bluuebunny/crossref_metadata_2025 huggingface dataset:
Flow cytometric analysis of megakaryocyte differentiation
Authors: Worthington Ronald E. , Nakeff Alexander , Micko Steven
Published: August 2005

Megakaryocytes were isolated quantitatively from rat bone marrow by centrifugal elutriation (CE). CE‐enriched megakaryocytes were stained supravitally for either (1) DNA content with Hoechst 33342, (2) surface membrane immunofluorescence with fluorescein isothiocyanate (FITC)‐conjugated antiplatelet antibody, or (3) both. The cells were then measured using a Becton Dickinson FACS IV flow cytometer. The following correlations were analyzed: (1) DNA content and light scatter, (2) light scatter and antiplatelet immunofluorescence, and (3) DNA content and antiplatelet immunofluorescence. Although the range of light scatter increased as a function of DNA content, discrete subpopulations of megakaryocytes with different light scatter properties were detected within each of the three principal ploidy classes (8C, 16C, and 32C). Other discrete megakaryocyte sub‐populations were revealed in the analysis of antiplatelet surface immunofluorescence as a function of degree of light scatter. The nonlinear relationship between the latter suggested that the degree of membrane immunofluorescence did not bear a simple relationship to cell size as reflected in light scatter. Megakaryocyte DNA content, on the other hand, varied in a linear fashion with membrane immunofluorescence, supporting the conclusion that there may be a proportional increase in the expression of platelet antigens with DNA content. The use of multiple markers, correlated multiparameter flow cytometry and multivectorial analysis to define differentiation on a single cell basis have revealed new complexities in this process. Flow cytometric analysis holds promise as a useful method for further characterization of megakaryocyte differentiation.
A new model for the quantitative analysis of cell movements in vitro: Definition of a shape change factor
Authors: Verschueren Hendrik , Van Larebeke Nicolas
Published: August 2005

Changes in shape, in addition to translocations, are an important aspect of cell motility. We propose a simple geometrical model for the quantitative analysis of shape changes undergone by cultured cells. The extent to which images of a given cell do not overlap at the beginning and end of a time interval is used as a measure of motility, and a translation step included to eliminate translocation effects. Initial findings suggest that the method is widely applicable.
Counterstaining human chromosomes for flow karyology
Authors: Meyne Julianne , Bartholdi Marty F. , Travis Gayle , Cram L. Scott
Published: August 2005

Isolated human metaphase chromosomes stained with the fluorochromes 4′‐6‐diamidino‐2‐phenylindole (DAPI) and chromomycin A3 (CA3), and counterstained with nonfluorescent netropsin (NTR), have been analyzed by dual‐laser flow cytometry. Counterstaining with NTR reduces DAPI fluroscence except at regions on chromosomes 1,9,15,26, and Y, corresponding to C‐band heterochromatin. Bivariate flow karyology of human chromosomes treated with this triple‐stain combination resolves chromosomes 1,9, and Y distinctly from the remaining chromosomes and resolves variations between chrosome homologues not detected by staining with propidium iodide (PI) or with the double stain combination Hoechst 33258 (HO) and CA3.
Use of somatic cell hybrids for quantitation of mutagenesis: Reduction in background mutants by fluorescence‐activated cell sorting (FACS)
Authors: Waldren Charles , Martin John , Sutherland Jeffrey , Cram Scott
Published: August 2005

Environmentally induced mutations, especially those involving large scale genetic damage such as deletions and chromosome loss, are of central importance in the production of human genetic disease and cancer. We have developed a methodology, the AL assay, that permits detection of such extensive genetic changes which often escape detection in other systems in which they are lethal. The AL assay employs a human‐Chinese hamster ovary cell hybrid that retains a single human chromosome, number 11. A set of specific cell surface antigens result from genes located on opposite arms of this chromosome. Exposure to mutagens produces mutants which form colonies in the presence of complement and specific antiserum that kill nonmutant cells. The frequency and pattern of marker loss provides a measure of single gene mutation, large and small deletion, and loss of the entire chromosome 11. We have employed the indirect fluorescein conjugated isothiocyanate (FITC) technique and fluorescence‐activated cell sorting (FACS) to remove spontaneous mutants from the initial population. The 100‐fold reduction in background thus far achieved should allow accurate analysis of mutation by ionizing radiation at doses of less than 10 rad.
Flow cytometric analysis of blood cells stained with the cyanine dye DiOC1[3]: Reticulocyte quantification
Authors: Jacobberger James W. , Horan Paul K. , Hare J. Donald
Published: August 2005

The fluorescent dye 3,3′‐dimethyloxacarbocyanine (DiOC1[3]) is taken up by all cells in mammalian blood which then fluoresce as follows: mature erythrocytes < immature erythrocytes ≃ platelets < leukocytes. A continuous fluorescence distribution can be generated for the red blood cells by flow cytometry and deconvolved into two arbitrary populations, mature and immature erythrocytes (mRBC and imRBC). This analysis mimics the established method of counting imRBC stained with the supravital dyes, new methylene blue, brilliant cresyl blue (BCB), and acridine orange (AO). However, the population of imRBC as quantified by DiOC1[3] flurorescence is a subset of reticulocytes (reticulocytes as determined by BCB assay). The advantages and disadvantages of using DiOC1[3], AO, or pyronine Y as reticulocyte stains are discussed.
Preservation of cells sorted individually onto microscope slides with a fluorescence‐activated cell sorter
Authors: Alberti S. , Stovel R. , Herzenberg L. A.
Published: August 2005

Fluorescence‐activated cell sorters permit analyses and separation of cell populations based on light scatter and surface immunofluorescence parameters. Since a sorter can deposit individually identifiable cells onto a microscope slide, it was considered of interest to combine the flow measurements with analyses available on cells adhering to a surface as in, for example, morphological studies, cytoplasmic immunofluorescent staining, and mRNA in situ hybridization. A necessary condition for these studies is the preservation of cell structures after sorting. We report here a procedure suitable for this purpose. The most important features of this procedure are A) reducing the saline content of the sorter sheath fluid to about 0.0015 M (one‐hundredth that of normal saline) to prevent cell damage due to hypertonicity during drying, and B) coating the substrate with a thin layer of newborn calf serum to promote the adherence of the cells to the substrate during subsequent fixing and staining.
Differentiation of HL‐60 promyelocytic leukemia cells monitored by flow cytometric measurement of nitro blue tetrazolium (NBT) reduction
Authors: Blair Owen C. , Carbone Rocco , Sartorelli Alan C.
Published: August 2005

Reduction of nitro blue tetrazolium (NBT) to insoluble blue formazan granules occurs during the stimulus‐induced respiratory burst of mature granulocytes and is routinely used as an indicator of the extent of granulocytic differentiation of HL‐60 acute promyelocytic leukemia cells. In the present study, the differentiation of HL‐60 leukemia cells induced by dimethylsulfoxide (DMSO) or retinoic acid was monitored by flow cytometric (FCM) measurement of forward and 90° light scatter of NBT treated cells. Two‐parameter correlated analysis permitted a distinction between cells with increased forward and decreased 90° light scatter (NBT−), and cells with decreased forward and increased 90° light scatter (NBT+). Fixation of NBT treated cells with 1% paraformaldehyde facilitated flow cytometric analysis, and allowed differences in NBT reduction to be quantitated. DMSO‐induced cells expressed an all‐or‐none reduction of NBT to formazan, compared with retinoic acid treated cells that exhibited a graded response. Three parameter flow cytometric analysis of HL‐60 leukemia cells stained with propidium iodide in combination with NBT allowed the determination of the cell cycle distribution of NBT‐treated cells.
A convenient on‐line device for reagent addition, sample mixing, and temperature control of cell suspensisons in flow cytometry
Authors: Omann Geneva M. , Coppersmith Ward , Finney David A. , Sklar Larry A.
Published: August 2005

We describe a simple and inexpensive device which permits the addition of up to three different solutions into a cell suspension which is on‐line in a flow cytometer. The mixing chamber houses a disposable plastic cuvette stirred with a magnetic stirrer. The sample chamber is attached to a circulating water bath, hence accurate temperature control is achieved.

Because the system is prepressurized and the sample line is very short, the delay time‐between the point of sample modification and the point of analysis is reduced to a few seconds. Thus reagents may be added rapidly, and kinetic measurements of high temporal resolution are possible. Because the temperature of the sample chamber is regulated, binding can be observed over longer time periods than was previously possible. We demonstrate the usefulness of this device in determining the binding of fluoresceinated hexapeptide to human neutrophils under conditions where the stimulus is infused into the cell suspension while on‐line in the cytometer.
DNase I sensitivity of nuclear DNA measured by flow cytometry
Authors: Roti Roti Joseph L. , Wright William D. , Higashikubo Ryuji , Dethlefsen Lyle A.
Published: August 2005

The DNase I digestion kinetics of DNA in isolated nuclei (from HeLa or murine mammary carcinoma, 67 cells) were assayed flow cytometrically by measuring the changes in ethidium bromide (EtBr) fluorescence following various digestion time intervals. The DNase I digestion curve was characterized by an initial 25–30% increase in fluorescence upon addition of the enzyme, a rapid reduction in fluorescence to approximately 50–55% in 30 minutes, and a limit digest of 45–50% beyond 45 minutes.

Throughout digestion, the DNA histogram retained its characteristic bimodal shape, showing that histogram rearrangement was not responsible for the changes in EtBr fluorescence. Irradiation with 5 ± 106 rads (137Cs‐γ‐rays) or exposure to 50 mM EDTA caused an increase in EtBr fluorescence similar to that caused by DNase I, suggesting that DNA nicking and/or chromatin loosening were responsible for this increase. Residual DNA assayed by the solubilization of 14C‐TdR (thymidine)‐labeled DNA indicated a similar kinetic pattern without the initial increase. However, at the limit digest, the fraction of DNA remaining trichloroacetic acid (TCA) insoluble (10%) was smaller than that measured by loss of EtBr fluorescence (50% of initial, 40% of maximum). Part of this difference was due to the presence of TCA soluble DNA trapped within the nuclear matrix (15–20%). This trapped DNA was released when the digested nuclei were exposed to 0.5–1.0 M NaCl just prior to EtBr staining.

Exposure of HeLa cells to three agents that are believed to cause changes in chromatin structure resulted in alterations in the DNase I digestion kinetics measured flow cytometrically. N‐butyrate exposure resulted in an increased rate of digestion. In contrast, heat shock and hypertonic shock resulted in a reduced rate of digestion.

The proliferating status of the cells was reflected in the DNase I sensitivity of nuclear DNA. Nuclei from quiescent 67 cells showed a 22% reduction in EtBr fluorescence after 10 minutes of digestion, whereas nuclei from proliferating 67 cells showed a 26% loss. Additionally, the initial increase in fluorescence intensity was greater for quiescent cells, possibly reflecting a greater degree of chromatin loosening. Thus, the flow cytometric assay of DNase I sensitivity has been demonstrated to be a useful probe for chromatin structure.
Flow cytometric analysis and sorting of human endometrial cells after immunocytochemical labeling for cytokeratin using a monoclonal antibody
Authors: Oud Peter S. , Henderik Jos B. J. , Beck Hans L. M. , Veldhuizen José A. M. , Peter Vooijs G. , Herman Chester J. , Ramaekers Frans C. S.
Published: August 2005

Endometrial cells in suspension were stained with propidium iodide and a monoclonal antibody against a cytokeratin intermediate filament protein specific for glandular and columnar cells (RGE 53). In this way columnar epithelial cells of the normal endometrium and of adenocarcinomas can be distinguished and separated by flow cytometry from non‐epithelial cells (fibroblasts and inflammatory cells) and squamous epithelial cells, all of which are negative for RGE 53. This makes it possible to analyse and also sort pure fractions of this particular tissue type for further studies. The use of propidium iodide allows simultaneous DNA flow cytometry of these columnar epithelial cells. Therefore, the use of antibodies to cytokeratin in combination with propidium iodide can be of help in analyzing and sorting pure fractions of both normal and malignant cells. This allows a more refined examination of complex cell mixtures using flow cytometry.
Flow cytometric determination of DNA content in malignant and benign bone tumours
Authors: Heliö H. , Karaharju E. , Nordling S.
Published: August 2005

The nuclear DNA content of 38 malignant and 25 benign bone tumours was measured by flow cytometry. The specimens were taken either from biopsies or from surgical specimens. Seventeen of 26 primary malignant bone tumours were aneuploid, 15 had a single aneuploid DNA content, and 2 had a biclonal abnormality. Thirteen of 15 osteosarcomas were aneuploid, but only 2 of 6 chondrosarcomas showed an aneuploid DNA content. Six of 12 metastatic malignant bone tumours were also aneuploid. All 25 benign tumours had a diploid DNA content. Cell cycle analysis showed that the proportion of cells in S‐ and G2M‐phases was higher in the malignant compared to benign tumours, indicating a higher proliferative activity. The increase was statistically significant (p < 0.05) both in diploid and in aneuploid tumours. Among five tumours studied after chemotherapy, four displayed a marked hyperdiploid abnormality. Aneuploidy and high proliferative activity both were highly associated with malignant bone tumours, suggesting that DNA flow cytometry may be an adjunct in the assessment of malignancy of bone tumours.
Flow cytometric enumeration of absolute lymphocyte number in peripheral blood using two parameters of light scatter
Authors: Smart Y. C. , Cox J. , Murphy B. , Enno A. , Burton R. C.
Published: August 2005

A method was developed to measure the absolute lymphocyte count (ALC) of whole blood using the Spectrum III automated flow cytometer. Ninety‐nine samples of human peripheral blood were analysed on the Spectrum and the Coulter Counter S Plus II, to allow for comparison of the two machines. Regression analysis was used to test the extent of agreement between the sets of measurements on the two machines. The results demonstrated that the slope of the regression line was not significantly different from one, indicating a high level of correlation between Spectrum and Coulter ALC's. However, the mean difference between Coulter and Spectrum ALC's was not equal to zero, with the Spectrum giving counts approximately 10% lower than those of the Coulter machine. This is attributed to the different ways by which the two machines define a lymphocyte, the Spectrum III by two parameters of light scatter and the Coulter S Plus II by the single parameter of cell volume.
Fluorescence studies of Hoechst 33342 with supercoiled and relaxed plasmid pBR322 DNA
Authors: Sandhu Laurie Cochran , Warters Raymond L. , Dethlefsen Lyle A.
Published: August 2005

The fluorescence properties of Hoechst 33342 (HO 33342) were examined with plasmid pBR322 in the supercoiled (Form I) or relaxed covalently closed circular (Form I°) conformation in order to determine whether qualitative or quantitative differences in fluorescence properties might provide an assay for topological states of DNA. It was found that HO 33342 exhibited a 30% greater fluorescence intensity with Form I pBR322, independent of the dye or DNA concentration. As the dye to DNA ratio was increased, a red shift of approximately 8 nm was observed for HO 33342 complexed with Form I or Form I°. The red shift in fluorescence emission occurred at higher HO 33342 concentrations with Form I vs. Form I° DNA; however, when Form I and Form I° were mixed in various proportions, neither the fluorescent intensity differences nor the HO 33342 concentration at which the wavelength shift occurred could be used to quantitate the relative proportions of topological states present. These results suggest that although the fluorescence properties of HO 33342 complexed with Form I DNA are different than those of HO 33342 complexed with Form I° DNA, the fluorescence assay is not sufficiently sensitive to quantitatively discriminate among a mixture of DNA in various topological states.
A method for simultaneous nuclear immunofluorescence and DNA content quantitation using monoclonal antibodies and flow cytometry
Authors: Clevenger Charles V. , Bauer Kenneth D. , Epstein Alan L.
Published: August 2005

A preparative technique for the two‐parameter flow cytometric study of nuclear antigen expression is reported. This method employs a brief sequential treatment of cells at 4°C first with 0.5% paraformaldehyde and second with 0.1% Triton X‐100 in phosphate‐buffered saline followed by cellular staining with indirect immunofluorescence and propidium iodide. Using this technique, cellular morphology is preserved, cell clumping is minimized, and high‐quality indirect immunofluorescence and DNA staining are obtained with a minimum of nonspecific labeling. Utilizing nuclear antigen‐specific monoclonal antibodies in conjunction with this technique, the cell‐cycle phase‐dependent expression of such antigens is examined. From these data, the utility of two‐parameter flow cytometry in the identification and quantification of cell‐cycle‐dependent modulation of nuclear antigens is discussed.
Flow cytometric evaluation of cell dispersion from human head and neck tumors
Authors: Th. Bijman J. , Th. Wagener D. J. , van Rennes H. , Wessels J. M. C. , van den Broek P.
Published: August 2005

The preparation of single‐cell suspensions from 25 human head and neck tumors is described. Dispersal was performed overnight at 4°C under slight agitation of the tissue suspensions using various combinations of enzymes and additives. The cell suspensions were examined for number of cells released, viability, amount of debris, and DNA distribution by means of flow cytometry (FCM).

It was shown that both trypsin/dithioerythritol (TD) and collagenase/DNase (CDse) were of value in dispersing single cells from tumor tissue. In contrast to CDse, incubation with TD appeared to be cytolytic to normal lymphocytes. In a number of cases, DNA‐FCM revealed ploidy abnormalities in a TD‐suspension, which were not discernible in the concurrent CDse‐suspension. Cell culture of primary cell suspensions corroborated the reliability of the DNA‐FCM measurements. Pretreatment with CDse improved tumor disaggregation by TD and indicated a different dispersal capacity.

Addition of Ca2+ and Mg2+ ions to the dispersal mixtures and preincubation of tumor slices in complete medium for 1 day before initiation of cell dispersion influenced favorably the quality of the cell suspension.
Detection of minimal disease in acute leukemia using flow cytometry: Studies in a rat model for human acute leukemia
Authors: Martens Anton C. M. , Hagenbeek Anton
Published: August 2005

The study was made using a rat model for human acute myelocytic leukemia (BNML), which shows striking similarities with human acute myelocytic leukemia (AML). A monoclonal antibody (MCA‐Rm124), raised against BNML cells, allowed the recognition of the leukemic cell fraction. The discriminative capacity of the monoclonal antibody is based on differences in labeling intensities between normal and leukemic cells. After i.v. cellular transfer of leukemia, the growth of the leukemic cell population in the bone marrow, the liver, and the spleen was monitored using MCA‐Rm124 and flow cytometry. For the bone marrow and the liver, a clonogenic assay for leukemic cells was used to quantify the cell content in these organs. A good correlation was found between the bioassay‐derived data and the flow‐cytometry‐derived data. The doubling times of the leukemic cell population were not equal for the two organs studied, indicating that a number of different processes contribute to the net cell production per organ.

Apart from their application in the detection of residual leukemia, monoclonal antibodies might be employed in the analysis of the growth kinetics of the “invisible” leukemic cell population.
Active analog pipeline delays for high signal rates in multistation flow cytometers
Authors: Parson J. D. , Hiebert R. D. , Martin J. C.
Published: August 2005

Flow cytometers with sequential measuring stations require alignment of event timing information to assure that multiparameter data for each cell are properly correlated. Information from the first detector must be delayed until signals are produced at the last detector, with adjustments to align them in time for simultaneous digitization after the last measurement is completed. By using an analog “pipeline” delay, the deadtime between the first and last measuring stations can be minimized. The described device is capable of acquiring and propagating many signals within the delay period, with good fidelity at high signal rates. An integrated circuit charge‐coupled device (CCD), which is an analog shift register, is shown to be useful as a signal delay in the time range from 22 μs to several milliseconds.
The optimal application of forward and ninety‐degree light scatter in flow cytometry for the gating of mononuclear cells
Authors: Thompson James M. , Gralow Julie R. , Levy Ronald , Miller Richard A.
Published: August 2005

Peripheral blood mononuclear cells from ten normal donors were labeled with a monoclonal antibody specific for monocytes and analyzed using a fluorescence activated cell sorter (FACS). Forward and 90° light scatter parameters were studied in order to apply optimal computerized gating to identify and exclude monocytes from lymphocyte populations. An average of 9.45% versus 1.22% of cells, within chosen lymphocyte gates established by forward angle and 90° scatter, respectively, were identified as monocytes. In samples from ten donors, the exclusion of monocytes from the lymphocyte population was more efficient using 90° scatter than forward scatter. Simultaneous use of forward and 90° scatter did not significantly improve the ability to accurately exclude monocytes, but did result in a significant increase in the improper exclusion of lymphocytes. Use of 90° scatter alone, forward scatter alone, and forward and 90° scatter simultaneously to identify lymphoid cells resulted in the exclusion of 12, 17, and 23% of lymphocytes from further analysis. The 90° scatter alone appears to be the optimal method to eliminate monocytes electronically from mononuclear cell populations in which lymphocytes are being studied.
Detection of distinct subpopulations of langerhans cells by flow cytometry and sorting
Authors: Vaigot P. , Czernielewski J. , Prunieras M.
Published: August 2005

Flow cytometry was found to be a very appropriate tool for the study of Langerhans cells (LC), which represent a minor cell population (2–3%) of human epidermis, and allowed us to obtain new phenotypic, functional and cell cycle data on these rare cells.

The phenotypic analysis of cell surface antigens demonstrates the existence of two subpopulations of LC: the former is HLA‐DR+ and OKT 6 + (about 90% of total HLA‐DR+ cells) and the latter is HLA‐DR+ and OKT 6− (about 10% of total HLA‐DR+ cells). These subpopulations of LC are both able to stimulate the proliferation of peripheral blood lymphocytes (PBL) in the presence of keratinocytes i.e., in mixed skin lymphocyte reaction (MSLR).

Analysis of the cell cycle could be performed on OKT 6+ LC. Results show that they can be found in the various phases of the cell cycle, suggesting that the large majority of Langerhans cells are able to proliferate in situ in normal human epidermis.
DNA image cytometry on machine‐selected breast cancer cells and a comparison between flow cytometry and scanning cytophotometry
Authors: Cornelisse Cornelis J. , Van Driel‐Kulker Anneke M.
Published: August 2005

A DNA image cytometry method, implemented on the LEYTAS image processing system, has been applied to acriflavine‐Feulgenstained breast cancer cytology specimens. An essential feature of the LEYTAS image cytometry method (LCM) is the automated selection of single nuclei according to predetermined specifications. Visual interaction has been used to reject remaining artefacts like overlapping nuclei. DNA profiles obtained with LCM have been compared with DNA profiles obtained by scanning cytophotometry (SCM) or flow cytometry (FCM). The resolution of DNA profiles obtained with LCM is similar to that from SCM but lower than that from FCM. However, a high correlation is found for the DNA indices measured with LCM and FCM (r = 0.97). The LCM profiles of aneuploid tumours generally showed lower accessory diploid fractions than FCM profiles due to the automated rejection of leukocyte nuclei. Also, LCM profiles frequently showed the presence of minor subpopulations of highly aneuploid/polyploid tumour cells that could not be identified by FCM. Therefore, LCM appears to be supplementary to FCM for studying tumour cell stemline heterogeneity.
Spot‐blot analysis of sorted chromosomes assigns a fructose intolerance disease locus to chromosome 9
Authors: Lebo Roger V. , Tolan Dean R. , Bruce Barry D. , Cheung Mei‐Chi , Kan Yuet W.
Published: August 2005

The aldolase B gene was mapped to chromosome 9 using a rapid gene mapping system. This system uses a dual‐laser sorter to identify and separate metaphase human chromosomes stained with either DIPI‐chromomycin or Hoechst‐chromomycin. Chromosome panels were constructed from a normal cell line by sorting 22 chromosome fractions directly onto nitrocellulose filters. Twelve labeled gene probes hybridized to the sorted chromosomal DNA fractions predicted by previous chromosome assignments. Eighteen newly cloned genes have been mapped using the same protocol.
The relationship between mean channel selection and the calculated coefficient of variation
Authors: Schuette W. H. , Carducci E. , Marti G. E. , Shackney S. E. , Eden M.
Published: August 2005

Calculated coefficients of variation (CV) taken from the quotient of the standard deviation (S.D.) and the mean value of measured distributions are often used as an indicator of system performance in linear flow cytometry (FCM). The ability of the calculated CV to estimate the true CV of the underlying experiment before grouping (channelization) is dependent on the relationship between the width of the data channels and the magnitude of the S.D. of the measured distribution. When the channel width is equal to the S.D. of a distribution, the calculated CV is approximately 20% larger than the true CV of an experiment. By the time the S.D. is only one‐half of a channel width, the calculated CV is unreliable. When the distribution S.D. is narrower than a channel's width, small changes in the distribution mean value will cause large variations in the calculated CV. As the true CV decreases, the calculation must be made with higher mean channel values. This dependence of calculated CV accuracy upon the relationship between S.D. and channel width places limitations upon mean channel selection that must be considered when using CV calculations for evaluating system performance, especially when looking for small improvements during optical alignment procedures. When an instrument is assumed to have a constant CV and the data are collected linearly, it is possible to improve the CV estimation accuracy by placing distributions in higher‐numbered channels. When linear data with a constant CV are processed logarithmically before channelization, the accuracy of linear equivalent CV calculations is dependent upon the slope of the logarithmic converter and the number of channels available. Under these conditions, CV determination accuracy is independent of mean channel selection.
A fast cell sampler for flow cytometry
Authors: Battye F. L. , Darling W. , Beall J.
Published: August 2005

A simple device has been developed for delivering samples into a flow cytometer. Designed with economy, simplicity, and flexibility in mind, this device, having only one moving part, can be used for sample volumes as small as 20 μl, for virtually any form of cell sample container, and for a wide range of cell concentrations. It consists essentially of a lever‐operated disc valve that allows the cell sample to be loaded into a loop of tubing and then to be injected into the cytometer nozzle under pressure from a saline source. The sampler has lifted the maximum analytical throughput of a FACS II cell sorter to better than 120 samples per hour.
Effect of 5‐fluoro‐2′‐deoxyuridine (FdUrd) on 5‐bromo‐2′‐deoxyuridine (BrdUrd) incorporation into DNA, measured with a monoclonal BrdUrd antibody and by the BrdUrd/hoechst quenching effect
Authors: Ellwart Joachim , Dörmer Peter
Published: August 2005

The purpose of this study was to improve the application of bromodeoxyuridine (BrdUrd) for the flow cytometric analysis of cell kinetics. In order to obtain a quantitative measure of the DNA synthesis rate (or the number of divided cells). BrdUrd should replace thymidine (dThd) completely in the newly synthesized DNA strands. The de novo synthesis of dThd monophosphate competing with BrdUrd incorporation was stopped by fluorodeoxyuridine (FdUrd). Cells of a human leukemic cell line (REH) were exposed O BrdUrd for either 20 min, 8h, or 24h. Bromodeoxyuridine incorporation was determined by a monoclonal antibody as well as by the BrdUrd/Hoechst (H) technique. Counter‐staining of the DNA was performed with propidium iodide or ethidium bromide. DNA fluorescences were measured in both techniques with a two‐parameter flow cytometer, the his‐tograms being analyzed by computer. It was found that FdUrd is required in the BrdUrd/H technique for replacement of dThd at low BrdUrd concentrations and long incubation times. With short incubation periods, as used for detection by the monoclonal anti‐BrdUrd antibody, FdUrd increases the incorporated BrdUrd amount when BrdUrd concentrations of 10 μU or less are applied.
Flow cytometric analysis of DNA replication during the differentiation of 3T3‐L1 preadipocytes
Authors: Gratzner Howard G. , Ahmad Patricia M. , Stein Jeanne , Ahmad Fazal
Published: August 2005

We have employed a flow cytometric method of monitoring DNA synthesis in order to study the stimulation of DNA replication during the induction of adipocyte differentiation in the preadipocyte cell line 3T3‐L1. When confluent monolayers of this cell line are treated with a mixture of methyl isobutyl xanthine, dexamethasone, and insulin (MDI), they undergo at least one round of cell division, which is followed by the de novo synthesis of many enzymes that are involved in the formation of lipid. If the MDI‐treated cells are incubated in iododeoxyuridine (IdUrd)‐or bromodeoxyuridine (BrdUrd)‐containing medium and subsequently stained with antibodies specific for these base analogues and with propidium iodide (PI), the kinetics of the induction of replication can be monitered. No differences in the patterns of IdUrd incorporation versus PI staining were observed between exponentially growing 3T3‐L1 cells and those that had been stimulated to replicate DNA with MDI. In addition, we developed a flow cytometric (FCM) method for staining fatty acid synthetase and localizing the antigen in the G1 phase. We have thus demonstrated the feasibility of this methodology for correlating by FCM the production of enzymes such as fatty acid synthetase with IdUrd and BrUrd incorporation. The technique should permit studies of the inhibition of differentiation of adipocytes by halogenated pyrimidines.
DNA Replication and differentiation in rat myoblasts studied with monoclonal antibodies against 5‐bromodeoxyuridine, actin, and α2‐macroglobulin
Authors: Kaufman Stephen J. , Robert‐Nicoud Michel
Published: August 2005

During the differentiation of skeletal muscle, mononucleate myoblasts proliferate, then stop replicating, spontaneously fuse, and express a large number of genes which encode the muscle phenotype. We have used monoclonal antibodies specific for 5‐bromodeoxy‐uridine, myoactin, and equine α2‐macroglobulin to follow and establish the sequence of events that surround the transition from a replicating to a differentiating population. Triple‐label immunofluorescence microscopy was used to visualize the changes in DNA synthesis, formation of myoactin fibers, and the cessation of endocytosis of α2‐macroglobulin that accompany myogenesis. Our results indicate that myoblasts cease actively endocytosing α2‐macroglobulin after stopping DNA synthesis but prior to fusion. Formation of myoactin fibers rarely occurs in mononucleate myoblasts and only in post‐mitotic cells, but they are common in multinucleate myotubes. We suggest that the regulation of DNA synthesis is critical to normal myogenesis and that detection of incorporated BrdUrd by immunofluorescence, in conjunction with other antibodies and nucleic acid probes, is a convenient method with which to study and sequence the molecular events in single cells as they relate to the transition in DNA synthesis that accompanies differentiation.
Cytokinetic properties of asynchronous and cytosine arabinoside perturbed murine tumors measured by simultaneous bromodeoxyuridine/DNA analyses
Authors: Pallavicini M. G. , Summers L. J. , Dolbeare F. D. , Gray J. W.
Published: August 2005

This paper describes the determination of cytokinetic properties of asynchronous and cytosine arabinoside‐(Ara‐C) treated KHT tumors growing in vivo using the bromodeoxyuridine (BrdUrd)/DNA analysis technique. The cytokinetic properties of asynchronously growing tumors were estimated by computer analysis of sequential BrdUrd/DNA distributions measured at 2‐ to 3‐h intervals after administration of a single i.p. injection of BrdUrd. The cytokinetic properties of the AraC‐treated tumors were estimated by computer analyysis of BrdUrd/DNA distribution sured at 2‐ to 3‐h intervals after Ara‐C treatment. BrdUrd was injected 30 min prior to tumor harvest. The cytokinetic properties of the cells rendered nonclonogenic by Ara‐C were followed in BrdUrd/DNA distributions measured at 2‐ to 3‐h intervals after Ara‐C treatment of tumors that were labeled with BrdUrd 30 min prior to Ara C injection. The Gl‐, S‐, and G2M‐phase durations were estimated to be 7.6, 10.9, and 2.0 h prior to Ara‐C; decreasing to 1.2, 4.1, and 1.4 after Ara‐C. The growth fraction was estimated to be 0.8 prior to Ara‐C. Complete recruitment of the normally noncycling subpopulation was observed after Ara‐C treatment. Ara‐C‐killed cells were removed from the tumor within 24 h following Ara‐C injection. These cytokinetic properties were similar to those reported in other studies.
The labelling index of human and mouse tumours assessed by bromodeoxyuridine staining in vitro and in vivo and flow cytometry
Authors: Wilson G. D. , McNally N. J. , Dunphy E. , Kärcher H. , Pfragner R.
Published: August 2005

Bromodeoxyuridine (BrdUrd) incorporation and flow cytometry were used to measure human tumour kinetic parameters in vitro and in vivo. The technique was validated by comparison of labelling index estimates of mouse tumours in vivo and in vitro using BrdUrd and flow cytometry Nvith tritiated thymidine (3HdThd) autoradiography. Similar labelling indices were obtained with both in vivo and in vitro incorporation into DNA of the two different precursors. Measurements of human tumour labelling indices were similar following in vitro incubation with either BrdUrd or 3HdThd.

The use of BrdUrd allowed the visualization of a population of S‐phase cells that did not appear to incorporate BrdUrd or 3HdThd. The human tumour labelling indices obtained with BrdUrd incorporation were similar to previously reported values using autoradiography studies. Preliminary studies demonstrated that significant human tumour labelling could be achieved with an intravenous injection of 500 mg BrdUrd.
Automatic classification of chromosomes as part of a routine system for clinical analysis
Authors: Lundsteen Claes , Gerdes Tommy , Maahr Jan
Published: August 2005

A procedure for automatic classification of G‐banded human chromosomes has been implemented on a semiautomated system for routine clinical analysis. Chromosomes represented by their density profiles are described by so‐called weighted density distribution (WDDs) by application of a number of weighting functions and classified by a parametric discriminant analysis. During 16 mo of routine use of the system, 2,794 metaphases (127,925 chromosomes) from amniotic fluid have been karyotyped by the system with an error rate of 8–9%. This corresponds to 4–5 errors per metaphase. These errors can immediately be corrected by the operator on a displayed karyogram with a light pen.
Effect of section thickness on quality of flow cytometric DNA content determinations in paraffin‐embedded tissues
Authors: Stephenson Robert A. , Gay Helen , Fair William R. , Melamed Myron R.
Published: August 2005

DNA content determinations were carried out by flow cytometry on nuclear suspensions prepared from the same paraffin embedded tissue block for each of eight surgically resected human carcinomas at section thicknesses of 5,10,20,30,40,50, and 100 μm. Flow cytometric DNA determinations were also obtained on fresh tissue specimens in four of the eight carcinomas. As section thickness decreased below 50 μm, there was a progressive increase in the histogram baseline noise at low DNA values and a decrease in the relative peak height of aneuploid DNA. The former was attributed to an increase of nuclear fragments in thinner sections, and the latter to the greater probability of transection of the larger aneuploid cells within a specimen. Both artifacts were minimized at section thickness of 50 μm or greater.
Flow cytometric discrimination of mitotic cells: Resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde
Authors: Larsen Jo̊Gen Knud , Munch‐Petersen Birgitte , Christiansen Jette , Jo̊gensen Knud
Published: August 2005

Cells in mitosis can be flow cytometrically discriminated from G1, S, and G2 cells by analysis of a nuclear suspension prepared with nonionic detergent, fixed with formaldehyde, and stained with mithramycin, propidium iodide, or ethidium bromide. With these DNA‐fluorochromes, the fluorescence is quenched by formaldehyde less in mitotic nuclei than in interphase nuclei. Mitotic nuclei have a 20–40% increased mithramycin fluorescence and 30–60% decreased light scatter in comparison to those of G2 nuclei. There is a high correlation (r = 0.95; P < 0.001) between microscope counts of mitotic figures in smear preparations of the initial cell suspension and the flow cytometrically estimated fraction of nuclei with increased mithramycin flourescence. Flow sorting (FACS) demonstrates that the mitotic nuclei are confined to the peak of increased mithramycin fluorescence and decreased light scatter. The method has been applied to cultures of Yoshida ascites tumor cells, JB‐1 retculosarcoma cells, and PHA‐stimulated human lymphocytes, incubated in the presence or absence of vinblastine for mitotic arrest. In a heteroploid mixture of fixed Yoshida (near‐diploid) and JB‐1 (hypotetraploid) nuclei, the mitotic fractions of the two cell lines could be estimated separately when analyzed with mithramycin flourescence versus light scatter or with mithramycin fluorscecence versus propidium iodide fluorescence.
Flow cytometric and radioisotopic determanations of platelet survival time in normal cats and feline leukemia virus‐infected cats
Authors: Jacobs R. M. , Boyce J. T. , Kociba G. J.
Published: August 2005

This study demonstrates the potential usefulness of a flow cytometric technique to measure platelet survival time in cats utilizing autologous platelets labeled in vitro with fluorescein isothiocyanate (FITC). When compared with a 51Cr method, no significant differences in estimated survival times were found. Both the 51Cr and FITC‐labeling procedures induced similar changes in platelet shape and collagen‐induced aggregation. Platelets labeled with FITC had significantly greater volumes compared with those of glutaraldehyde‐fixed platelets. These changes were primarily related to the platelet centrifugation and washing procedures rather than the labels themselves. This novel technique potentially has wide applicability to cell circulation time studies as flow cytometry equipment becomes more readily available.

In a preliminary study of the platelet survival time in feline leukemia virus (FeLV)‐infected cats, two of three cats had significantly reduced survival times using both flow cytometric and radioisotopic methods. These data suggest increased platelet turnover in FeLV‐infected cats.
Cell size, DNA, and cytokeratin analysis of human head and neck tumors by flow cytometry
Authors: Bijman J. Th. , Wagener D. J. Th. , Wessels J. M. C. , van den Broek P. , Ramaekers F. C. S.
Published: August 2005

Cell subsets have been discriminated in cell suspensions derived from 37 human head and neck tumors by means of light scatter, DNA, and cytokeratin flow cytometry (FCM). Cell dispersion was performed overnight at 4°C in two different enzyme mixtures, i.e., trypsin/dithioerythritol and collagenase/DNase, under slight agitation of sliced tumor tissue. Cells were examined before and after fractionation on a discontinuous low‐density bovine serum albumin (BSA) gradient.

Forward and right‐angle light scatter FCM of 23 tumor specimens revealed four main subpopulations with different size and structure. Fractionation of primary cell suspensions on a BSA gradient at unit gravity separated debris, small cells and large cells. DNA FCM of the enriched populations demonstrated a relation between large cells and DNA aneuploidy. Epithelial cells, as recognized by cytokeratin antibodies, were also related with large cells.

The results demonstrated the usefulness of light scatter, DNA, and cytokeratin analysis of crude and fractionated tumor cell suspensions for assessment of the efficacy of a particular dispersion technique and to obtain information of the cell subsets dispersed.
A pulse generator simulating slit‐scan chromosome analysis signals
Authors: Weier H.‐U. L. , Eisert W. G.
Published: August 2005

A simple circuit is described for generating a variety of electronic pulses to test hardware and software for slit‐scan chromosome analysis in a flow cytometer. The pulse shape can be changed to have different numbers of local minima, thereby simulating fluorescence pulses from acrocentric, monocentric, and dicentric chromosomes. Long pulses simulate aggregates of chromosomes. The pulse repetition rate as well as the pulse amplitude is variable. Although the circuitry is built with only three integrated circuits, the pulse‐to‐pulse variation in shape and height is quite small. After digitization of the analog signals, the constructed histograms of pulse integrals show a relative coefficient of variation below 1%. This signal generator provides a valuable tool for a number of electronic test applications that would otherwise require expensive standard particles analyzed in a well‐tuned flow cytometer.
A protocol for papanicolaou staining of cytologic specimens following flow analysis
Authors: Berkan Todd K. , Reeder Jay E. , Lopez Peter A. , Gorman Kevin M. , Wheeless Leon L.
Published: August 2005

A protocol has been developed for restaining cytologic specimens that have been analyzed on a multidimensional slit‐scan flow system. The technique involves Papanicolou staining of cells on a membrane filter that has been previously stained with acridine orange and fixed with glutaraldehyde buffer. The specimen and staining solutions were sequentially added to a 5‐micrometer pore size, 47‐mm diameter Gelman “Metricel” filter while it remained in a glass filtration apparatus.

The practice of retaining the filter in the filtration apparatus throughout the staining procedure minimizes cell loss and eliminates specimen cross contamination when compared with conventional filter dip staining. The availability of this postflow specimen Papanicolaou staining protocol permits accurate determination of the performance characteristics of a multidimensional slit‐scan flow system and should be useful whenever staining of a limited number of cells with minimal cell loss is desired.
Griffonia simplicifolia I isolectin as a functionally monovalent probe for use in flow cytometry
Authors: McCoy J. Philip , Shibuya Naoto , Riedy Mary C. , Goldstein Irwin J.
Published: August 2005

The inherent tendency of lectins to agglutinate cells has limited their use as reagents for the detection of carbohydrate groups on cell surfaces by flow cytometry. In the current study, we demonstrate a method for theuse of a fluoresceinated tetrameric isolectin (Griffonia simplicifolia I‐A3B, FITC‐GS I‐A3B) as a functionally monovalent, nonagglutinating probe in flow cytometry. This isolectin contains three A subunits and one B subunit. Both types of subunits bind α‐D‐galactopyranosyl (α‐D‐galp‐) end groups with similar affinities; however, the A subunits have a 1,000‐fold greater affinity for N‐Acetyl‐D‐galactosamine (GalNAc) than does the B subunit. The addition of low (1–2 mM) concentrations of GalNAc to the FITC‐GS I‐A3B isolectin results in blockage of the three A subunits without significantly affecting the B subunit; this yields a functionally monovalent probe for the detection of cell surface α‐D‐Galp end groups. This approach has been used to examine two types of cells: Ehrlich ascites tumor cells and rat alveolar macrophages, both of which are known to express cell surface α‐D‐Galp end groups. Lectin binding, as determined by number of positive cells and fluorescence intensity, was dependent upon concentration of the lectin and haptenic sugar. Specificity of the staining was demonstrated by the ability of methyl α‐D‐galactopyranoside (Met α‐D‐Galp) to abolish the binding of the lectin to the cells. Elimination of both GalNAc and Met α‐D‐Galp from the staining solution resulted in agglutination of the cells, indicating that the A subunits were active in the absence of GalNAc.
Isolation of viable type II alveolar epithelial cells by flow cytometry
Authors: Wilson Julie S. , Steinkamp John A. , Lehnert Bruce E.
Published: August 2005

We developed a new method for isolating viable type II cells from fractionated and unfractionated lung cell suspensions by flow cytometry using acridine orange (AO). Fischer‐344 rat lungs were dispersed into single‐cell suspensions by a technique that yields a high number of cells (4–5 × 108 cells/lung, =85% viable), =11% of which are type II cells. Elutriated fractions from the lung cell preparation and parent, unfractionated cell suspensions were incubated with 1.0–0.02 μg/ml AO and analyzed by flow cytometry. Parameters analyzed included axial light loss (ALL) and red fluorescence (RF). Based on their unique RF, attributable to AO staining of type II cell lamellar bodies, and their ALL characteristics, type II pneumocytes were sorted from elutriated fractions to greater than 95% purity. Using the same approach, type II pneumocytes were sorted from unfractionated lung cell suspensions at ≤85% purity. The viabilities of the type II alveolar epithelial cells isolated by this method range from 85% to 95%, and the ultrastructural features of the sorted cells were unaltered by AO labeling or sorting.