bluuebunny/crossref_metadata_2025_split huggingface dataset:
Centromeric index measurement by slit‐scan flow cytometry
Authors: Lucas J. N. , Gray J. W. , Peters D. C. , Van Dilla M. A. 
Published: August 2005

AbstractWe report here the application of slit‐scan flow cytometry (SSFCM) in the classification of muntjac, Chinese hamster, and human chromosomes according to centromeric index (CI) and total flurescence. Chromosomes were isolated from mitotic cells, stained with propidium iodide and processed through the SSFCM where flurescene profiles were measured. The centromere for each profile was taken us the point of maximum difference between the measured profile and a standard profile having no centrmeric dip. The areas under the profile on eithr side of the centromere were then calculated and the CI was calculated as the ratio of the larger area to the total area under the profile. Relative DNA contents for each chromosome were taken to be proportioned to the total fluorescence. Mean CI's for muntjac chromosomes 1, 2, and × + 3 were 0.52, 0.88, and 0.73, respectively; CI's for Chinese hamster M3‐1 chromosomes 1, 2, 5, 8, and M2 were 0.53, 0.55, 0.77, and 0.86, respectively; and average CI's for chromosome groups 4 + t (X;5), 6 + 7 + Y, 9 + M1, and 10 + 11 were 0.56, 0.82, 0.82, 0.58, and 0.60, respectively. These results were, on average, within 4.4% of CI measurements made by image cytometry. CI's measured for human chromosomes 9 through 12, were, on average, within 2.0% of those made by image cytometry.
Immunofluorescence analysis of bacillus spores and vegetative cells by flow cytometry
Authors: Phillips A. P. , Martin K. L. 
Published: August 2005

AbstractA commercially available flow cytometer (Cytofluorograf) was used for the immunofluorescence (IF) analysis of spores of Bacillus anthracis, Bacillus cereus, and Bacillus subtilis, using fluroescein‐labelled antispore conjugates. The cytometer was modified to allow analysis of known numbers of bacteria. In attempting to identify the region of the cytometer fluroescence histogram associated with the presence of stained spores, evidence was produced for signal components due to antibody bound to extracellular antigens. Under some reaction conditions these components were large enough partially or completely to obscure the fluroescence distribution imputed to the spores. The results support the hypothesis that the fluroescence histogram for a bacterial suspension can be modified by subtracting the histogram of the cell‐free centrifugation supernatant to provide a fluorescence distribution more representative of the bacteria themselves. Spore and vegetative forms of B. anthracis could be differentiated in the flow IF assay by comparing the peak and area (integral) values of the photomultiplier output. The 90° scatter histograms of the stained spores and their cell‐free supernatants were so alike in shape that it was not possible to ascrible a unique peak to the spores themselves. Overall, these results confirm the considerable potential of flow cytometry for the rapid and quantitative IF assay of bacterial populations.
Cytofluorometric analysis for estrogen receptors using fluorescent estrogen probes
Authors: Kute Timothy E. , Linville Constance , Barrows George 
Published: August 2005

AbstractEstrogen receptor (ER) analysis of breast cancer tissue has been shown to be very useful in predicting which patients will respond to hormone therapy and have a better prognosis. The ER assay is, however, tedious and time consuming. Measurement of ER by flow cytometry would be rapid and based on either an average fluorescence‐E2 probe intensity per cell or the percentage of the ER+ cells per cell suspension.Analysis of E2 modified structures for relative binding affinity to the ER determined by competition studies and for fluorescence uptake into cell suspensions determined by flow cytometry was performed. Lack of high affinity to the ER and purity of the compound were major problems for the fluorescein‐labeled estrogen in fluorescein‐E2 compounds demonstrated by HPLC very little estradiol derivative in the parent compounds compared to total components present. A second type of fluoresceinated estrogen which has a peptide bond between the steroid and the chromophore was also tested. It was less contaminated but was unable to get into the cell and showed no binding activity to the ER. A pure plant fluorescent estrogen, coumestrol, has Ka of 6 × 108 M−1 for the ER and is a single component as determined by HPLC.Specific fluroescent uptake of coumestrol was performed on ER+ and ER− viable cell suspensions. When these coumestrol‐cell suspensions were excited at 350–360 nm and the blue emission was measured using flow cytometry, the result was a fluroescence uptake that was not highly displaceable by exess nonfluorescence E2 probes. Further studies on the modification of several parameters of the incubation step to enhance specific fluorescent uptake were also unsuccessful. Coumestrol was, however, a good competitor for both cellular and nuclear uptake of 3H‐E2.
Preparation and stability of sixteen murine tissues and organs for flow cytometric cell cycle analysis
Authors: Burns E. Robert , Bagwell C. Bruce , Hinson William G. , Pipkin James L. , Hudson Jerry L. 
Published: August 2005

AbstractThree different technical protocols were used to prepare samples for flow cytometric (FCM) analysis. Each protocol developed worked best for only certain organs. Protocol I involved mincing small pieces of fresh tissue in the propidium iodide (PI) staining solution and filtering through packed glass wool. The organs that were prepared by protocol I were: submandibular gland, urinary bladder, liver, thymus, bone marrow, spleen, lung, kidney and testis. Protocol II involved exposure of the organ to 0.5% acetic acid for 48 h prior to mincing in the PI. The organs that were prepared by protocol II were: uterus, rectum, colon, ileum, and heart. Protocol III utilized an exposure to 0.5% acetic acid, pepsinization, and then staining with PI. The tissues that were prepared by protocol III were the epithelium of the anterior surface of the cornea and the epithelium of the surface of the tongue. A total of 16 different organs and tissues were successfully prepared. For each organ, averaged DNA histograms were analyzed by nonparametric and parametric programs and the results (phase fractions) are presented in tabular form. Several of the organs used came from animals exposed to 1.0 mg/kg vincristine (VC) for 5–6 h to test the capability of the different protocols to detect the enlargement of the G2 + M compartment by the accumulation of VC‐arrested mitotic figures. The stability of the many different sample preparations was tested by comparing averaged DNA histograms obtained on the day of sample preparation to averaged DNA histograms of the same set of samples after storage at 4°C, with or without fixation in 10% phosphate‐buffered formalin, for days to weeks. After staining with propidium iodide, fixation of the sample with a final concentration of 2–3% phosphate‐buffered formalin, was the procedure adopted to assure sample stability. The demonstration of sample stability permits sample preparation to occur at one site followed by transport of the samples to the FCM laboratory at another geographical location. The major findings of this work were (a) technical protocols were developed which resulted in acceptable nuclear suspensions for FCM from 16 different murine organs or tissues, (b) the stability of these samples can be assured by fixing the PI stained nuclear suspension with formalin, and (c) each different protocol was capable of detecting and preserving at least some of the mitotic figures arrested and collected by vincristine.
The use of trout erythrocytes and human lymphocytes for standardization in flow cytometry
Authors: Jakobsen A. 
Published: August 2005

AbstractA method of standardization of flow cytometric ploidy measurements using trout erythrocytes and human lymphocytes is described. The sources of errors of the ratio between the modal channel number of erythrocytes and lymphocytes were investigated. The sample standard deviation was 0.3%, and the variation between persons of the same sex was approximately 0.5%. A distinct difference (1.82%) between the two sexes indicated that small deviations of DNA content can be detected.The standardization method was applied to the analysis of biopsy specimens from bladder tumors, normal bladder mucosa, and lymphocytes from the same patients. In diploid populations the standard deviation of the DNA indices was 1.5–2% for normal bladder mucosa but approximately 2.5% for tumors. These values indicate that deviations amounting to 4–5% from the diploidy are detectable by a single analysis of one sample.
Maturation rates and transition probabilities of cycling cells
Authors: Sahar Elhanan , Wage Michael L. , Latt Samuel A. 
Published: August 2005

AbstractThe kinetics of cell cycling are analyzed in terms of equations incorporating a DNA synthesis rate continuously varying along S phase and a portion of G1 phase through which the cells advance in a probabilistic fashion. These equations are solved analytically for several steady state as well as nonsteady state populations of cells. Experiments are described illustrating how these results can be applied to obtain both the rate of DNA synthesis at each point along S‐phase and the probability of transition through the nondeterministic portion of G1.
Increased uptake of vital dye hoechst 33342 during S phase in synchronized HeLa S3 cells
Authors: Frankfurt Oskar S. 
Published: August 2005

AbstractUptake of the vital dye Hoechst 33342 (HO342) was studied in HeLa S3 cells synchronized with hydroxyurea or thymidine. Cell monolayers were incubated with 0.05–0.5 μg/ml HO342 for 1–10 min at 37°C, and the fluorescence intensity of DNA‐bound intracellular dye was then measured in a flow cytometer. Modal fluorescence intensity normalized to DNA content was used as a measure of the dye uptake. No efflux of the dye from vitally stained cells was observed during 2 hr at 37°C in a monolayer or at 20°C in a suspension. HO342 uptake increased within in 1 hr after the renewal of progression (early S phase), achieved a maximum level in 2 hr (mid‐S phase) and returned to the inital level in 4 hr (late S‐G2 phase). The pattern of increased uptake of HO342 was similar in cells synchronized with either hydroxyurea or with thymidine. To estimate the relationship between fluorescence and dye uptake, the amount of dye released by Triton X‐100 after vital staining was measured. The method for this measurement was based on the simultaneous addition of chicken erythrocytes (“indicator cells”) with Triton X‐100. Fluorescence intensity of chicken erythrocytes was significantly higher in the mixture containing asynchronous or late S‐G2 cells. Thus the fluorescence after vital staining with HO342 reflected HO342 uptake and, possibly, fluctuations of membrane permeability. The period of maximal HO342 uptake in synchronized cells was also characterized by a a high sensitivity of cytotoxic effects of adriamycin. Uptake of HO342 at nonsaturating concentrations may be used as a marker of cell sensitivity and resistance to a number of cytotoxic compounds.
A rapid, vital staining procedure for flow cytometric analysis of human reticulocytes
Authors: Sage Burton H. , O'Connell James P. , Mercolino Thomas J. 
Published: August 2005

AbstractA fluorescent staining procedure based upon thioflavin T is described. This dye permits rapid, vital, nonprecipitative staining of human reticulocytes. Flow cytometric analysis of thioflavin T stained whole blood specimens results in reticulocyte counts that correlate well with new methylene blue reticulocyte counts and give coefficients of variation approaching the theoretical limit. The shape of the fluorescence histogram is qualitatively similar to the reticulocyte age profile. Paired new methylene blue and thioflavin T reticulocyte counts are presented with illustrative cases of reticulocyte maturity distributions.
Analysis of malaria parasite‐infected blood by flow cytometry
Authors: Jacobberger James W. , Horan Paul K. , Hare J. Donald 
Published: August 2005

AbstractThe use of flow cytometry in the quantitative analysis of blood from mice infected with Plasmodium vinckei has been studied. Several fluorescent dyes responsive to cell membrance potential were screened and one dye, 3,3′‐dimethyloxacarbocyanine (DiOC1(3)), was chosen for further study. Mature red blood cells (mRBC), immature RBC (imRBC), and parasitized RBC (pRBC) could be recognized and counted in the flow cytometer. When infected blood was separated on a Percoll gradient and fractions analyzed by flow cytometry using DiOC1(3), distinct populations of pRBC were recognized, the frequency of which varied with density. These subpopulations could not be correlated with distinct morphologic stages but varied with the size or age of the growing parasite. Methods combining the use of DiOC1(3) with a DNA specific‐dye, Hoechst 33342, are discussed as an approach to more complete analysis of the blood of malaria‐infected animals.
The mutual effect of hydrogen ion concentration and osmotic pressure on the shape of the human erythrocyte as determined by light scattering and by electronic cell volume measurement
Authors: Porath‐Furedi Asher 
Published: August 2005

AbstractThe measurement of the fluctuations of the scattered light were the source of information on the flatness of erythrocytes. Data on cell volume, together with the measure of scattered light fluctuations, defined the cell shape. The measurements were repeated in a wide range of osmotic pressures and pH values in order to affect both the hemoglobin and the cytoskeleton. Major volume changes were detected at low osmotic pressures and pH. The erythrocyte volume was determined by electronic volume measurement. The cell flatness is maximal at physiological conditions.
Multidimensional slit‐scan prescreening system: Preliminary results of a single blind clinical study,
Authors: Wheeless L. L. , Patten S. F. , Berkan T. K. , Brooks C. L. , Gorman K. M. , Lesh S. R. , Lopez P. A. , Wood J. C. S. 
Published: August 2005

AbstractA multidimensional slit‐scan flow system was developed to serve as an automated prescreening instrument for gynecological cytology. A 2‐year single blind clinical study was carried out to evaluate system performance. Cellular material was collected by scraping the uterine cervix and stained in suspension with acridine orange. Seven hundred and forty specimens (701 patients) including 156 abnormal specimens representing a broad spectrum of abnormality were analyzed. Approximately 50,000 cells were analyzed for each specimen. The system false‐positive rate was 17.6% while the false‐negative rate was 2.8%. All misclassified abnormals were specimens with cellular changes consistent with a slight dysplasia of nonkeratinizing type. The instrument in its present configuration appeared sensitive to the entire spectrum of abnormality existing in the female genital tract and it classified as abnormal any specimen containing on the order of 0.1% (or greater) abnormal cells.
Erythrocyte deformability and size measured in a multiparameter system that includes impedance sizing
Authors: Bator Jenny M. , Groves Michael R. , Price Brandon J. , Eckstein Eugene C. 
Published: August 2005

AbstractA Coulter Counter was modified to allow the photographing of erythrocytes as they exit the sizing orifice. These photographs provide the necessary information to determine the shape of individual cells. This data was combined on a cell‐by‐cell basis with the corresponding impedance signal to determine accurately both the cell size and deformed state of each cell. The particle shape is required if the size is to be determined accurately. Normal, diabetic and sickle cell samples were used in this study. The cell size was corrected according to theory that relates the impedance signal with cell shape. Although there was no objective test to determine the efficacy of this calculation in terms of improving the size measured, the reduction in the associated distribution width is taken as indicative of an improvement in the measuring process.The data presented represent the first attempt to relate cell size with deformed state on a cell‐by‐cell basis and indicates the potential usefulness of multiparameter deformability and size measurements.
Filipin as a flow microfluorometry probe for cellular cholesterol
Authors: Muller Claude P. , Stephany David A. , Winkler David F. , Hoeg Jeffrey M. , Demosky Stephen J. , Wunderlich John R. 
Published: August 2005

AbstractThe polyene antibiotic filipin, which forms specific complexes with 3β‐hydroxysterols, displays spectral properties compatible with its use in flow microfluorometry (FMF). The purpose of this study was to test the suitability of filipin as an FMF probe for unesterified cellular cholesterol. The following experimental conditions appeared optimal for cells with an average unesterified cholesterol content of less than 3 nmol per 106 cells: 2 × 106 fixed cells (1–4% p‐formaldehyde, 30 min, 21°C) stained for 2–4 h with 100 μg/ml filipin and excited at 350. 7/356. 7 nm. Fluorescence emission (Em) was measured above 510 nm. Less suitable conditions involved excitation at 488 nm or using cells which had not been fixed. Fixation preserved the live‐dead cell discrimination provided by forward light scatter measurements, so that dead cells could be excluded from the FMF analysis of cellular cholesterol. Under the above conditions FMF showed that in all cases the fluorescence intensity of filipin‐stained cells was clearly increased above autofluorescence levels of the unstained control cells. The increase in fluorescence signal in different filipin stained cell types correlated (P ⩽ .001) with the cellular content of unesterified cholesterol determined by an independent enzymatic assay. The sensitivity of the FMF assay was in the femtomole (10–15) range. Mixing experiments with cells of different cholesterol levels showed that the technique distinguishes cell populations with distinctive levels of unesterified cholesterol. We therefore concluded that filipin is a useful FMF probe for determining relative levels of unesterified cholesterol in cells.
Flow cytometric analysis of DNA aneuploidy in primary and metastatic human solid tumors,
Authors: Frankfurt O. S. , Slocum H. K. , Rustum Y. M. , Arbuck S. G. , Pavelic Z. P. , Petrelli N. , Huben R. P. , Pontes E. J. , Greco W. R. 
Published: August 2005

AbstractDNA histograms were measured by flow cytometry for 656 human solid tumors (365 primary and 291 metastatic). The proportion of aneuploid cells in cell suspensions obtained by mechanical disaggregation was significantly higher than those obtained after enzymatic disaggregation (collagenase + DNAse) of the same tumor. A strong correlation was observed between the values of DNA‐indices measured after staining with propidium iodide and with 4′,‐6‐diamidino‐2‐phenylindole (r = 0.97). Aneuploid cells were observed in 430 tumors (66%); 30 of these had two aneuploid stemlines, and two had three aneuploid stemlines. The overall frequency of aneuploidy was 61% among primary and 71% among metastatic tumors. The median value of the DNA index was 1. 67 for 224 primary aneuploid tumors and 1.68 for 206 metastatic aneuploid tumors. For most diseases, the largest proportion of aneuploid primary and metastatic tumors had DNA‐indices in the hypertriploid region. No major differences in frequency and degree of aneuploidy was observed between primary and metastatic tumors. For carcinomas of the bladder and prostate, frequency of aneuploidy was higher among poorly differentiated, than among moderately and welldifferentiated tumors. For carcinomas of the breast and for sarcomas, tumors with DNA indices of > 2.0 were observed mostly in the poorly differentiated group. For patients with carcinomas of the bladder and prostate most tumors at earlier stages of disease were diploid; whereas most tumors at later stages of disease were aneuploid. For patients with carcinomas of the ovary, colon, and kidney, no relationship between stage of disease and aneuploidy was evident.
Development and use of metaphase chromosome flow‐sorting methodology to obtain recombinant phage libraries enriched for parts of the human X chromosome
Authors: Lalande Marc , Kunkel Louis M. , Flint Alan , Latt Samuel A. 
Published: August 2005

AbstractMetaphase chromosomes isolated from human lymphoblastoid cell lines containing structurally abnormal X chromosomes have been stained with the bisbenzimidazole dye Hoechst 33258 and analyzed on a FACS II flow system equipped with a 5‐W all‐lines argon ion laser. The chromosomal fluorescence has been highly resolved at flow rates of 1,000–3,000 chromosomes per second.With the goal of obtaining recombinant DNA libraries from parts of the human X chromosome, fluorescence populations enriched for a dicentric X (Xpter‐> Xq24::Xq24‐> Xpter) chromosome and an isochromosome of the long arm of the X [i(Xq)] have been identified. The dicentric X chromosome has been resolved as a discrete peak in the fluorescence flow histogram. In contrast, the fluorescence intensity of the isochromosome is indistinguishable from that of chromosomes 3 and 4. Recombinant DNA libraries from the flow‐sorted chromosomes have been constructed in the lambda phage, Charon 21A, and consist of 1.6 × 105 and 0.7 × 105 plaque‐forming units in the case of the dicentric X and the isochromosome, respectively. Ninety percent of the phage in both recombinant libraries contain inserts which hybridize to highly repetitive human DNA sequences. The recombinant phage library from the flow‐sorted dicentric X chromosome, which could be assigned to a discrete fluorescence peak, has been further characterized and shows at least a tenfold enrichment for X chromosome‐specific DNA sequences as determined by Southern blot hybridization of cloned fragments.
Preparation of chromosome suspensions for flow cytometry
Authors: Engh Ger Van Den , Trask Barb , Cram Scott , Bartholdi Marty 
Published: August 2005

AbstractCertain variables in the preparation of chromosome suspensions for flow cytometric analysis have been investigated. The optimal conditions have been determined. The results of this series of experiments have been incorporated to yield a preparation protocol that gives chromosome profiles with a low amount of small particle debris and few chromosome clumps. The method reduces variability that results from sample preparation. Chromosomes are optimally isolated in a hypotonic solution buffered to pH 8.0. MgSO4 and dithiothreitol added to the buffer reduce the number of clumps and small fluorescent particles. The presence of MgSO4 also stabilizes the chromosomes and precludes the need for other stabilizing agents such as propidium iodide. When the swelling buffer developed in this investigation is used, unstained chromosomes are stable for at least 1 wk if stored at 4°C. The preparation procedure can be used with the DNA stains, propidium iodide, Hoechst 33258, and mithramycin. Preliminary experiments show that this procedure can also be used for bivariate analysis of human and Chinese hamster chromosomes. The importance of this improvement for studies on chromosome damage caused by irradiation or mutagens is discussed.