bluuebunny/crossref_metadata_2025 huggingface dataset:
The relative effects of different types of growth factors on DNA replication, mitosis, and cellular enlargement
Authors: Zetterberg Anders , Engström Wilhelm , Dafgård Eva
Published: August 2005

It was recently demonstrated that growth in cell size can be dissociated from DNA synthesis and mitosis. 3T3 cells starved to quiescence in low serum concentration can be stimulated to undergo DNA synthesis and one cell division without growing in size (unbalanced growth) (42–44). We report here that in cells stimulated to undergo unbalanced growth, the cell nucleus undergoes balanced growth, i. e., nearly doubles in size prior to mitosis. The reduced ability to grow in cell size under unbalanced growth conditions is thus mainly ascribable to the cytoplasm. Furthermore, the extent to which cells grow in size prior to mitosis is dependent on the serum concentration in the tissue culture medium (44).

This data suggests that some macromolecular factor or factors in serum are required for growth in cell size prior to mitosis. We report in this study that epidermal growth factor (EGF) alone exerts a small but significant stimulatory influence on DNA synthesis and mitosis but does not affect cellular enlargement. In contrast, insulin added at supraphysiological concentrations does not stimulate quiescent cells to enter S phase but instead stimulates growth in cell size in the small fraction of dividing cells. Furthermore, cells stimulated to proliferate by EGF could be induced to undergo balanced growth when insulin was added concomitantly. Finally, platelet‐derived growth factor (PDGF) stimulates quiescent sparse 3T3 cells to undergo DNA synthesis and mitosis. PDGF also exerts a limited but significant effect on cellular enlargement. However, PDGF alone could not induce a complete balanced growth, i. e., a doubling in cell size prior to mitosis.
The DNA‐based human karyotype
Authors: Mayall Brian H. , Carrano Anthony V. , Moore Dan H. , Ashworth Linda K. , Bennett Deborah E. , Mendelsohn Mortimer L.
Published: August 2005

Image cytometry and computer analysis are used to determine the relative DNA content and the DNA‐based centromeric index of the 24 chromosomes of the human karyotype. A two‐step procedure is used. Chromosomes of cells in metaphase first are stained with quinacrine and identified visually by their fluorescent Q‐band patterns. They then are stained for DNA using gallocyanin‐chrome alum. The chromosome images are scanned and recorded as digital values of optical density by an CYDAC image cytometric microscope system, CYDAC. The digital images are processed by computer to measure for each chromosome the relative DNA stain contents of the whole chromosome and of the p and q arms and the DNA‐based centromeric index. About ten cells are analyzed for each of the donors, who are phenotypically normal men and women. The chromosome measurements are pooled by chromosome type for each donor and are compared among donors. The means of the chromosome measurements give the DNA‐based human karyotype. Analysis of the DNA‐based data shows that some chromosomes or portions of chromosomes vary significantly among donors. These variants do not correlate with detectable morphologic polymorphisms, such as Q‐ or C‐band variants; thus they represent new and otherwise undetectable chromosome polymorphisms whose genetic basis and clinical significance are yet to be determined.
The relationship between thrombomodulin expression and cell cycle stages in cultured rabbit endothelial cells
Authors: Debault Lawrence E. , Esmon Naomi L. , Esmon Charles T.
Published: August 2005

Cultured rabbit endothelial cells have significant but variable amounts of thrombomodulin (TM), both on their surface as well as inside the cell. To determine if variations in TM antigen is cell cycle related, cells with very high levels of TINI antigen were identified and staged according to the intracellular distribution and relative amounts of the antigen, using immunofluorescence techniques. After staging, the nuclear DNA content of each of these cells was determined by measuring the propidium iodide (PI) fluorescence intensity cytophotometrically. Stages 1, 2, and 3, which exhibited TM immunofluorescence in the golgi area, clustered to the G1 phase of the cell cycle. Cells without discernible golgi fluorescence (stages 4 and 5) but with variable amounts of cytoplasmic and surface fluorescence appeared to have little or no relationship to the cell cycle.
Quantitative histochemistry of the angiotensinase A (APA) in the renal glomeruli of rats after stimulation of the renin‐angiotensin system,
Authors: Kugler P. , Schiebler T. H.
Published: August 2005

Quantitative histochemical measurements of aminopeptidase A(APA, angiotensinase A) were clone kinetically in the kidney glomeruli of rats after short‐term experiments (treatment with furosemide as well as captopril for 2, 4, and 6h). The APA activities increased after treatment with furosemide or captopril. Highest activities were determined after 4h using furosemide and 6h using captopril. It is concluded that glomerular APA activities correspond to the renin/angiotensin plasma levels and that the fast changes of APA activities are well demonstrable by kinetic densitometric measurements in situ.
The effects of naturally occurring metabolites (L‐cysteine, vitamin C) on cultured human cells exposed to smoke of tobacco or marijuana cigarettes,
Authors: Leuchtenberger Cecile , Leuchtenberger Rudolf
Published: August 2005

The effects of vitamin C on the growth of human lung cultures and of vitamin C or L‐cysteine on a human breast cancer culture (SK‐Br‐3) were assessed with and without exposure to fresh smoke from tobacco or marijuana cigarettes. When grown in the presence of vitamin C, lung cultures exposed or not exposed to either type of smoke showed a stimulation of growth and a significant decrease in mitotic abnormalities. However, abnormalities were much more marked in marijuana‐exposed cultures than in tobacco‐smoke‐exposed ones. Nonexposed or tobacco‐smoke‐exposed breast cancer cultures, when grown in the presence of vitamin C, also showed acceleration of growth of epithelial cells, significant reduction in mitotic abnormalities, and occurrence of pseudoglandular structures, indicating differentiation. These alterations not only disappeared, but the cultures also became fibroblastic when they were returned to media without vitamin C. In contrast, vitamin C did not reduce mitotic abnormalities in marijuana‐smoke‐exposed breast cancer cultures, but stimulated abnormal growth and dedifferentiation. In nonexposed, tobacco, or marijuana‐smoke‐exposed breast cancer cultures, L‐cysteine evoked an acceleration of fibroblastic growth, which was not altered when the cultures were returned to media without L‐cysteine.
Flow‐Cytometric DNA analysis in primary breast carcinomas and clinicopthological correlations
Authors: Ewers Sven‐Börje , Baldetorp Eva Långström , Killander Dick
Published: August 2005

Flow‐cytometric DNA analyses of single cell nuclei were performed on nuclear suspensions prepared from biopsies of primary breast carcinomas in 638 patients. Propidium‐iodide‐stained cell nuclei were analysed in an Ortho 50‐H Cytofluorograph. The patients were staged by the TMN classification. Sixty percent of all the patients had aneuploid primary breast carcinomas; of size T1, T2, T3, and T4 tumors, 51%, 63%, 67%, and 75% were aneuploid, respectively. The proportions of aneuploid tumors in each of the tumor stages SI, SIl, SII1, and SIV were 47%, 62%, 67%, and 69%, respectively. This trend to increasing aneuploidy proportions with more advanced disease was significant in contrast to the degree of aneuploidy found in relation to axillary nodal tumor involvement. Multiple aneuploid cell populations were found in 109 (17%) tumors.

With a mean follow‐up time of 16 months, 92 patients have relapsed out of 540 completely staged patients with unilateral breast cancer with no distant metastases at the time of initial treatment. When the influence of various treatments and tumor stage are not considered, the recurrence rate was twice as high among patients with aneuploid primary tumors than among patients with euploid tumors. The differences in relapsing rates among patients with euploid and aneuploid primary tumors decreased with more advanced disease. Out of 170 patients with T1 tumors, 17 relapsed and 16 of these were aneuploid. No such difference in relapse rate in relation to ploidy was, however, found in patients with more advanced primary disease.
Cytophotometrical and biochemical characterization of nonpalpable, mammographically detected mammary adenocarcinomas
Authors: Fallenius Anders G. , Skoog Lambert K. , Svane Gunilla E. , Auer Gert U.
Published: August 2005

Fifty nonpalpable, mammographically detected, invasive breast carcinomas were analysed with respect to DNA distribution pattern, steroid receptor content, and histopathological criteria. No significant histomorphological differences were found as compared to palpable breast carcinomas. In contrast, DNA distributions of palpable and nonpalpable tumors differed. Ninety percent of these relatively small breast carcinomas were found to exhibit nuclear DNA amounts within the diploid and tetraploid regions of normal breast epithelium. In earlier findings in palpable breast carcinomas, 55% are of the diploid‐tetraploid type. The mean cellular content of the estrogen receptor was 1. 0 fmole/μg DNA in this group of mammographically detected carcinomas, which is significantly higher than in routinely detected, ie, larger, breast carcinomas. It is suggested that, despite the histomorphological findings, nonpalpable, mammographically detected breast carcinomas are dominated by biologically highly differentiated, slowly proliferating carcinomas with a favourable prognosis.
Morphologic changes in rat urothelial cells during carcinogenesis: II. Image cytometry
Authors: Young Ian T. , Vanderlaan Martin , Kromhout Lois , Jensen Ronald , Grover Abigail , King Eileen
Published: August 2005

Improved early detection of neoplasia by screening of urothelial cells requires an understanding of the features distinguishing normal and neoplastic cell populations. We have begun a program of study based upon a rat model system for the controlled observation of early‐stage lesions produced by the carcinogen N‐butyl‐N‐(4‐hydroxybutyl)‐ nitrosamine. Cells dissociated directly from normal and malignant urothelium were characterized by conventional cytopathology techniques and by quantitative microscopy (for nuclear texture and nuclear and cytoplasmic size, shape, and stain content) to derive a comprehensive picture of bladder tumor development. By following the changes that occur in the dissociated urothelial cells we have found that the nuclear area, total nuclear stain, nuclear shape, and the nuclear chromatin change significantly over a 48‐wk interval as the lesions progress toward malignancy.
Classifying breast carcinomas with DNA measurements and morphometry
Authors: Boon Mathilde E. , Auer Gert U. , van Kaam Han , Schwinghammer Henriette
Published: August 2005

The relation between the DNA distribution pattern and the variables DNA content and nuclear area was investigated in 73 breast carcinomas. According to the DNA patterns, the tumors were subclassified into groups I, II, III, and IV (Auer's classification). The Auer's classes correlate with prognosis. In this investigation it was shown that Auer's classes strongly correlate with the standard deviation of DNA content and mean nuclear area. The carcinomas were subclassified using data of DNA content and nuclear area. For the establishment of this subclassification, multivariate analysis was applied. It was evident that Auer's group III was a hybrid group. Therefore a second classification rule was calculated without the data of Auer's group III. This second classification rule was even more successful in determining the Auer's group of each individual case. Our investigation shows that it is possible to subclassify breast carcinomas objectively using DNA content and nuclear area of the tumor cells.
A new disaggregation device for cytology specimens
Authors: Oud Peter S. , Zahniser David J. , Haag Ditty J. , van Boekel Maria C. G. , Hermkens Huub G. , Herman Chester J. , Vooijs G. Peter
Published: August 2005

A new means of disaggregating cytology specimens in suspension using an immersible rotor device is described. The new rotor is compared to an automated syringing apparatus using cervical samples. Similar results using both devices are obtained for both normal and abnormal specimens.
Application of flow cytometry to studies on the distribution, persistence, and regeneration rate of Epstein‐Barr virus receptors
Authors: McCune Bryan , Volsky David J.
Published: August 2005

Flow cytometry has been applied to study the persistence and regeneration rate of Epstein‐Barr virus (EBV) receptors and other membrane proteins in normal and tumor cells. EBV receptors were detected in a binding assay utilizing fluorescein isothiocyanate‐conjugated virions (FITC‐EBV). After stripping receptors from the cell surface with trypsin, binding of FITC‐EBV was rapidly regenerated in the lymphoma cell line Loukes, reaching 75% of the control levels by 3 h. Fresh human lymphocytes regenerated receptors at a much slower rate, reaching 50% of control levels by 24 h. The regenerating receptors did not reappear simultaneously on all the cells. Subpopulation of new receptor‐positive cells increased gradually during the incubation at 37°C. Reappearance of receptors was inhibited in the absence of protein synthesis. Conversely, receptors persisted in the nontrypsinized cells in the absence of protein synthesis. These results show that certain parameters of membrane receptor turnover, such as the change in receptor distribution within a cell population, receptor persistance, and regeneration rate, can be measured at the single‐cell level by flow cytometry.
Effects of temperature on the yeast cell cycle analyzed by flow cytometry
Authors: Vanoni Marco , Vai Marina , Frascotti Gianni
Published: August 2005

The effects of temperature (in the range 15–36°C) on growth and the nuclear and budding cycle have been studied in populations of the yeast Saccharomyces cerevisiae exponentially growing in batch on yeast nitrogen base (YNB) glucose medium. The maximal rate of exponential growth is achieved at 30°C, and a transition point is apparent at about 20°C. At all tested temperatures DNA replication begins when cells are still unbudded and both the budded period and the postreplicative period have the same temperature dependence. A temperature compensatory mechanism seems to operate in S phase, during which duration remains relatively constant, in the range 21–36°C, while duration of G2+M phases shows a much more pronounced temperature dependence. The results are discussed in terms of a cell‐cycle model for budding yeast.
Flow karyology of serially cultured chinese hamster cell lineages
Authors: Bartholdi Marty F. , Ray F. Andrew , Cram L. Scott , Kraemer Paul M.
Published: August 2005

Flow karyology of serially cultured Chinese hamster cell lineages has been observed to be influenced by the degree of cellular heterogeneity in culture. The minimum coefficient of variation (CV) and debris fraction obtainable vary as a cell lineage evolves from a primary cell culture to an established cell line. The cell lineages pass through a stage of decreased cellular heterogeneity from which flow karyotypes can be obtained with lower CV and debris fraction. The influence of cellular heterogeneity on flow karyology is observed with constant preparative protocol and constant instrument performance, and can be an additional source of variability.
Flow cytometric analysis of immunogold cell surface label
Authors: Böhmer Ralph‐M. , King Nicholas J. C.
Published: August 2005

Goat antimouse immunoglobulin antibodies conjugated to colloidal 40‐nm gold particles were used to label mouse spleen lymphocytes. The labeled cells were analysed with a flow cytometer, equipped with an argonion laser and a (0.5 mW) helium‐neon laser. The right‐angle (90°) light scatter signal of the red (632.8 nm) helium‐neon light was enhanced more than tenfold by the gold label. Dual labeling with gold and fluorescein isothiocyanate (FITC) showed no interference between the two labels. Thus immunogold provides a nonfluorescent cell surface label that can be combined with other cell labels for multiparametrical cell analysis.
A proposal for a flow cytometric data file standard
Authors: Murphy Robert F. , Chused Thomas M.
Published: August 2005

The increasing complexity of multiparameter data collection and analysis in flow cytometry and the development of relatively inexpensive arc‐lamp‐based flow cytometers, which increases the probability that laboratories or institutions may have more than one type of instrument, creates a need for sharable analysis programs and for the transport of flow cytometric data files within an installation or from one institution to another. To address this need, we propose a standard file format to be used for all flow cytometric data. The general principles of this proposal are: (1) The data file will contain a minimum of three segments, TEXT, DATA, and ANALYSIS; (2) The TEXT and ANALYSIS segments consist of KEYWORDS, which are the names of data fields, and their values; (3) All TEXT is encoded in ASCII; (4) KEYWORDS and their values may be of any length; (5) Certain KEYWORDS will be standard, i.e., having specified formats to be recognized by all programs. The structure of the DATA segment will be uniquely defined by the values of KEYWORDS in the TEXT area. It may be in any bit resolution, facilitating compatibility between machines with different word length and/or allowing bit compression of the data. The structured nature of the TEXT area should facilitate management of flow cytometric data using existing data base management systems. The proposed file format has been implemented on VAX, PDP‐11, and HP9920 based flow cytometry data acquisition systems.
Application of a high‐resolution TV‐microscope system to estimate the sequence of centromere separation in muntjak chromosomes
Authors: Gerlach B. , Solleder E. , Haucke M. , Harms H. , Schmid M. , Aus H. M.
Published: August 2005

The sequence of centromere separation in muntjak fibroblasts was studied using a high‐resolution TV‐microscope system. Computer programs that identify the chromosomes as well as measure the centromere separation are described. Important prerequisites for this study are the high sampling rate and the digital processing of the two images from the red and green channel of a color TV‐camera. The study demonstrates that the centromeres do not separate in a random sequence and that the separation sequence is not solely controlled by the amount of constitutive heterochromatin in the chromosomes.
Enumeration of cytoplasmic μ immunoglobulin positive acute lymphoblastic leukemia cells by flow cytometry: Comparison with fluorescence microscopy
Authors: Zipf T. F. , Bryant L. D. , Koskowich G. N. , MacGregor S. E. , Chin L. , Johnson H.
Published: August 2005

The pre‐B phenotype of childhood acute lymphoblastic leukemia, in which the μ immunoglobulin molecule is expressed in the cytoplasm of the blast cells, has been shown to have independent prognostic significance. Patients with this phenotype of the disease tend to have a poorer outcome on contemporary treatment regimens than do those with the other B‐precursor pheonotypes. For these clinical studies, the detection of the pre‐B phenotype has depended upon fluorescent‐microscope based techniques. A flow‐cytometry based technique for the detection of the cytoplasmic μ immunoglobulin molecule is presented here and the results compared in detail with those of fluorescent microscopy. The results show that the two techniques are equivalent. The flow cytometry method has the advantage of a standardized control, is less labor intensive, and is observer‐independent.
Proliferative characteristics of primary and metastatic human solid tumors by DNA flow cytometry
Authors: Frankfurt O. S. , Greco W. R. , Slocum H. K. , Arbuck S. G. , Gamarra M. , Pavelic Z. P. , Rustum Y. M.
Published: August 2005

The percentage of cells in S‐phase (S‐index) was calculated from DNA histograms of 453 primary and metastatic human solid tumors (predominantly bladder, breast, colorectal, renal, prostate, ovarian and lung carcinomas, melanomas, and sarcomas). S‐indices varied widely among both primary and metastatic tumors (1–48%); there was no significant difference in S‐indices between primary and metastatic tumors. The S‐indices for aneuploid tumors were signifycantly higher than for diploid tumors. When data for all aneuploid tumors were analyzed collectively, there was no significant relationship between S‐index and DNA ploidy index. However, for colorectal and ovarian carcinomas S‐indices increased, and for lung carcinomas S‐indices decreased with elevation in the degree of DNA ploidy. Lung carcinomas had the highest S‐indices. Comparison of flow cytometry (FCM) and cytology data indicated that for most diploid tumors S‐indices reflect the proportion of S‐phase cells among a mixed population of normal and tumor cells. For most aneuploid tumors, the proportion of tumor cells estimated cytologically was similar to the proportion of aneuploid cells estimated by FCM. For a small proportion of aneuploid tumors a comparison of cytology and FCM data indicated the presence of a predominant diploid tumor stemline and a minor stemline with aneuploid DNA content. There was a wide spread in the values of S‐indices within tumor groups defined by degree of differentiation and stage of disease at surgery.
An easy‐to‐build‐timer for kinetic measurements in flow cytometry
Authors: Beumer Tom , Lenssinck Hans , Pennings Arie , Haanen Clemens
Published: August 2005

Measuring kinetics of any time‐dependent intracellular process on a cell‐to‐cell basis is a powerful extension of flow cytometry (FCM) possibilities. A digital timer is described that is easy to build and that fits with all commercial or custom made multiparameter equipment. Of all possible applications of this timer (phagocytosis kinetics, staining of cell components, enzyme reactions, etc.), one example is given: enzymatic intracellular precipitation owing to myeloperoxidase in human peripheral blood cells.
Alignment and focusing unit for dual‐laser excitation in the fluorescence‐activated cell sorter
Authors: de Josselin de Jong J. E. , Hartwijk P. , Verkerk T. , Jongkind J. F.
Published: August 2005

The two laser beams in a dual‐laser fluorescence‐activated cell sorter FACS‐II can be aligned and focused independently on the sample stream with an additional unit, which can be fitted easily on the optical bench of the FACS. The unit consists of two spherical lenses, which have been mounted in separate holders and can be moved in three directions by way of micrometer gauges. The lenses, which have different focal lengths, have been cut off on one side so each laser beam only passes one lens. The setup has been tested using the flow analysis of a suspension of double‐stained chicken red blood cells. The histograms of both fluorescence signals showed normal distributions with a coefficient of variation of approximately 6%. After willful interference with the adjustments, the laser beams could be readily readjusted within five minutes.
Automatic cell identification and enrichment in lung cancer: V. Adenocarcinoma and large cell undifferentiated carcinoma
Authors: Tyrer Harry W. , Pressman Norman J. , Albright Craig D. , Frost John K.
Published: August 2005

The aims of this study were to develop a protocol for the identification and enrichment of cancer cells from sputum obtained from patients with adenocarcinoma of the lung (n = 6) and large‐cell undifferentiated carcinoma of the lung (n = 2), and to compare these findings with the results from our previous studies on other cell types from lung cancer. The hypotheses tested were: (1) Cancer cells in sputum can be preserved following flow sorting. (2) Enrichment for cancer cells from acridine orange (AO)‐stained specimens can be achieved. (3) Discrimination of cancer cells from noncancer cells is by AO green fluorescence and discrimination of lymphocytes from other cell types is by AO red fluorescence. (4) Cancer cells are consistently enriched in the AO high green and red fluorescence region, although, for a given cell type, maximal enrichment is patient‐dependent. (5) Finally, cancer cell enrichment and lymphocyte exclusion can be done simultaneously.

Cells from sputum were initially fixed, stained with AO, sorted on a dual parameter flow sorter, and classified into six groups corresponding to two ranges of green and three ranges of red fluorescence intensities. Cells of each region were stained by the method of Papanicolaou and differential counts were performed to determine the relative frequencies (i.e., purities) of leukocytes, macrophages and unsorted (i.e., control) samples.

The average purity of leukocytes (81%), macrophages (6%), squamous cells (11%), and cancer cells (2%) varied markedly from sample to sample. However, the largest enrichment values (i.e., ratio of purity of a cell type in a sorted sample to its purity in the unsorted control sample) achieved for cancer cells consistently occurred for each patient sample in the region corresponding to high green and high red fluorescence intensities. Experimentally, a cancer cell average enrichment of sixteen‐fold was obtained by this method. Additionally, fluorescence intensity ranges which increased the enrichment for macrophages by cell sorting typically excluded leukocytes and squamous cells, and vice versa. Finally, red fluorescence intensity was the primary discriminatory parameter for all cell types studied, although the additional use of green fluorescence intensity significantly increased cancer cell enrichment rates.

In conclusion, sputum samples can be enriched for atypical and malignant cells, cells from these samples can be well preserved following machine processing, and sputum samples from patients with adenocarcinoma of the lung and large‐cell undifferentiated carcinoma have similar enrichment and preservation results to samples obtained from other types of lung cancer.
Cell‐cycle, protein content, and nuclear size in acute myeloid leukemia
Authors: Ffrench M. , Bryon P. A. , Fiere D. , Vu Van H. , Gentilhomme O. , Adeleine P. , Viala J. J.
Published: August 2005

Simultaneous analysis of DNA and cellular proteins provides information on cell proliferation and metabolism. Cellular protein content coupled with nuclear geometric parameters can be used to evaluate cellular maturation and differentiation. In this study, leucoblasts from 50 cases of adult acute myeloid leukemia were analyzed by flow cytometry, and semiautomatic morphometry was performed on bone marrow smears. Ethanol‐fixed bone marrow blast cells were stained for DNA with propidium iodide (PI) and for proteins with fluorescein isothiocyanate (FITC). On the resulting FITC versus PI histograms we defined the cells with low protein content which are associated with a nonproliferating subpopulation (LPC fraction). Low protein content fraction and S‐phase are correlated (p < 0.01). The LPC fraction values are more dispersed than S‐phase values and thus should indicate more clearly eventual differences between cellular populations. This hypothesis has been tested with the prognostic significance of cell‐cycle variables: The LPC fraction was significantly higher in the complete remission group than in the other (p < 0.01), while S‐phase did not show any difference.

The peak value of the protein content histograms is significantly lower in the granulocytic leukemias (M1, M2, M3) than in the leukemias with a monoblastic component (M4, M5). Furthermore, we showed that the differentiation and the maturation of the myeloid blast cells modify the nuclear size. The combination of these two parameters provides useful information for cytological classification.
A single‐parameter analog level discriminator: A cheap and powerful extension of FCM data acquisition
Authors: Beumer Tom , Lenssinck Hans , Pennings Arie , Haanen Clemens
Published: August 2005

Modern flow cytometers offer the possibility to measure more parameters of individual cells than the normally available data acquisition equipment can store. To enlarge the number of parameters that is taken into account while recording data, an analog level discriminator is described that is easy to build and easy to implement on any existing FCM system. An example of gated data acquisition in light scattering measurements on nucleated human bone marrow cells in given.

The level discriminator may give a cheap and yet valuable extension of expensive data acquisition equipment.
Flow cytometry of leukocytes after intravenous fluorescein angiography
Authors: Felberg Norman T. , Haimowitz Anna J.
Published: August 2005

Blood specimens were obtained for T lymphocyte analysis from patients undergoing intravenous fluorescein angiography (IVFA). Mononuclear cells were prepared by differential density centrifugation and analyzed by flow cytometry (FCM). Prior to IVFA, there was very little autofluorescence in the mononuclear cell population. After IVFA, there was an increase in the fluorescence of the mononuclear cells. Though barely visible by fluorescence microscopy, the fluorescence was uniformly distributed throughout the cytoplasm. By FCM, fluorescein was detected in 27% of the lymphocytes, 61% of the monocytes, and 75% of the granulocytes.

Care must be taken when interpreting flow‐cytometric lymphocyte subset data because of the increased nonspecific fluorescence of mononuclear cells in patients who have undergone IVFA.
A comparison of different focus functions for use in autofocus algorithms
Authors: Groen Frans C. A. , Young Ian T. , Ligthart Guido
Published: August 2005

A number of functions for the autofocusing of microscopes and other optical instruments are to be found in the literature. In this article we compare 11 of them to determine, in an objective manner, which functions are most suitable for implementation with real‐time video acquisition systems. Three different images, each representing a typical class of image, are used in the comparison. Among the best focus functions found in our study are the squared magnitude gradient, the squared Laplacian, and the normalized image standard deviation.
Preparation and bivariate analysis of suspensions of human chromosomes
Authors: van den Engh G. J. , Trask B. J. , Gray J. W. , Langlois R. G. , Yu L.‐C.
Published: August 2005

Chromosomes were isolated from a variety of human cell types using a HEPES‐buffered hypotonic solution (pH 8.0) containing KCl, MgSO4, dithioerythritol, and RNase. The chromosomes isolated by this procedure could be stained with a variety of fluorescent stains including propidium iodide, chromomycin A3, and Hoechst 33258. Addition of sodium citrate to the stained chromosomes was found to improve the total fluorescence resolution. High‐quality bivariate Hoechst vs. chromomycin fluorescence distributions were obtained for chromosomes isolated from a human fibroblast cell strain, a human colon carcinoma cell line, and human peripheral blood lymphocyte cultures. Good flow karyotypes were also obtained from primary amniotic cell cultures. The Hoechst vs. chromomycin flow karyotypes of a given cell line, made at different times and at dye concentrations varying over fourfold ranges, show little variation in the relative peak positions of the chromosomes. The size of the DNA in chromosomes isolated using this procedure ranges from 20 to over 50 kilobases. The described isolation procedure is simple, it yields high‐quality flow karyotypes, and it can be used to prepare chromosomes from clinical samples.
Flow cytometric immunofluorescence of rat anterior pituitary cells
Authors: Michael Hatfield J. , Hymer W. C.
Published: August 2005

We have developed a flow cytometric immunofluorescence technique for the quantification of growth hormone (GH), prolactin (PRL), and luteinizing hormone (LH) producing cells. The procedure requires about 24 hours and can objectively count 50,000 cells in about 3 minutes. It is based on indirect‐immunofluorescence (fluorescein) of intracellular hormone using an EPICS V cell sorter. The fluorescein distributions are gated on DNA content (propidium iodide) to eliminate counting cell clumps. Cells from the same suspensions were stained immunocytochemically and counted microscopically (1,000–2,000 cells/sample). Immunofluorescence and immunocytochemistry correlated to within a few percent for GH and PRL cells. Cell suspensions from adult males and females with or without castration and a diethylstilbestrol (DES)‐induced primary pituitary tumor were used to test the method. A major finding of this study was the objective identification of two populations of PRL producing cells, i.e., lightly and intensely stained cells. On the other hand, the fluorescence distribution of PRL cells from DES‐induced pituitary tumors did not fall into two distinct populations but, rather, represented a broad continuum. This method should prove useful in studying the dynamics of pituitary cell populations under various physiological and pharmacological conditions.
Gene mapping by chromosome spot hybridization
Authors: Collard J. G. , de Boer P. A. J. , Janssen J. W. G. , Schijven J. F. , de Jong B.
Published: August 2005

A method is described for the localization of cloned single‐copy genes to flow‐sorted chromosomes. Chromosomes were sorted directly onto nitrocellulose filters and the chromosomal DNA was subsequently hybridized with gene‐specific radioactively labeled DNA probes. Mild aspiration of the filters during sorting was applied to collect the deflected chromosomes in a small spot. Sorting of 10,000–30,000 chromosomes was sufficient to detect gene‐specific hybridization with single‐copy DNA probes. Using this technique, we have sublocalized the human c‐myb oncogene to 6q21–q23 by sorting translocated chromosomes with breakpoints in the q21 and q23 region of chromosome 6. Chromosome spot hybridization appears to be a rapid and simple method to assign cloned genes to chromosomes. Hybridization of an unlocalized gene probe to spots of chromosomes pre‐enriched by velocity sedimentation can quickly narrow the choice of chromosomes which need to be sorted. Conversely, individual chromosomes in a flow karyotype can be identified by hybridizing sorted chromosomal DNA with chromosome‐specific DNA probes.
Detection of Escherichia coli in blood using flow cytometry
Authors: Mansour James D. , Robson Jillian A. , Arndt Carolyn W. , Schulte Thomas H.
Published: August 2005

A rapid method for the detection of Escherichia coli in blood has been developed. The method employs blood cell lysis, staining of bacteria with ethidium bromide, and detection of stained bacteria using flow cytometry. The detection protocol requires less than 2 h sample handling time and is not dependent on bacterial growth. This method has been applied to human donor blood specimens seeded with various E. coli concentrations and to two rabbit model systems. Bacterial detection is evident from the in vitro human blood studies at levels of 10 E. coli/ml and from in vivo rabbit model studies at < 100 E. coli/ml.
The G0 ⇆ G1 transitions of human lymphocytes as monitored by quantitative 14C‐uridine autoradiography and high‐resolution image analysis
Authors: Giaretti Walter , Abmayr Wolfgang , Dörmer Peter , Santi Leonardo
Published: August 2005

Human lymphocytes, transferred in culture into medium conditioned with phytohemaglutinin, were studied with the combined use of quantitative 14C‐uridine autoradiography and high‐resolution image analysis. During the first 24 h before the onset of DNA synthesis it was possible to study pure G0 and G1 populations. The return of the proliferating cells into the G0 state was clearly monitored in an RNA synthesis rate/DNA content plot during 1‐week growth. On days 3–6, the proliferation decreased progressively, and more cells accumulated in the quiescent G0 state. Geometric, densitometric, and textural parameters evaluated from the Feulgenstained nuclei of the same cells indicated that the decondensation/condensation processes of the nuclear chromatin during the G0 °CG1 transitions paralleled the RNA synthesis activity. The results help to associate descriptors of nuclear morphology, as derived from image analysis, with functional parameters during activation and return to quiescence of human lymphocytes.
A method to estimate the DNA content of whole nuclei from measurements made on thin tissue sections
Authors: Bins M. , Takens F.
Published: August 2005

A microdensitometry method that allows estimation of the distribution of the DNA content of nuclei in thin tissue sections is described. The method is based on a theoretical model of Feulgen‐stained spherical nuclei, of different sizes, in each of which the DNA is present as a homogeneous solution. In thin sections of nuclei of different sizes, the fraction of DNA per section is inversely proportional to the radius of the nucleus. Histograms of the product of DNA content and radius per nuclear section are independent of nuclear size but depend on total DNA content. The distribution of the total DNA content of nuclei in a section can be estimated from such a histogram. The results of the measurements of a Feulgen‐stained rat liver section are described.
Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea
Authors: Evenson Donald P. , Higgins Paul J. , Grueneberg Dorre , Ballachey Brenda E.
Published: August 2005

The effects of the mutagenic agent ethylnitrosourea (ENU) on spermatogenic function and sperm chromatin structure were studied by flow cytometry and the results compared with sperm head morphology measurements. Groups of mice received daily exposures ranging from 0 to 75 mg/kg body weight °C5 days and were sacrificed 28 days later. Fresh testicular cell suspensions and epididymal sperm were stained with acridine orange (AO) and measured by flow cytometry. Sperm nuclei were isolated, fixed, rehydrated, and then either subjected to thermal stress or not prior to staining with AO. Body weights were unaffected by the chemical exposure while the testicular weights were reduced by about 50%. Two‐parameter (DNA, RNA) flow cytometry measurements showed a dose‐response relationship in the loss of certain cell types, particularly the elongated spermatids, from the testes of treated animals. Flow cytometric analysis of both heat‐stressed and non‐heat‐stressed nuclei showed a relationship between dosage and the coefficient of variation of α t {red/(red + green fluorescence)} measurements of AO stained nuclei, thereby demonstrating that alterations of chromatin structure occurred in response to ENU. Enzymatic digestions with RNAse, DNAse, and nuclease S1 suggest that the increase in red fluorescence is due to an increase of single‐stranded DNA induced by heat or acid treatment of chemically altered chromatin structure. The lowest daily dosage used (5 mg/kg) caused no significant changes in ratios of testicular cell types, a questionable increase in abnormal sperm head morphology and a detectable change in chromatin structure expressed as α t. This report shows that our technique for assaying sperm nuclear chromatin structure appears to have the same level of sensitivity to ENU induced nuclear alterations as the sperm head morphology test.
The use of flow cytometry and cell sorting in a human colon carcinoma model
Authors: Noguchi Philip D. , Cunningham Robert E. , Ridge Jeanette , Muller Jacqueline
Published: August 2005

We investigated the growth characteristics of a human colon carcinoma cell line, WiDr, grown in culture flasks and on chick embryonic skin (CES). WiDr cells labeled in vitro with bromodeoxyuridine (BrdU) and analyzed by combined propidium iodide/Hoechst 33258 fluorescence showed evidence of more BrdU incorporation in early S phase as compared to late S phase. When inoculated on the CES, WiDr cells multiplied and invaded the underlying skin. Morphologic examination showed that with extended culture WiDr cells on the CES undergo progressive structural organization with the development of acini and basal lamina, structures similar to those in in vivo tumors. WiDr cells were labeled with monoclonal antibody to carcinoembryonic antigen (CEA) and the brightest 2% of the population was sorted. When subsequently grown on the CES, the sorted cells formed significantly more acinar structures at 3 and 6 days of culture than an unsorted population grown for a comparable time.
Flow cytometric analysis of fluorescein‐conjugated estradiol (E‐BSA‐FITC) binding in breast cancer suspensions
Authors: Benz Chris , Wiznitzer Israel , Lee Sin Hang
Published: August 2005

With greater utilization of histochemical methods for detecting estrogen binding (ER) in tumor cells, there is an increasing need to quantitate objectively these fluorescently stained cells. This study utilizes flow cytometry (FCM) to examine the binding specificity and kinetics of 17β‐estradiol‐6‐CMO‐BSA‐FITC (E‐BSA‐FITC) in two human mammary carcinoma cell lines, MCF‐7 and 47‐DN. Cells are rendered permeable to this ligand by freeze‐thawing, a process analogous to the routine staining of frozen tumor sections with E‐BSA‐FITC for the clinical detection of ER. FCM quantification of E‐BSA‐FITC binding intensity demonstrates a saturable dose‐reponse that is specifically reduced in the presence of diethylstilbestrol (DES) in doses known to saturate Type I ER. Scatchard analysis suggests that E‐BSA‐FITC binding occurs with receptors of varying affinities (Kd). Lineweaver‐Burk plots show that the DES inhibition is competitive for a high‐affinity receptor binding to E‐BSA‐FITC with a Kd of approximately 50 nM.

This report also compares both FCM and biochemical methods of quantitating ER under two conditions of tumor cell growth potentially encountered in clinical specimens: quiescent versus actively proliferating cells, and cells pretreated with the antiestrogen tamoxifen. By FCM analysis, the cells with greater proliferating activity contain tenfold more specifically bound E‐BSA‐FITC, and tamoxifen pretreatment reduces this specific binding by 50%. These FCM measurements correlate well with biochemical results and suggest that this new methodology may supplement the detection of E‐BSA‐FITC binding by fluorescence microscopy.
Dual laser flow cytometry: Focal length compensation when focussing through a single lens
Authors: Fellner‐Feldegg Hugo
Published: August 2005

Dual laser operation of flow cytometers, using a single focussing lens for both beams, requires compensating for chromatic aberration of the lens. By using a prefocussing lens at a fixed position in one of the laser beams, complete focal length compensation is obtained without any loss in system performance.
The LLNL high‐speed sorter: Design features, operational characteristics, and biological utility
Authors: Peters D. , Branscomb E. , Dean P. , Merrill T. , Pinkel D. , Van Dilla M. , Gray J. W.
Published: August 2005

This report describes a dual‐beam, high‐speed sorter (HiSS) with a droplet production rate of 220,000 s−1 now in routine use at the Lawrence Livermore National Laboratory. The system can process and sort objects at rates in excess of 20,000 s−1. We report here on the development of HiSS, describe its operational characteristics, and evaluate its utility for analysis and purification of human chromosomes, human erythrocytes, and living Chinese hamster ovary cells.
Flow cytometric fluorescence emission spectrum analysis of hoechst‐33342‐stained DNA in chicken thymocytes
Authors: Watson James V. , Nakeff Alexander , Chambers Stephen H. , Smith Paul J.
Published: August 2005

Hoechst‐33342‐stained chicken thymocytes were analysed simultaneously on two fluorescence wavelength bands (green and violet) in our custom‐built flow cytometer, and two major subsets were identified. In one subset (33% of the total) the emission spectrum remained constant with time, with little change in the respective green and violet fluorescence intensities. In the other subset (42% of the total) the green fluorescence increased during staining, resulting in a considerable change in the green‐to‐violet ratio, due to a change in the “shape” of the fluorescence emission with time. The data indicate that two binding sites, or two types of binding at the same site, exist in DNA for this dye and that these have different binding energies and, consequently, different fluorescence emission properties.
DNA analysis and sorting of rat testis cells using two‐parameter flow cytometry
Authors: van Kroonenburgh Marinus J. , Beck Johannes L. , Scholtz Johannes W. , Hacker‐Klom Ursula , Herman Chester J.
Published: August 2005

By use of two‐parameter flow cytometry of rat testis cell suspensions stained with mithramycin for DNA (the peak amplitude of the fluorescence signal versus total fluorescence intensity integrated over time), eight cell compartments could be distinguished without pre‐enrichment of the samples. Cells in these compartments were identified by sorting and subsequent microscopic examination.
DNA distribution in non‐small‐cell lung carcinomas and its relationship to clinical behavior
Authors: Volm Manfred , Mattern Jürgen , Sonka Jaroslav , Vogt‐Schaden Marlies , Wayss Klaus
Published: August 2005

A study of 187 surgical specimens of tumors of patients with non‐small‐cell lung carcinomas was carried out by means of flow cytometry. Eighty‐four percent of the tumors were classified as tumors with abnormal DNA stemlines (DNA aneuploidy). Patients with tumors demonstrating DNA aneuploidy had significantly shorter survival times than those with tumors demonstrating DNA diploidy (p = .009).

Cell cycle analysis was possible in 122 tumors. Patients whose tumors had 0–8% S‐phase cells died later than patients whose tumors had 9–16% S‐phase cells (p = .018). In addition, patients with tumors with a low fraction of labeled S‐phase cells (autoradiography) had a better prognosis than patients with tumors with a high proportion of labeled S‐phase cells (p = .041).
Short‐term cryopreservtion of human breast carcinoma cells for flow cytometry
Authors: Stone Kenneth R. , Bruce Craig R. , Palmer James O. , Rivkin Saul E. , McDivitt Robert W.
Published: August 2005

A procedure is described for short‐term cryopreservation of primary human tumor cells and tissue slices for later analysis by flow cytometry. Cells were mechanically dispersed into a freezing medium, which was then frozen at either –20°C or –70°C for delayed cell cycle analysis. The results show that a correlation coefficient of greater than 0.95 exists between cell cycle kinetic analyses performed immediately after surgical excision of the tumor and on cells frozen from 1 to 30 days at –70°C in this freezing solution. Somewhat lower levels of correlation exist for cells frozen at –20°C in this freezing medium. This procedure has also been successfully used to preserve freshly isolated breast carcinoma cells shipped from distant laboratories for analysis in the flow cytometer, thus expanding the data base on certain types of breast carcinoma.