bluuebunny/crossref_metadata_2025 huggingface dataset:
Flow cytometric determination of circulating immune complexes with the indirect granulocyte phagocytosis test
Authors: Terstappen L. W. M. M. , de Grooth B. G. , Nolten G. M. J. , ten Napel C. H. H. , van Berkel W. , Greve J.
Published: August 2005

A method for the determination of circulating immune complexes (CIC) was adapted for flow cytometric analysis. Human granulocytes were used to phagocytose IgG‐bearing CIC of serum from systemic lupus erythematosus (SLE) patients. A method for labeling the phagocytosed CIC with FITC‐conjugated anti‐human IgG was developed where the granulocytes remain in suspension during fixation and labeling. The fluorescence per cell, measured with a flow cytometer, is a measure of the total amount of the phagocytosed IgG. The results indicate that a rapid and quantitative method for the detection and measurement of phagocytosed CIC is possible using the flow cytometer.
Application of DNA flow cytometry to paraffin‐embedded archival material for the study of aneuploidy and its clinical significance
Authors: Hedley David W. , Friedlander Michael L. , Taylor Ian W.
Published: August 2005

By using a recently developed flow cytometric method we have analyzed cellular DNA content of paraffin‐embedded histological material from cancer patients. This method allows the retrospective study of tumors from patients whose clinical outcome is already known, and we have applied it to ovarian cancers, stage II breast cancers, and to metastatic adenocarcinoma of unknown primary site. In addition to knowledge of patient survival, comprehensive information was available about other prognostic determinants and treatment received, and we have used multivariate analysis in an attempt to determine the prognostic significance of cellular DNA content. In ovarian cancer, it is a major prognostic variable except in stage IV disease, whereas in metastatic adenocarcinoma of unknown primary site cellular DNA content has no influence on survival. For stage II breast cancer the situation is more complex and requires larger numbers to be studied. However, aneuploid tumors tend to have more extensive involvement of axillary lymph nodes and a poorer overall disease‐free survival. This influence of DNA content on disease‐free survival appears to be confined to premenopausal patients, and has no effect on patient survival following disease recurrence. Although we need to study more patients and more turnor types, taken together the results so far show a generally more favorable prognosis for patients with diploid tumors, except in the presence of recurrent or metastatic disease. The better prognosis associated with diploid tumors could be due to the fact that they are more commonly found in earlier clinical stages rather than to their being inherently less aggressive than aneuploid tumors.
Estimation of nonproliferating cells in the neural retina of embryonic chicks by flow cytometry
Authors: Morris Valerie B. , Taylor I. W.
Published: August 2005

Nonproliferating cells (N cells) in the neural retina of embryonic chicks were estimated after isolating them in the 2C peak of a DNA distribution by exposure to the cell‐cycle inhibitor ICRF 159. ICRF 159 inhibits cell division but not DNA synthesis, so proliferating cells can leave the 2C peak but not reenter it. Cells left in the 2C peak after exposure to ICRF 159 were assumed to be N cells. The effectiveness of ICRF 159 in inhibiting cell division but not inhibiting DNA synthesis was demonstrated in neural retinae from stage 17 embryos which showed no evidence of a 2C peak after 6‐h exposure to ICRF 159 and which were thus shown to have no N cells. A test to detect escape from the cell division block in older embryos with an N cell population in the neural retina showed some escape after longer exposures to ICRF 159. The escape was suppressed by a second dose of ICRF 159, given some hours after the first.
Analysis of the expression of cloned HLA class I genes in mouse transfected L cells by quantitative flow cytometry
Authors: Le Bouteiller Philippe P. , N'Guyen Catherine , Lemonnier François A. , Jordan Bertrand R. , Caillol DanièLe , Mishal Zohair
Published: August 2005

By using a calibrated cell sorter and monoclonal antibodies conjugated to fluorochromes, a quantitative analysis of the levels of expression of HLA class I molecules at the surface of cloned murine L cells transfected with purified A3, B7, or CW3 genes was performed and compared with radioimmunoassay data. We selected clones of heterogenous levels of HLA class I expression, which were shown to remain constant over a period of 4 mo in absence of HAT selection and not to be correlated to the DNA copy number of the corresponding integrated gene.
Differences between labeling index and DNA histograms in assessing s‐phase cells from a homogenous group of chronic phase CML patients
Authors: Raza Azra , Bhayana Ranjan , Ucar Kalust , Kirshner Jeffery , Preisler Harvey D.
Published: August 2005

The reliability of DNA histogram analysis in accurately estimating S‐phase cells from human tumors was tested by comparing the results to those of simultaneously obtained tritiated thymidine labeling index (LI) studies. Patients with chronic myelocytic leukemia (CML) during chronic phase were selected for study because the Philadelphia chromosome (Ph) was the only cytogenetic abnormality in each case and, since it is a balanced translocation, the frequently encountered problem of aneuploidy in human neoplastic cells was avoided. Unfortunately, when 30 CML patients were studied simultaneously by DNA histogram analysis and LI studies, the correlation coefficient between the two results was only r = 0.611. A comparison of three different mathematical programs for DNA histogram analysis showed that none was completely satisfactory. We conclude that DNA histogram analysis does not provide the same data as autoradiographically processed labeling index studies even in patients with Ph‐positive CML during the chronic phase when the situation is not complicated by additional aneuploidy.
Flow cytometric analysis of head and neck carcinoma DNA index and S‐fraction from paraffin‐embedded sections: Comparison with malignancy grading
Authors: Johnson Tod S. , Williamson Kenneth D. , Cramer Michael M. , Peters Lester J.
Published: August 2005

Archival, paraffin‐embedded, pathology specimens representing pretreatment tissue biopsies from 73 patients with epidermoid carcinoma of the head and neck were analyzed for DNA Index and %S‐phase cells by flow cytometry and were scored for quantitative histomorphology. The DNA fluorescence/light scatter size patterns derived from paraffinembedded specimens were shown to be essentially the same as those from mechanically disaggregated, ethanol‐fixed cells obtained from the same tissue specimen. Patterns ranged from lymphocyte‐like to highly abnormal DNA Index cytokinetic patterns. The DNA Index values ranged from 0.70 to 3.50 (median 1.42), with an aneuploidy frequency of 63/73 (86%). DNA distribution %S ranged from 4% to 45% (mean 19), with the microscopic malignancy grading showing broad heterogeneity (mean 2.1, range 1.0–3.0, where 1.0–1.7 = well differentiated, 1.8–2.3 = moderately differentiated, 2.4–3.0 = poorly differentiated). Cross‐comparison of these data showed that (1) the tumor %S was dependent on DNA Index (higher %S at higher ploidy), (2) low to high malignancy tumors were randomly distributed between diploid/near diploid tumors and high‐degree DNA abnormality tumors, and (3) proliferative activity values broadly overlapped between low to high malignancy scored tumors. However, those carcinomas characterized by high DNA Index (≥ 1.50) and high %S‐phase fractions (≥ 20) had a five fold higher incidence of high‐degree malignancy, invasive tumors than diploid/near diploid (%S ⩽19) tumors.
Droplet Sorting of Large Particles
Authors: Jett James H. , Alexander Robert G.
Published: August 2005

The systematics of droplet formation conditions for orifices with diameters up to 200 μm are described. Sorting recovery experiments indicate that particles up to 44 μm in diameter can be recovered by charged droplet deflection of two drops with at least 75% recovery. By reducing the jet velocity, a deflection of greater than 1 cm was obtained for all droplet sizes.
Characterization of monoclonal antibodies to bromodeoxyuridine
Authors: Vanderlaan Martin , Thomas Cynthia B.
Published: August 2005

The characteristics of three mouse monoclonal antibodies to halogenated uridine derivatives are presented. Two, IU‐1 and IU‐2, are produced by hybridomas derived in our laboratory, and the third is the B‐44 hybridoma described by Gratzner (7) and obtained commercially from Becton‐Dickinson Monoclonal Center. Hybridomas IU‐1 and IU‐2 were derived from the fusion of spleen cells from a Biozzi High Responder mouse immunized with iododeoxyuridine (IdUrd) conjugated to bovine serum albumin and SP2/0 mouse myeloma cells. This paper presents methods and results for enzyme‐linked immunosorbent assays (ELISA) against whole cells labeled with bromodeoxyuridine (BrdUrd), ELISA against BrdUrd‐labeled DNA, and a competition ELISA for free BrdUrd. All three antibodies show similar binding affinities and specificities. The IU antibodies react with BrdUrd and IdUrd when the nucleosides are either free in solution or incorporated into single‐stranded DNA (ss‐DNA). The antibodies do not recognize either halogenated base in doublestranded DNA (ds‐DNA), nor do they react with uracil or bromocytidine. Weak binding to thymidine, 5‐fluorodeoxyuridine, and unsubstituted ss‐DNA occurs.
Cytochemistry for bromodeoxyuridine/DNA analysis: Stoichiometry and sensitivity
Authors: Dolbeare F. , Beisker W. , Pallavicini M. G. , Vanderlaan M. , Gray J. W.
Published: August 2005

This report describes an improved immunochemical procedure for staining cells in suspension for amount of incorporated bromodeoxyuridine (BrdUrd) and total DNA. In this procedure, cellular DNA is partially denatured by extracting the cells with 0. 1 M HCl and then heating them to 80°C in a 50% formamide solution. The cells are then immuno‐fluorescently stained using a monoclonal antibody against BrdUrd in single‐strand DNA (ssDNA) and counterstained for DNA content with propidium iodide (PI), a dye that fluoresces preferentially when bound to double‐strand DNA (dsDNA). We show that the relative amounts of immunofluorescently stained BrdUrd in ssDNA and PI in dsDNA can be altered reciprocally by changing the formamide concentration, denaturation time, and denaturation temperature. We show that this new immunochemical staining procedure allows more complete DNA denaturation so that fivefold lower levels of BrdUrd incorporation can be quantified. In addition, we show that the BrdUrd‐linked immunofluorescence achieved using the new denaturation procedure is more linearly related to cellular BrdUrd content than that achieved after acid DNA denaturation. However, cell loss is sufficiently severe with the thermal denaturation procedure that it may not be applicable to all cell types.
Cell cycle analysis using numerical simulation of bivariate DNA/bromodeoxyuridine distributions
Authors: Yanagisawa Masashi , Dolbeare Frank , Todoroki Takeshi , Gray Joe W.
Published: August 2005

This report describes a mathematical model of cell proliferation for simulation of bivariate DNA/bromodeoxyuridine (BrdUrd) distributions. The model formulates the change with time in the frequency of cells with any DNA content and in the amount of incorporated BrdUrd, according to given cytokinetic parameters, i.e., durations and dispersions of cell cycle phases and DNA synthesis rate during S‐phase. We have applied this model to sequential DNA/BrdUrd distributions measured for Chinese hamster ovary cells asynchronously grown in vitro, 1 for 30 min in 10 μM BrdUrd followed by growth in BrdUrd‐free medium for 0 to 24 h, or (2) during continuous incubation in 3 μM BrdUrd plus 30 μM thymidine for 2 to 24 h. The matches between the experimental and simulated distributions give the G1, S, G2M, and total cell cycle durations (and coefficients of variation) of 5.6h (0.08), 7.0h (0.07), 1.4h (0.16), and 14.0h (0.05), respectively. The model is shown to be useful for quantitative interpretation of the bivariate distributions.
Macrophage‐induced cytostasis: Kinetic analysis of bromodeoxyuridine‐pulsed cells
Authors: Stevenson Anita P. , Crissman Harry A. , Stewart Carleton C.
Published: August 2005

The effect of tumoricidal macrophages on the cell cycle progression of six different cell lines was studied using an anti‐bromodeoxyuridine (BrdUrd) monoclonal antibody to follow the traverse of BrdUrd‐labeled cells. Exponentially growing cultured mammalian cells, from six different cell lines, were prepulsed with BrdUrd before exposure to tumoricidal macrophages. The cultured cells were then analyzed as a function of time for DNA content (by propidium iodide staining) and for BrdUrd incorporation (using a fluoresceinisothiocyanate [FITC]‐conjugated anti‐BrdUrd monoclonal antibody). The position of the cells in cycle and the progression of the BrdUrd‐labeled cohort was followed using flow cytometry. The cell lines examined were: Colon 26, BALB/c‐3T3, ST3T3 (a spontaneously transformed, tumorigenic clone of 3T3), WCHE5 (a clone of whole Chinese hamster embryo cells), RIF (a radiation‐induced fibrosarcoma), and A101D (a human melanoma).

The bivariate distributions showed that for all six cell lines the BrdUrd‐labeled cohort in the control cultures progressed around the cell cycle during the first 12h of culture, as the cells exponentially increased. In contrast, when each cell line was incubated with tumoricidal macrophages, the BrdUrd‐labeled cohort did not progress through cell cycle but remained in S phase throughout the 12‐h culture period. There was also no evidence for progression of cells out of G1. The data show that cells were arrested in every phase of cell cycle. This study suggests that cytostasis, as manifested by the termination of progression in all phases of the cell cycle, is a universal phenomenon induced by tumoricidal macrophages.
Phorbol ester enhances deoxynucleoside incorporation while inhibiting proliferation of K‐562 cells
Authors: Colamonici O. R. , Trepel J. B. , Neckers L. M.
Published: August 2005

Recently a new technique has been developed to study bromodeoxyuridine incorporation into DNA as a function of the cell cycle phase distribution. We have made the observation that treatment of the hematopoietic cell line K‐562 results in a complete and immediate cessation of cell growth, althoughtritiated thymidine incorporation is markedly elevated for as long as 5 days. We have made use of that newly described technique to study this phenomenon.
Cell cycle kinetic measurements in an irradiated rat rhabdomyosarcoma using a monoclonal antibody to bromodeoxyuridine
Authors: Nüsse Michael , Afzal S. M. Javed , Carr Betsy , Kavanau Kris
Published: August 2005

Cell cycle kinetics after X‐irradiation were studied in a solid rat rhabdomosarcoma using a monoclonal antibody to bromodeoxyuricline (BrdUrd) In cells in which the DNA was labeled by BrdUrd. It could be shown that this tumor was composed of about 80% diploid host cells, and only 20% of the cells in the dissociated tumor were actually tetraploid tumor cells. When rats were injected intraperitoneally with BrdUrd to label S‐phase cells in the tumor, only a fraction of both types of cells became labeled with Brdtlrd during s‐phase, even 24 h after injection. The diploid BrdUrd‐labeled cells progressed rapidly into cycle; 4 h after injection of BrdUrd, labeled diploid s‐phase cells could be observed. Only 25% of the tetraploid S‐phase cells could be labeled by a single injection of BrdUrd (160 mg/kg body weight). These labeled tetraploid cells progressed through the cell cycle with similar velocities as did labeled diploid cells. Using a “Mini Osmotic Pump” containing bromodeoxycytidine (BrdCyd) at high concentration (0.3 mol/L) that released BrdCyd continuously into the organism where it was converted to BrdUrd, it could be shown that after 2 days about 60% of cells in S‐phase and 70% of cells in S‐phase were labeled. The fraction of labeled S‐phase cells in irradiated tumors (D = 10 and 20 Gy) was enhanced between 10 and 50 h after irradiation clue to a radiation‐induced G2 block in cycling tetraploid tumor cells. No effect of irradiation on incorporation of BrdUrd in S‐phase could be observed in the continuous labeling experiments in contrast to the pulse labeling with BrdUrd, where a small retardation of DNA synthesis was observed in the irradiated tumors during the first 15 h after irradiation. When dissociated cells of the tumor were plated in medium containing 20 μmol/L BrdUrd, only the tetraploid tumor cells were able to incorporate BrdUrd and to enter cycle.
A method to measure the duration of DNA syntheses and the potential doubling time from a single sample
Authors: Begg A. C. , McNally N. J. , Shrieve D. C. , Kärche H.
Published: August 2005

A method is described whereby the DNA synthesis time, Ts, call be calculated using data of a single sample of cells taken several hours after labelling with bromodeoxyuridine (BrdUrd). The method involves a simple calculation using flow cytometry data of BrdUrd incorporation (green fluorescence, FITC‐labelled anti‐BrdUrd‐DNA antibody) and total DNA content (red fluorescence, propidium iodide). The movement of BrdUrd‐labelled cells through the S phase can be Ouantified by measuring their mean red fluorescence relative to that of G1 and G2 cells. Assuming the movement of the labelled cells toward G2 is linear with time, T., can be calculated by measuring their relative movement at any one time. The method was tested on cells in vitro and on bone marrow and tumor cells in vivo. Reasonable agreement was seen with published estimates of TS for these tissues.
Cell kinetic studies of in situ human brain tumors with bromodeoxyuridine
Authors: Hoshino Takao , Nagashima Tadashi , Murovic Judith , Levin Ellen M. , Levin Victor A. , Rupp Stephen M.
Published: August 2005

At the time of surgery, 18 patients with various brain tumors were given a 1‐h i.v. infusion of bromodeoxyuridine (BrdUrd), 170–200 mg/m2. At an infusion rate of 200 Mg/M2/h, serum BrdUrd levels of 8 μM were achieved. After the infusion, tumor tissue was obtained and divided into two portions. One portion was fixed in 70% ethanol, embedded in paraffin, and sectioned; the sections were deparaffinized, denatured with 2 N HCl, and reacted with monoclonal antibodies against BrdUrd (anti‐BrdUrd MAb). BrdUrd‐labeled nuclei were demonstrated satisfactorily by an indirect peroxidase method. The other portion was dissociated into single cells with a DNase enzyme cocktail and reacted with FITC‐conjugated anti‐BrdUrd MAb to determine the percentage of BrdUrd‐labeled cells or with chromomycin A3 for DNA analysis. The single‐cell suspensions were analyzed by flow cytometry. The fraction of S‐phase cells in the tissue sections was similar to both the percentage of BrdUrd‐labeled nuclei and the S‐phase fraction determined by flow cytometric analysis. The results obtained with BrdUrd‐labeled nuclei were similar to those obtained from previous autoradiographic studies of various brain tumors exposed to a pulse of 3H‐thymidine. Since BrdUUrd is not radioactive and is nontoxic at the dosage used, these techniques, together with the histopathological diagnosis, may help to predict the biological malignancy of individual tumors.
Double labeling and in vitro versus in vivo incorporation of bromodeoxyuridine in patients with acute nonlymphocytic leukemia
Authors: Raza Azra , Ucar Kalust , Preisler Harvey D.
Published: August 2005

A monoclonal antibody against bromodeoxyuridine (BrdUrd) was produced, and a rapid slide technique (RPMB technique) was developed for the estimation of S‐phase cells in a population using this antibody. Bone marrow cells from patients with acute nonlymphocytic leukemia (ANIA) were studied by both the RPMB technique and tritiated thymidine (3HdThd) labeling index studies. The percentage of S‐phase cells obtained by each method was compared in 50 samples, and the correlation coefficient was r = 0.89.

A “double label” method is also described in which cells were simultaneously incubated with either BrdUrd and 3HdThd or BrdUrd and tritiated cytosine arabinoside (3HAra‐C). The samples were first processed by the RPMB technique and then by autoradiography. Results showed only black grains overlying the nuclei of fluorescent cells in each group. An automated microphotometer was used to quantitate grains and fluorescence from each cell. This demonstrated an almost direct relationship between grains and fluorescence from BrdUrd + 3HdThd slides, whereas different patterns of relationship were noted from BrdUrd + 3HAra‐C slides of leukemic patients. Their implications are discussed in the text.

Finally, intravenous infusions of BrdUrd was given to five leukemic patients. S‐phase cells were recognized distinctly within 5 min of starting the infusion. The percentage of Sphase cells was almost identical from in vivo and in vitro samples. Various possibilities of studying the biological behavior of acute leukemias and analyzing cell cycle characteristics are discussed.
Numerical evaluation of changes in the cytoplasmic microtubule complex of C3H mouse cells by optical diffractometry and of changes in cell shape by fourier analysis
Authors: Mareel Marc M. , De Bruyne Georges K. , Ostrowski Kazimierz , Rozycka Monika , Strojny Pawel , Dziedzic‐Goclawska Anna , Lenczowski Stanislaw , Grzesik Wojcech , Kieler Jorgen
Published: August 2005

Mo mouse cells in culture on glass were treated with taxol, or nocodazole, or incubated at 4°C to alter their cytoplasmic microtubule complex (CMTC). From each treated group and from an untreated group, 30 cells stained with an antiserum against tubulin, were photographed under the photomicroscope, and negatives were analysed by optical diffractometry. Differences between groups of cells were tested by variance analysis. Phasecontrast micrographs of the same cells were used for Fourier analysis of cell shape. Both types of analyses provided numerical objective data about changes in the CMTC and in cell shape that were typical for the kind of treatment. We conclude that optical diffratometry of immunostained cells and Fourier analysis of cell shape are complementary to photomicroscopy for the study of the CMTC in cell populations cultured on an artificial substrate.
On‐line three‐parameter data uptake, analysis, and display device for flow cytometry and other applications
Authors: Kachel V. , Schneider H.
Published: August 2005

Multiparameter flow cytometric measurements are of growing interest in the study of complex features of biological cells. With state of the art instrumentation, three‐parameter (3‐P) data handling is relatively complicated and time consuming and the display methods are not satisfactory.

As an alternative, an inteactive 3‐P analyzing module, Cytomic 123 is described, which displays 3‐P fields during and immediately after data uptake in the form of a cubic array of 32,768 channels. The fields can be randomly rotated by hardware and software. The event frequencies is the field are primarily visualized by brightness modulation of the display dots. Additionally, the display of the field may be confined to user selected ranges of event frequencies, which may also be superposed to mixed frequency displays. A set of preprogrammed functions is available for the following tasks: (a) uptake of 3‐P histograms combined with on‐line control of the transducer pulses, (b) automatic uptake of a series of 2‐P time correlated histograms in the cube, (c) generation and numerical evaluation of sections and projections of cube histograms, (d) interactive generation and evaluation of spatial subfields for integration, or as sorting matrix by successive erosion of section planes, or reprojection of projection windows, and (e) isometric display of sections and projections and exchange of data sets with other Cytomic modules or other data systems, especially the Cytomic 12 module, whose 2‐P capabilities can be used.

The module is built with low cost Z80 microprocessor eurocards. A standard oscilloscope serves as a display unit.
Flow cytometric analysis of membrane permeability properties influencing intracellular accumulation and efflux of fluorescein
Authors: Prosperi E. , Croce A. C. , Bottiroli G. , Supino R.
Published: August 2005

A flow cytometric investigation has been made on the membrane permeability properties that mediate intracellular turnover of fluorogenic substrates. The accumulation and efflux of fluorescein, consequent to the enzymatic turnover of fluorescein diacetate, were assessed in the presence of metabolic inhibitors and after treatment with membrane‐active compounds.

The metabolic poisons KCN and rotenone greatly inhibited only the fluorescein efflux, reducing the rate constant to as little as one‐tenth in relation to control cells; in the presence of glucose such inhibition was partially removed.

Glucose availability also affected fluorescein efflux: an increase of the rate constant was observed in cells treated with 20 mM glucose, and a decrease was measured in cells incubated for 1 hr in glucose‐free buffer.

Membrane‐active compounds Triton X‐100 and hydrocortisone reduced fluorescein accumulation. Hydrocortisone strongly blocked also the efflux; the addition of glucose did not restore the rate significantly.

The major evidence of these results is that fluorescein efflux is dependent on membrane integrity and on availability of metabolic energy. Fluorescein accumulation is only partially related to permeability properties regulating FDA uptake, due to the influence that treatments exhibit at the same time on FDA hydrolysis and/or fluorescein release.
Realtime acquisition, storage, and display of correlated three‐parameter flow cytometric data
Authors: Stewart Stephen S. , Price Gerald B.
Published: August 2005

A microprocessor‐based system which performs realtime correlated acquisition, storage and display of multiparameter (3‐parameter) data from a flow cytometer (FACS‐III) is presented. List‐mode techniques are not employed.

The 3‐parameter data is collected and correlated, then displayed along with cell‐frequency as a realtime 3‐parameter colour scattergram, while the experiment is in progress; in addition, correlated and uncorrelated higher‐resolution projectons of the 3‐parameter data are collected and stored. The data projections may also be displayed: as 1‐parameter histograms, or as 2‐parameter colour or grey‐scale scattergrams. Examples of 2‐and 3‐parameter colour scattergrams are presented.

The speed and some characteristics of the realtime acquisition and display software are examined; methods to increase the realtime speed are discussed.
Fluorescence spectra of cells stained with a DNA‐specific dye, measured by flow cytometry
Authors: Steen Harald B. , Stokke Trond
Published: August 2005

Fluorescence spectra of ethanol‐fixed rat thymocytes stained with the DNA‐specific dye Hoechst 33258 have been measured in an arc lamp‐based flow cytometer including a grating monochromator in front of the fluorescence detector. Spectral resolution was 5–10 nm. Increasing dye concentration was found to yield an increasing shift of the fluorescence spectrum toward longer wavelengths, thus supporting previous work on soluble DNA that indicated several different binding modes of this dye. The results show that similar data may be obtained for all commonly used DNA‐specific dyes. It appears that this type of spectral information may be used to probe the structure of cell chromatin.
An application of mathematical morphology to analysis of the size and shape of nuclei in tissue sections of non‐Hodgkin's lymphoma
Authors: Lesty C. , Raphael M. , Nonnenmacher L. , Leblond‐Missenard V. , Delcourt A. , Homond A. , Binet J. L.
Published: August 2005

Using principles from the theory of mathematical morphology, a semiautomatic analysis of the size and shape of cell nuclei on tissue sections was carried out on a Leitz Texture Analysis System (Leitz‐TAS). The four parameters proposed here are more discriminatory than conventional shape evaluation by the form factor (FF), which is based on the ratio of perimeter squared to area. The parameters quantified, respectively, nuclear elongation (ND), narrow (R1) and wide (R2) irregularities, and the distribution of R1 and R2 along the nuclear contour (ID). The properties of these parameters were tested nucleus‐by‐nucleus on 24 nuclear models.

The methodology was then illustrated by a study of lymph node nuclei in non‐Hodgkin's lymphoma (NHL). Prior to analysis, 45 lymphomas were classified into five categories of nuclear size and shape according to the International Working Formulation (IWF). Two hundred nuclei were measured on each lymph node section. Statistical interpretation was based upon an analysis of the nuclear surface area on sections and upon the mean values of R1, R2, and ND, the standard deviations of R1 and R2, and the percentage of cleaved nuclei detected by ID. The mean value of R2 discriminated best between the two sets of populations with regular and irregular nuclear contours, respectively. Parameters R1, ND, and ID permitted the distinction of certain NHL cases among populations with irregular nuclei. Nuclear invaginations decreased in depth as the nuclear area became greater. The median surface area was well correlated to the IWF, and the skewness coefficient (third statistical moment of the nuclear surface area distribution) was related to the number of nuclear size or shape sub‐populations.
Flow cytometry analysis of recombinant Saccharomyces cerevisiae populations
Authors: Srienc Friedrich , Campbell Judith L. , Bailey James E.
Published: August 2005

A new fluorescent stain has been developed for detecting cloned β‐galactosidase activity in individual cells of Saccharomyces cerevisiae by flow cytometry. The staining reaction is based on enzymatic cleavage of α‐naphthol‐β‐D‐galactopyranoside by intracellular β‐galactosidase and trapping of the liberated naphthol by hexazoniumpararosaniline yielding a fluorescent, insoluble end product. This stain, in connection with an appropriate host strain, has been applied for detecting plasmids encoding inducible β‐galactosidase in unstable recombinant cell populations carrying plasmids with different origins of replication. The method enables rapid determination of the fraction of plasmid‐containing cells as well as quantitation of intracellular β‐galactosidase content by kinetic enzyme assay. Inducibility of the marker enzyme is important for maintaining correlation between enzyme and gene content.
Flow cytometric DNA and cytogenetic studies in human tumors: A comparison and discussion of the differences in modal values obtained by the two methods
Authors: Tribukait B. , Granberg‐öhman I. , Wijkström H.
Published: August 2005

The numerical chromosome values in 53 human tumors were determined and compared with the modal DNA values as measured by flow cytometry. In tumors with chromosome counts in the diploid and tetraploid range, the modal DNA values were found to correspond to the modal values based on the chromosome counts. In tumors with chromosome counts in the triploid range, however, the modal DNA values were about 15% higher than expected.

In order to explain this difference, the ratio between large and small chromosomes in the karyotyped metaphases was assessed. In addition, the DNA content of individual chromosomes, including markers and minutes, was calculated as a reflection of the DNA content of the whole cell. The ratio of large to small chromosomes did not deviate from the normal ratio found in cells with diploid, triploid, and tetraploid chromosome counts. Neither difficulties in karyotyping nor shortcomings in the flow cytometric methodology could be used to explain the discrepancy between the expected and empirical modal DNA values. Some of the chromosomes in triploid tumors may, therefore, contain an increased amount of DNA.
Count‐Dependent filter for smoothing bivariate FCM histograms
Authors: Schuette W. H. , Shackney S. E. , Marti G. E.
Published: August 2005

A data‐smoothing filter has been developed that permits the improvement in accuracy of individual elements of a bivariate flow cytometry (FCM) histogram by making use of data from adjacent elements, a knowledge of the two‐dimensional measurement system point spread function (PSF), and the local count density.

For FCM data, the PSF is assumed to be a set of two‐dimensional Gaussian functions with a constant coefficient of variation for each axis. A set of space variant smoothing kernels are developed from the basic PSF by adjusting the orthogonal standard deviations of each Gaussian smoothing kernel according to the local count density. This adjustment in kernel size matches the degree of smoothing to the local reliability of the data. When the count density is high, a small kernel is sufficient. When the density is low, however, a broader kernel should be used. The local count density is taken from a region defined by the measurement PSF.

The smoothing algorithm permits the reduction in statistical fluctuations present in bivariate FCM histograms due to the low count densities often encountered in some elements. This reduction in high‐frequency spatial noise aids in the visual interpretation of the data. Additionally, by making more efficient use of smaller samples, systematic errors due to system drift may be minimized.
Mitochondrial uptake of rhodamine 123 by rabbit articular chondrocytes
Authors: Benel L. , Ronot X. , Kornprobst M. , Adolphe M. , Mounolou J. C.
Published: August 2005

Rhodamine 123 was used to stain and analyze by flow cytometry the mitochondria of rabbit articular chondrocytes. Stationary primary cultures and exponentially growing subcultures were compared to enzymatically released chondrocytes from cartilage. The increase in mitochondrial fluorescence, when chondrocytes are transferred from cartilage to culture environment, is suggestive of some change in chondrocyte adaptation and/or differentiation in these conditions.
Construction and analysis of an EMBL‐3 phage library containing partially digested human chromosome 21‐specific DNA inserts (15–20 kb)
Authors: Janssen J. W. G. , Collard J. G. , Tulp A. , Cox D. , Millington‐Ward A. , Pearson P.
Published: August 2005

In the mouse‐human hybrid cell line SCC 16–5, chromosome 21 is the only human chromosome present. Fractions highly enriched for this chromosome were obtained by applying the chromosome velocity sedimentation technique to this cell line. DNA prepared from these chromosomal fractions was partially digested with Mbo I, size fractionated on an NaCl gradient, and cloned in the EMBL‐3 phage vector.

The phage library thus prepared was highly enriched for human chromosome 21‐specific recombinant DNA sequences 15–20 kb long. Of the approximately 21,000 phage clones obtained, at least 99% were recombinant. Following phage plaque filter hybridization and Southern blotting, it was found that half of the recombinants were positive for human repetitive DNA. Almost all phages harbored highly or middle repetitive human or mouse DNA sequences owing to the large size of the recombinant inserts. In this library, the human chromosome 21 is represented approximately four times. All human recombinants studied thus far contained DNA inserts originating from chromosome 21 only. The employed cloning strategy is discussed with regard to utility, purity, quality, and completeness of chromosome‐specific recombinant DNA libraries.
Flow karyotyping of human melanoma cell lines
Authors: Arkesteijn Ger J. A. , Van Dierendonck Jan‐Hein , Vossepoel Albert M. , Cornelisse Cornelis J.
Published: August 2005

Single‐laser flow cytometry has been used to study the feasibility of flow karyotyping of human solid tumors. As a model, seven human melanoma cell lines have been used with varying numerical chromosome composition as verified by FCM DNA content measurements and chromosome countings. For all seven cell lines, flow karyotypes that showed a variety of consistent deviations from the normal diploid flow karyotype could be obtained although the resolution of the flow system and varying debris continuum limited the number of resolvable peaks. The predominant changes observed involved the regions normally representing chromosomes 3–8, 9–12, and 13–16. It is concluded that at present the preparation procedure is the main limiting factor for exploring the full potential of flow karyotyping for cytogenetic analysis of solid‐tumor cell lines.
Optimization of flow‐cytometric discrimination between reticulocytes and erythrocytes
Authors: Schmitz Franz J. , Werner Eckhard
Published: August 2005

An automatized technique to count reticulocytes by means of flow cytometry is described. Blood samples were stained by the fluorescent dye acridine orange without the use of fixative. Scatter and red fluorescence of the blood cells were measured in a flow cytometer. A discrimination between reticulocytes and erythrocytes was only achieved by using logarithmic amplification. The discrimination was better in peak mode than in area mode. The optimum dye concentration was 0.5 mg/liter acridine orange. At lower dye concentrations, not all reticulocytes were measured, whereas at higher dye concentrations the degree of discrimination between reticulocytes and erythrocytes decreased. There was a suitable discrimination between reticulocytes and erythrocytes. The reticulocyte numbers were scored by flow cytometry as well as by microscope for blood samples with 0.1–14% reticulocytes. The correlation between both methods was close.
Simultaneous separate detection of low angle and large angle light scattering in an arc lamp‐based flow cytometer
Authors: Steen Harald B.
Published: August 2005

A device is described for simultaneous separate detection of the light scattering of cells at low and large scattering angles in an arc lamp‐based flow cytometer with epi‐illumination through an oil immersion microscope objective. Light scattering was measured in a dark field configuration that allows separate detection of light scattering greater than 2° and 15°, respectively. Dual parameter light scattering histograms of a blood cell suspension containing various types of leukocytes were closely similar to that obtained with a commercial laser‐based instrument with light scattering detection at forward and right angles. The sensitivity of the device was sufficient to measure polystyrene particles with 0.25‐μm diameter. A potential application may be differentiation of bacteria.
The induction of the human T‐cell growth factor receptor precedes the production of RNA and occurs in the presence of inhibitors of RNA synthesis
Authors: Redelman Doug , Wormsley Susan
Published: August 2005

The receptor for T‐cell growth factor (TCGF) is an activation antigen that is present in low amounts on a small fraction of resting T lymphocytes. The TCGF receptor on human T cells can be detected with the anti‐Tac monoclonal antibody within 7–12 h of stimulating the cells with phytohemagglutinin (PHA). In the current studies, we examined human lymphocytes cultured alone, with PHA, or with PHA plus sufficient actinomycin‐D to inhibit RNA synthesis. After varying intervals, aliquots of the lymphocytes were stained with acridine orange (AO) or pyronin‐Y(PY) to measure RNA and/or with anti‐Tac plus FITC goat anti‐mouse lg. Tac expression began to increase after 6–8 h incubation with PHA, whereas increases in PY or AO staining were not detected until 12 h or later. Furthermore, the initial increase in Tac expression was not affected by sufficient actinomycin‐D to block all detectable nucleic acid synthesis. Therefore, it appears that the initial expression of TCGF receptors detected after lymphocyte activation does not require de novo production of RNA.
Fluorescence‐activated sorting of individual cells onto poly‐L‐lysine‐coated slide areas
Authors: Kanz L. , Bross K. J. , Mielke R. , Löhr G. W. , Fauser A. A.
Published: August 2005

Cells sorted by a fluorescence‐activated cell sorter are collected onto small areas of a glass slide. These collection areas have been coated with poly‐L‐lysine to attach the cells firmly to the glass surface. This simple procedure proved to be suitable to sort single cells and small cell populations with preservation of cytomorphology and viability without modifying the cell sorter. Additional studies on sorted cells may be performed, as shown by preoxidase‐anti‐peroxidase analysis of cellular antigens and by mRNA in situ hybridization.
Improved high‐affinity monoclonal antibody to lododeoxyuridine
Authors: Vanderlaan Martin , Watkins Bruce , Thomas Cynthia , Dolbeare Frank , Stanker Larry
Published: August 2005

Three monoclonal antibodies (Mabs), IU‐1, IU‐4, and B‐44 were evaluated in enzyme immunoassays (ELISA) and by flow cytometry for their abilities to recognize bromodeoxyuridine (BrdUrd)‐ and iododeoxyuridine (IdUrd)‐substituted DNA's, nucleotides, and nucleosides. IU‐4 is a new Mab, derived from mice immunized with 5‐iodo‐2′‐deoxyuridine‐5′‐monophosphate (IdUMP) conjugated through the phosphate group to albumin. This immunogen was selected to resemble IdUMP in DNA. In competition ELISA assays, IU‐4 prefers IdUrd to BrdUrd and prefers halogenated nucleotides over the corresponding nucleosides. In both ELISA and flow analysis, IU‐4 recognizes IdUrd in DNA at substitution frequencies at least as low as one IdUrd one per 1,000 normal bases. The high affinity of IU‐4 for IdUrd‐DNA contrasts with IU‐1 and B‐44, which show a strong binding dependency on the frequency of base substitution and require DNA that is essentially fully substituted with BrdUrd for binding in both flow and ELISA assays. The high affinity of IU‐4 for IdUrd in DNA and its independence of IdUrd residue spacing make it a superior reagent for the quantitative labeling of halogenated thymidine analogues in whole cells.
Spectral bandwidth in automated leukocyte classification
Authors: Zahniser D. J. , Brenner J. F. , Selles W. D.
Published: August 2005

Investigators have repeatedly pointed out the importance of spectral information in the automated classification of white blood cells. In general, monochromatic images recorded through two or three color filters are used to extract this information. Although it has generally been thought that the use of narrow band filters provides “cleaner” color information than is obtainable through wide band filters, the choice has not been fully investigated and the question is far from being settled. The use of wide band filters has the clear practical advantage of increased light levels at the detector, resulting in higher signal‐to‐noise ratio with less demand on light source design.

In order to investigate this issue, a series of 681 leukocytes of the most frequently occurring types were digitized by the use of both narrow (10 nm) and wide (90 nm) band filters. Parameters were extracted independently from both sets of images. These parameters were then used to develop a classifier for each set of images. The choice of features and classifier results indicate that there are no major performance differences between the two types of filters.
DNA microspectrophotometry of bone sarcomas in tissue sections as compared to imprint and flow DNA analysis
Authors: Bauer Henrik C. F. , Kreicbergs Andris , Tribukait Bernhard
Published: August 2005

The aim of the present study was to establish an upper limit of diploidy for microspectrophotometric (MSP) DNA measurements in sections of mesenchymal tissue analyzing DNA data of a large number of normal cell populations. The reliability of this upper limit of diploidy for discriminating between diploid and hyperploid bone sarcomas was tested by analyzing the same tumors by MSP in imprint preparations and flow cytometry (FCM).

The median DNA value of control cells in tissue sections was given arbitrary value of DNA index (DI) 1.0, denoting the diploid DNA content. The proportion of cells with DNA values exceeding DI 1.25 (>DI 1.25) was determined for each normal cell population. The maximum percentage of cells with DNA values exceeding DI 1.25, encountered by analysis of 91 normal cell populations in tissue sections, was 31%. This percentage was set as an upper limit of diploidy. Hence, tumors with a higher percentage of cells >DI 1.25 were classified as hyperploid. When we applied this criterion, 31 of 36 sarcomas analyzed by MSP in tissue sections were hyperploid, which was in complete agreement with FCM and MSP in imprints of the same tumors.

Apart from discriminating between diploid and hyperploid tumors, an attempt was made to determine peak DNA values of sarcomas analyzed in tissue sections. Peak DNA values, as defined by a minimum of 30% of the cells within a class width of DI 0.25, could be determined for 23 of 36 tumors. These peak DNA values correlated well with corresponding peaks obtained by FCM.

In conclusion, reliable discrimination between diploid and hyperploid tumors can be made by MSP in tissue sections provided the wide distribution of DNA values, inherent in the method, is taken into account. In selected cases, peak DNA values may be determined.
Electron microscopy microsampling of isolated nuclei sorted by flow cytometry
Authors: Zini Nicoletta , Matteucci Alessandro , Squarzoni Stefano , Galanzi Angela , Rizzoli Riccardo , Papa Stefano
Published: August 2005

This paper describes an efficient method to concentrate for electron microscopic examination minute quantities of subcellular particles obtained by cytofluorimetric sorting. The advantages of this micromethod, based on diafiltration on Millipore filters under constant positive nitrogen pressure, are discussed.
Design and application of a versatile triple‐laser cell and chromosome sorter
Authors: Lebo Roger V. , Bruce Barry D. , Dazin Paul F. , Payan Donald G.
Published: August 2005

A high‐resolution triple‐laser sorter was designed and constructed to provide flexible switchover and high‐resolution sorting of cells or chromosomes with any combination of one, two, or three lasers. These features provide a central facility instrument that currently serves multiple users and analyzes different stain combinations with minimal switchover effort between experiments. Improved optics and mounts that focus the three laser beams independently are able to resolve beads and chromosomes better than our previously reported dual‐laser sorter. An improved signal collection unit with electronically controlled reference positions can be focused more quickly and precisely for any signal combination. A removable dye laser extends the range of usable fluorochrome labels. A rapid sheath switchover permits sorting of sterile cells and sterile chromosomes sequentially without additional sterilization or reservoir sheath change. Improved dual‐laser chromosome resolution is at least as good, analyzing 8,000 chromosomes/s, compared to the previous dual‐laser bench at 2,000/s. Stimulated and unstimulated peripheral blood lymphocytes were analyzed according to simultaneous measurements of cell surface receptors labeled with a fluoresceinated neuropeptide and a Texas red‐labeled antibody as well as DNA content during the cell cycle. These results demonstrate the broad range of potential applications of this triple‐laser system.