bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multiple labeling using two‐color immunofluorescence with only one light source, two fluorescence photomultiplier tubes, and two light scatter detectors
Authors: Mansour Issam , Jarraya Mohamed A. , Gane Pierre , Reznikoff Marie F. 
Published: June 2005

AbstractMultiple labeling is necessary for the detailed phenotyping of cells in many biological systems in human and animal species. In a previous report, we described an approach permitting the study of three labels simultaneously by using the two‐color immunofluorescence and one light source (Mansour et al., Cytometry 11:636–641, 1990). This approach allowed enumeration of cell subpopulations positive for only one label and negative for many others (X+A−B− …︁). We here present an improvement of the previous approach to allow analysis of double positive phenotypes (X+Y+A−B− …︁), using only two fluorescence photomultiplier tubes arid light scatter detectors. It consists of a two‐step analysis that does not require additional material than that used in the former technique. Briefly, all antibodies are conjugated to only, two flurochromes: either FITC or PE. For the analysis of the X+Y+A−B− …︁ phenoytpe, the Y, A and B labels are all coupled to the same dye (FITC, e. g.) and the × label to the other dye (i. e, PE). In a first step, cells are labeled with X, A, and B, and in a second step, the second positive (Y) label is added. Two examples are supplied: CD56+CD2+CD3−CD16− decidua infiltrating cells and CD3+TCR∂+CD4−CD8−peripheral blood lymphocytes. This technique is useful for qualitative as well as quantitative analysis, with cytometers that do not have the appropriate hardware to do true three‐color immunofluorescence analysis. © 1994 Wiley‐Liss, Inc.
An experimental model of affinity cell separation
Authors: Nordon Robert E. , Milthorpe Bruce K. , Schindhelm Klaus , Slowiaczek Peter R. 
Published: June 2005

AbstractCell affinity separations are based on the selective attachment of cell pheno‐type using antibody or lectins specific for cell surface markers. The major physico‐chemical factors which influence ligand‐mediated cell adhesion dynamics and the efficiency of cell affinity separation have been examined. Uniform cell detachment forces were generated with a parallel‐plate flow cell (plate separation 100 μm, surface area 3 cm2). Hydrodynamic shear stress was used to measure cell adhesion strength and to separate cells on the basis of surface affinity. Human cell lines grown in tissue culture were separated on a flat derivatised glass immunoadsorbent which formed the floor of the flow chamber. Flow‐cell residence time, detachment shear stress, temperature, and ligand density were shown to influence cell attachment probability. An understanding of the physical basis of ligand‐mediated cell adhesion provided a rationale for optimisation of affinity cell separation. At room temperature attachment of positive cells was rapid (<2 min) and adhesion strength was directly related to immunoadsorbent ligand density. Purity and recovery of enriched fractions were dependent on the separation shear stress and could be optimised using this parameter. Enrichment factors were greater than 100‐fold, with at least 90% of positive cells recovered in enriched fractions. Enrichment purity and yields did not decline at higher loading densities (105 cells/cm2). Selective immunoadsorbent surface chemistry is a prerequisite for efficient affinity cell separation. Purity and recovery may be optimised by fractionating enriched and depleted cell populations with uniform fluid shear stress. © 1994 Wiley‐Liss, Inc.
Enumeration of CD34+ hematopoietic stem cells for reconstitution following myeloablative therapy
Authors: Roscoe R. A. , Rybka W. B. , Winkelstein A. , Houston A. M. , Kiss J. E. 
Published: June 2005

AbstractThe CD34+ cell fraction of bone marrow and blood contains the hematopoietic stem cells required for marrow reconstitution following myeloablative therapy. Because they are present in small numbers, accurate quantification is often difficult. We have developed a reproducible and sensitive flow cytometric method for CD34+ enumeration of both bone marrow harvests and peripheral blood stem cell collections. The total numbers of harvested cells are enumerated by particle counting. A measured aliquot is stained with two FITC‐labeled anti‐CD34 antibodies, one directed against 8G12 and the other against QBend epitope. To eliminate cells committed to mature lineages (lin+), the suspension is counterstained with a cocktail of PE‐labeled antibodies including CD3 (T cells), CD19 (B cells), CD11b (neutrophils), and CD14 (monocytes). Particles < μm in diameter are excluded by use of a standard bead gate. Regions are established using unstained U937 cells to set the vertical axis and PE stained U937 cells for the horizontal axis. Because of the low numbers of CD34+ cells, 20,000 events/sample are analyzed. Dilutions of KG‐1A tumor cells (CD34+) in U937 cells showed a threshold of detection of 0.1% CD34+ lin‐ cells. Duplicate samples varied by <10%. Initial studies indicate that this procedure can be reliably used to measure CD34+ lin‐cells in blood, pheresis products, and bone marrow harvests. This CD34 enumeration procedure should result in increased consistency in enumerating this stem cell population. © 1994 Wiley‐Liss, Inc.
Tumor cell kinetics in mixed populations
Authors: Ritter Mark A. , Albrechtson Jennifer L. , Kim Youngmi J. , Kinsella Timothy J. 
Published: June 2005

AbstractThe delayed biopsy, relative movement method provides a rapid estimate of in situ potential doubling times in human tumors. The accuracy of the estimate, however, can be adversely influenced by normal diploid cells that are present in tumor biopsy specimens and have DNA profiles that overlap their tumor cell counterparts. Interference with the measurement of tumor cell kinetic parameters can result, the magnitude of which depends upon the DNA index and the proportion and labeling index of these normal cells.Two approaches that address this problem were explored in vitro: (1) A normal cell subtraction method was developed in which adjacent normal tissue and tumor are biopsied concurrently and are separately processed for flow cytometric analysis. Results are integrated to allow an approximate correction for the normal, diploid population's perturbation of the measurement of tumor labeling index and relative movement. This approach was examined in this present study through extensive testing in mixtures of various proportions of two in vitro cell lines. (2) The percent S phase method, developed by Durand, was examined in these same population mixtures, as well as separately in each population. Both methods provide reliable estimates of kinetics in mixed in vitro populations. The relative advantages and disadvantages of each, as well as the potential limitations if extended to in vivo measurements, are discussed. © 1994 Wiley‐Liss, Inc.
DNA content distribution of mouse cells following infection with polyoma virus
Authors: Lehman John M. , Laffin Judith , Friedrich Thomas D. 
Published: June 2005

AbstractInfection of primary to tertiary mouse embryo fibroblasts or mouse kidney cells with polyoma virus leads to stimulation of cellular DNA synthesis. When either confluent or growing mouse cells were infected, the monolayer cells were found to accumulate cells with a DNA content of S and G2/M phases of the cell cycle as assayed by flow cytometry. A similar pattern of DNA content was also observed in cells in the supernatant, which are probably cells replicating virus and dying. When compared with control cells, the infected monolayer and supernatant cells exhibited a population (5–27%) with a >G2 DNA content. The increase in DNA content of these >G2 cells was calculated to be an average of 26.7%, which is probably due to viral DNA. Polyoma contrasts with another papovavirus, SV40, which stimulates cells into DNA synthesis, with the majority of cells attaining a >G2/tetraploid DNA content, suggesting that there are differences in polyploidization between these two viruses. © 1994 Wiley‐Liss, Inc.
Fluorescence array detector for large‐field quantitative fluorescence cytometry
Authors: Wittrup K. D. , Westerman R. J. , Desai R. 
Published: June 2005

AbstractA prototypic fluorescence array detector (FAD) has been designed and constructed which is capable of quantifying single‐cell fluorescence emissions from a statistically significant population of cell‐sized objects (over 103) on a solid substrate. The system is comprised of a cryogenically cooled CCD, 50 mW air‐cooled argon ion laser, and optics that image a large (1 × 1 cm) field at 1:1 (no magnification). The CCD is effectively treated as a two‐dimensional array of 2.7 × 105 independent 20 × 20 μm photodetectors, with each cell‐sized object imaged across only a few CCD pixels. Algorithms have been developed for focusing, image segmentation, shading correction, and noise rejection; performance data for the FAD with fluorescent calibration beads are presented. The FAD is a simple alternative to microscope‐based imaging cytometry, allowing large‐field imaging without a scanning stage. © 1994 Wiley‐Liss, Inc.
Apparatus for high‐precision repetitive sequential optical measurement of living cells
Authors: Deutsch M. , Weinreb A. 
Published: June 2005

AbstractA system is described which permits the repetitive spectroscopic measurement of individual cells within a population of many cells, while the location of each cell is preserved during various manipulations of the cells and/or their surrounding medium. The central mechanical feature of the system is the cell carrier, a matrix of apertures in which the cells become trapped. The detector electronics operate in a preset photon counting mode, permitting the measurement of low and high light intensities with the same degree of precision. The present instrument configuration is specifically designed for the accurate and precise measurement of the polarization of fluorescence of probes within living cells for application to routine performance of the Cercek SCM test for cancer. With modifications, the apparatus can be applied to a wide range of other clinical and research tasks. © 1994 Wiley‐Liss, Inc.
Lissajous‐like patterns in scatter plots of calibration beads
Authors: Doornbos R. M. P. , Hoekstra A. G. , Deurloo K. E. I. , De Grooth B. G. , Sloot P. M. A. , Greve J. 
Published: June 2005

AbstractFlow cytometric measurements of light scattering of polystyrene calibration beads revealed remarkable Lissajous‐like loops in two‐parameter scatter plots. The existence of such loops is shown to be in qualitative agreement with Lorenz‐Mie scattering theory of homogeneous spheres. The occurrence of these patterns reflects the extreme particle size dependency of perpendicular light scattering of homogeneous spheres. These effects may give an explanation for the frequently observed phenomenon that polystyrene spheres reveal a relatively large CV in the perpendicular light scattering signals, whereas the CV of the forward light scattering signal is small. We conclude that one should be careful to optimize the perpendicular light scattering channel by minimizing the CV, because there is no linear relationship between instrument alignment quality and the CV of the perpendicular light scattering signals of calibration beads. © 1994 Wiley‐Liss, Inc.
Flow cytometric method for the measurement of epidermal growth factor receptor and comparison with the radio‐ligand binding assay
Authors: Brotherick I. , Lennard T. W. J. , Wilkinson S. E. , Cook S. , Angus B. , Shenton B. K. 
Published: June 2005

AbstractA method for the purification, conjugation, and use of EGF‐R antibody to quantify EGF receptors on tumour cells by flow cytometry is described. The quantification of both internal and external EGF receptors was determined by treatment with saponin, rendering the cells permeable to the EGF‐R antibody. Using QCS bead standards, the number of EGF‐R binding sites per cell was assessed. Results were compared with conventional EGF‐R quantification using a radio‐ligand binding assay. A high degree of correlation was found between the two methods. The flow cytometric method for EGF‐R quantification described is sim‐ple, rapid, and reproducible. The assay may be of particular value in measuring EGF‐R on urgent clinical samples or those that are too small (such as breast aspirates) for measurement by the radio‐ligand binding assay. Advantages of this assay over the radio‐ligand binding assay include reduction in use of radio‐labelled iodine compounds, a decrease in analysis time, and reduced cost and quantity of material needed for assay. In addition, flow cytometry offers the possibility of selecting cell phenotypes by gating as well as live/dead cells by using multi‐parameter flow cytometry. © 1994 Wiley‐Liss, Inc.
DNA strand breakage and DNA structure influence staining with propidium iodide using the alkaline comet assay
Authors: Olive P. L. , Banáth J. P. , Fjell C. D. 
Published: June 2005

AbstractThe alkaline comet assay is used to detect DNA single‐strand breaks in individual cells embedded in agarose, lysed to denature DNA and remove proteins, and briefly exposed to an electric field to allow broken DNA to migrate. Total DNA fluorescence, measured by staining individual comets with propidium iodide, is reduced 30–40% by low doses of ionizing radiation, N‐methyl‐N‐nitrosoguanidine (MNNG), etoposide, or hydrogen peroxide. Three possible explanations were examined: 1) these agents produce small fragments of DNA that are lost during lysis and electrophoresis, 2) the analysis of comet images is less efficient once the DNA is small enough to migrate in the electric field, and 3) DNA staining by propidium iodide is affected by changes in DNA structure caused by strand breaks and exposure to alkali. Our results indicate that the decrease in fluorescence after low doses is best explained by a change in ability of DNA, which has been denatured by alkali and subsequently renatured, to interact with fluorescent dyes. This change in fluorescence has the practical consequence of improving the ability of the alkaline comet assay to distinguish damaged from undamaged cells. © 1994 Wiley‐Liss, Inc.
Expression of proliferation‐associated antigens (PCNA, p120, p145) during the reentry of G0 cells into the cell cycle
Authors: Bolton Wade E. , Freeman James W. , Mikulka Warren R. , Healy Cynthia G. , Schmittling Robert J. , Kenyon Norma S. 
Published: June 2005

AbstractFlow cytometric bivariate analysis was used to evaluate the expression of PCNA, p120, and p145 during the G0 reentry of CHO‐K1 cells into the cell cycle. CHO‐K1 cells were placed in a G0‐like state using serum depletion and stimulated to reenter the cell cycle by replating into fresh, serum‐containing medium. At discrete intervals after stimulation, replicate samples were stained for either PCNA, p120, or 145; stained for DNA (Coulter DNA‐Prep); evaluated on the EPICS Profile I; and analyzed on the EPICS ELITE workstation. PCNA stained less than 10% of the G0 cells; in contrast, however, 30–35% of the G0 cells were positive for p120 and p145. Eight hours after stimulating G0 cells to reenter the cell cycle (during G0/G1), p120 reached 88% positivity, while p145 and PCNA were 63% and 30% positive, respectively. Cells in S phase (12 and 16 h following G0 stimulation) were greater than 90% positive for all three antigens. PCNA had the greatest change throughout the G0 reentry process, both in percentage positive and quantitatively (mean channel fluorescence).This report indicates that all three proliferation‐associated antigens studied are differentially expressed during the reentry of G0 cells into the cell cycle. Furthermore, these antigens may be useful in the early detection of G0 recruitment. © 1994 Wiley‐Liss, Inc.
Quantitative determination of particle concentrations in experimental and marine environmental samples
Authors: Robins David B. , Bedo Alain W. 
Published: June 2005

AbstractAs the application of flow cytometry increases across the environmental sciences, there is a need to expand instrument flexibility. In particular, some of the major applications within aquatic research demand an ability to quantify the particles under investigation, expressing them as numbers per unit of sample volume. Two of the principal areas of flow cytometric investigations are the characterisation of naturally occurring particulate assemblages in the oceans and investigations into the feeding behaviour of “filter feeding” organisms on the mixtures of such particles. In both cases some of the most useful data derived from flow cytometry require that analyses be determined in terms of particle concentrations. Therefore, a simple and practical modification is described, which allows known sample volumes to be analysed on flow cytometers. The method has a high degree of flexibility in sample volume and is cost effective and reliable. It involves introducing a standard chromatography “loop injection valve” into the sample‐handling fluidics system. Use of fluorescent beads shows that a range of praticle sizes and concentrations can be used with this system. Tests, also using fluorescent beads and an algal culture, show that replicate 100 μ1 sample volumes can be analysed and counted with a coefficient of variation of less than 3%. © 1994 Wiley‐Liss, Inc.
Staining of Escherichia coli for flow cytometry: Influx and efflux of ethidium bromide
Authors: Jernaes Mette W. , Steen Harald B. 
Published: June 2005

AbstractIn an attempt to develop procedures for nucleic acid staining of bacteria for clinical routine assays, the uptake of ethidium bromide (EB) in wild‐type Escherichia coli was studied using flow cytometry. Phosphate‐buffered saline (PBS) containing EDTA or Tris significantly increased the net uptake of EB compared to PBS only. However, in the majority of the cells, the net uptake reached a constant level that was only a few percent of that of fully permeabilized cells, apparently due to the activity of a metabolically driven efflux pump. When cells were exposed to cold shock (0°C for 30 min) in the presence of Tris or EDTA, the net uptake of dye was similar to that of fully permeabilized cells, whereas it was about half that value in PBS. When cold shock was given in growth medium, the cells split up into four subpopulations, with a net dye uptake ranging from that of fully permeabilized cells to less than 1% of that value. As expected, metabolic inhibitors (Na‐azide, 2‐deoxy‐D‐glucose, and CCCP) reduced efflux activity. However, fluorescence of metabolically inhibited cells never exceeded more than about half the value of that of dead cells, possibly reflecting conformational changes in DNA structure as a result of cell death. © 1994 Wiley‐Liss, Inc.
Optimizing the detection of cell surface antigens on elicited or activated mouse peritoneal macrophages
Authors: Hendrzak Jill A. , Wallace Paul K. , Morahan Page S. 
Published: June 2005

AbstractBlocking conditions that are optimal for the detection of surface antigens on resident peritoneal macrophages (PMϕ) by flow cytometry are not ideal for elicited or activated PMϕ. A blocking step of 10% goat serum can be used routinely to detect the F4/80 and Mac‐1 antigens on resident PMϕ. In contrast, high concentrations (33–50% each) of combined goat and mouse sera were required to reduce nonspecific binding and to improve the detection of the F4/80 antigen on PMϕ elicited by thioglycollate broth (TG) or activated by maleic anhydride divinyl ether copolymer (MVE‐2). However, even low concentrations of goat serum masked the expression of the Mac‐2 antigen on TG and MVE‐2 PMϕ. Thus, within a given elicited or activated PMϕ population, different blocking conditions may be necessary to detect different surface antigens optimally. In addition to blocking, the use of isotypic controls that match the monoclonal antibody isotypes was found to be necessary for the optimal detection of antigen expression on TG and MVE‐2 PMϕ. © 1994 Wiley‐Liss, Inc.
Quality control for evaluation of the S‐phase fraction by flow cytometry: A multicentric study
Authors: Silvestrini Rosella ,   SICCAB Group For Quality Control Of Cell Kinetic Determinations
Published: June 2005

AbstractFlow cytometric measurement of the S‐phase fraction (FCM‐S) may be clinically useful in human cancers to identify high‐risk patients. However, its clinical application requires a methodologic standardization, a high feasibility, and the reproducibility of results within the same and among different institutions. The interlaboratory quality control program for FCM‐S determination (promoted by the Italian Society of Basic and Applied Cell Kinetics) was carried out on DNA diploid and aneuploid cell lines and human brest cancers. FCM‐S was quantified by using four mathematical models characterized by different rationales and differences in the degree and shape of background subtraction functions. A statisfactory agreement in FCM‐S values among different institutions was observed for cell lines. In human breast cancers, a high reproducibility of FCM‐S estimates obtained by the different institutions was observed by using the same model. Conversely, different models gave different FCM‐S estimates. Intermodel differences were more evident in aneuploid than in diploid tumors. These findings could explain the contrasting results on the prognostic role of FCM‐S in clinically comparable series of breast cancers and should be considered in future efforts to define the optimal and most reproducible mathematical approach to quantify cells in the S‐phase. © 1994 Wiley‐Liss, Inc.
Ploidy pattern and cell cycle in breast cancer as detected by image analysis and flow cytometry
Authors: Luzi Pietro , Bruni Alessandra , Mangiavacchi Paola , Cevenini Gabriele , Marini Dolly , Tosi Piero 
Published: June 2005

AbstractBoth image analysis (IA) and flow cytometry (FCM) may be applied to detect ploidy pattern and cell cycle fractions. However, they have different performance characteristics and may yield different results. The two approaches are applied in this study to 66 breast cancers: IA on imprints and FCM on fresh tissue. The percent coefficient of variation (CV) ranged from 2.0 to 7.0 (mean 5.5; SD 1.1) in IA and from 2.0 to 7.0 (mean 4.4; SD 1.1) in FCM. The values were well correlated. With regard to ploidy pattern, the agreement between the two methods was 92.4%; disagreements were due to four cases being aneuploid by IA but not detected by FCM and one case being aneuploid by IA but tetraploid by FCM. This suggests that IA is capable of detecting aneuploidy with more sensitivity than FCM. In diploid cases, the percent values of cells in GO/G1, S‐phase (SPF), and G2M phase were concordant and well correlated. In aneuploid cases, IA was more sensitive than FCM in detecting aneuploid fraction as well as G2M phase, whereas FCM was more sensitive than IA in detecting SPF. A good correlation was found between the DNA indexes (Dls) obtained with the two methods. © 1994 Wiley‐Liss, Inc.