunofluorescence. This technique relies upon the use of specific fluorescent dye (LDS‐751) to stain nucleated cells, eliminating erythrocytes from analysis. Using LDS751, forward angle light scatter, and 90° side scatter, a granulocyte gate, monocyte gate, and lymphocyte gate were identified.

Analysis with multiple FITC conjugated monoclonal antibodies demonstrated greater than 95% purity of a flow cytometrically identified granulocyte population in whole blood without physical manipulation of the blood.

Utilizing 2'7' dichlorofluorescein diacetate (DCFH‐DA), we were able to measure granulocyte intracellular reactive oxygen species production. Dose response curves were obtained for the effect of granulocyte agonists phorbol myristate acetate, FMLP, and heat fixed Staphylococcus aureus on reactive oxygen species production. The techniques described in this paper should be useful for measuring granulocyte activation in vivo with flow cytometry.
Multiparametric flow cytometric analysis of radiation‐induced micronuclei in mammalian cell cultures
Authors: Schreiber Georg A. , Beisker Wolfgang , Bauchinger Manfred , Nüsse Michael
Published: August 2005

A new flow cytometric method is presented that quantities the frequency of radiation‐induced micronuclei in mammalian cell cultures with high precision. After preparing a suspension of main nuclei and micronuclei stained with ethidium bromide and Hoechst 33258, both types of particles are measured simultaneously in a flow cytometer using forward light scatter and three fluorescence emission intensities excited by UV, 488 nm, and by energy transfer from Hoechst 33258 to ethidium bromide. Nonspecific debris overlapping the micronucleus distribution especially in the low fluorescence intensity region was discriminated from micronuclei by calculating ratios of the different fhuorescences. The frequencies of radiation‐induced micronuclei measured with this new technique agreed well with results obtained by conventional microscopy. The lower limit of the DNA content of micronuclei identified by this technique was found to be about 0.5%–0.75% of the DNA content of G1‐phase nuclei. Dose effect curves and the time‐dependent induction of micronuclei were measured for two different mouse cell lines.
Comparative evaluation of several DNA binding dyes in the detection of apoptosis‐associated chromatin degradation by flow cytometry
Authors: Telford William G. , King Louis E. , Fraker Pamela J.
Published: August 2005

Mouse thymocytes readily undergo apoptosis‐associated DNA degradation upon exposure to glucocorticoids or ionizing radiation. It has been previously shown that flow cytometric cell cycle analysis of propidium iodide‐stained apoptotic thymocytes results in the appearance of a distinct cell cycle region (the A0 region) below the G0/G1 region. Cells in this region were shown to be undergoing apoptosis, and determination of apoptosis by flow cytometric analysis was proposed as a superior method for evaluating thymocyte apoptosis. In this study, a variety of DNA binding dyes with diverse primary binding mechanisms were evaluated for their ability to detect glucocorticoid and ionizing radiatiom‐induced apoptosis in mouse thymocytes. Apoptotic thymocytes stained with DNA binding dyes from the phenanthridinium, acridine, actinomycin, chromomycinone, anthracycline, and bisbenzimidazole groups all demonstrated clearly defined A0 regions with percentages comparable to those obtained for propidium iodide. These results indicate that the appearance of the A0 region is not dependent on a particular dye binding characteristic and may be the consequence of extensive changes in chromatin structure resulting in a significant degree of dye exclusion.
Novel derivation of total cell cycle time in malignant cells using two DNA‐specific labels
Authors: Bokhari S. A. J. , Raza A.
Published: August 2005

Cell cycle kinetic analysis in vitro has conventionally been accomplished by labeling S‐phase cells using two DNA specific labels given sequentially and separated from each other by a certain time interval. By counting the cells labeled by both versus those labeled by either one of the two label, and using the formulas described by Wimber and Quastler, approximate values for durations of S‐phase (Ts) and the total cell cycle (Tc) can be determined. More recently, instead of radioisotope labeled thymidine, two thymidine analogues have been used to label Sphase cells in vivo in a variety of human tumors based on the same principles. In the present report, new formulas are proposed for the calculation of Ts and Tc which are simpler to apply since only one type of labeled cells (those exiting Sphase as identified by containing only the first label) need to be differentiated from the remaining population for Tc calculations.
Flow cytometric DNA content of fresh tumor specimens using keratin‐antibody as second stain for two‐parameter analysis
Authors: van der Linden J. C. , Herman C. J. , Boenders J. G. C. , van de Sandt M. M. , Lindeman J.
Published: August 2005

Studies concerning flow cytometric assessed DNA content reveal problems in interpretating DNA histograms of tumor specimens. The main problems are histograms with a broad coefficient of variation in the G0/G1 fraction; a high G2M fraction and samples with a low percentage of tumor cells. Therefore, in the present study, 382 fresh tumor specimens of carcinomas were analysed routinely, double labeled with, on the one hand, propidium‐iodide for assessing DNA content and, on the other, a monoclonal keratinantibody for marking epithelial and tumor cells.

Of the 311 tumor samples, using single parameter analysis 165 (54%) were classified as DNA aneuploid arid 146 (46%) as DNA “euploid.” By double parameter analysis, 224 (72%) samples were keratin positive and 87 (27%) keratin negative and, of the 224 keratin positive tumors, 175 (78%) were DNA aneuploid and 49 (220) DNA euploid.

The DNA histograms of single and double parameter analysis were compared and it was concluded that in 24 cases (11%) keratin labeling was necessary to recognize DNA aneuploidy. In another 23 (10%) cases, keratin labeling was helpful in assessing DNA aneuploidy. Finally when the results of the 311 samples were combined, 215 (68%) were scored as DNA aneuploid and 99 (32%) DNA euploid. Thus the overall gain in assessing DNA aneuploidy using the double labeling technique is 14%.

In conclusion, it is shown that keratin labeling on fresh tumor cell suspensions of epithelial tumors is of additional value in establishing DNA content. Because single parameter DNA assessment is adequate in approximately 60% of the tested samples, the double labeling technique can be performed routinely, or after initial single parameter DNA assessment. Histograms having a broad CV and/or a high G2M are good candidates for the double labeling technique. Using this technique, DNA‐content assessment becomes more reliable.
Analysis of distribution of tumor‐ and preneoplasia‐infiltrating lymphocytes using simultaneous hoechst 33342 labeling and immunophenotyping
Authors: Loeffler David A. , Kukuruga Mark A. , Juneau Paul L. , Heppner Gloria H.
Published: August 2005

Hoechst 33342 in vivo staining was combined with immunofluorescent staining of cell surface antigens to quantify the distribution, relative to blood supply, of lymphocytes in a preneoplastic mammary lesion, the murine C4 hyperplastic alveolar nodule (HAN), and the C4 adenocarcinoma which develops from C4 HAN. The vascular supply to lymphocytes expressing Thy 1. 2, L3T4, Ly2, and ASGM1 cell surface antigens was evaluated in both tissues. The distribution of ASGM1 + cells, which include natural killer cells, differed between the two tissues, being significantly increased in the 20% brightest Hoechst‐stained lymphocyte fraction in HAN but not in C4 tumor. Distribution of T lymphocytes did not differed between the two tissues. The combination of in vivo Hoechst 33342 with in vitro immunofluorescence provides a simple method to evaluate the distribution with regard to blood supply of lymphocyte subsets in solid tumors and preneoplastic lesions.
Flow cytometric two‐color staining technique for simultaneous determination of human erythrocyte membrane antigen and intracellular malarial DNA
Authors: Pattanapanyasat Kovit , Webster H. Kyle , Udomsangpetch Rachanee , Wanachiwanawin Wanchai , Yongvanitchit Kosol
Published: August 2005

A novel fixative and permeabilization method is described which allows simultaneous flow cytometric detection of red blood cell membrane antigen and intracellular malaria parasites. To illustrate the method, red blood cells from patients with paroxysmal nocturnal bemoglobinuria were infected with Plasmodium falciparum and maintained in synchronous red blood cell culture. The infected red blood cells were immunolabeled with antibodies directed to the complement regulatory protein decay‐accelerating factor (DAF) followed by subsequent fixations in paraformaldehyde and then glutaraldehyde in phosphate‐buffered saline. Finally, DNA of the intraerythrocytic parasites was stained with propidium iodide. Using this technique, cellular morphology was well preserved, no cell aggregation was observed, and high‐quality indirect immunofluorescence and parasite DNA staining were obtained with negligible nonspecific labelling. Simultaneous measurement of parasite DNA and red blood cell membrane determinants makes possible the investigation of alterations of red cell membrane proteins in association with development of intracellular malaria parasites.
Comparison of flow cytometry and epifluorescence microscopy for counting bacteria in aquatic ecosystems
Authors: Monfort Patrick , Baleux Bernard
Published: August 2005

Flow cytometry was used to count bacterial cells from diverse origins: one strain of E. coli, one sample of lake water, and 18 samples of estuary water. To verify the accuracy and the precision of this technique, total bacteria counts made by flow cytometry were compared with counts by direct observation using epifluorescence microscopy. The results of this study showed that flow cytometry was a reliable technique for counting a mixture of bacteria in samples from aquatic ecosystems.
Integration of a barcode reader with a commercial flow cytometer
Authors: Robinson J. Paul , Maguire D. , King G. , Kelley S. , Durack G.
Published: August 2005

This report describes the application and installation of a barcode reader on a standard EPICS Elite flow cytometer. The barcode reader system eliminates keyboard entry of sample information on the cytometer. The system automates the transfer of sample information already present in our laboratory database to the cytometer at run time.

The system uses a standard “off‐the‐shelf” bar code wand with a personal computer keyboard interface and requires no additional software at run time. No typing of sample information is required by the operator at any stage of normal sample operation at the cytometer. All operations are automatically coded into the cytometry software using the macro functions of the software. Tubes are inserted into the tribe reader and sample information is transferred automatically into the cytometer. We have found that the system allows rapid and continuous operation of routine clinical and research samples. This automated data entry also reduces the possibility of data input errors.