bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell kinetics in normal and neoplastic mammary tissues by flow cytometric analyses
Authors: Kute Timothy E. , Linville Constance , Mehta Rajendra G. , Moon Richard C.
Published: August 2005

The cell cycle kinetics of normal mammary tissue and cancer mammary tissues were studied by flow cytometry to determine the effect of estrogen and progesterone and antiestrogens on proliferative activity. The proliferative activity was higher in rat mammary tissue during the estrus and pregnant stages compared to the nonestrus or lactational stages. Upon 5 days administration of estrogen and progesterone to the lactating animal, the low proliferative activity was increased to the values found in the pregnant stage. Steroidal effect on proliferative activity was also shown using organ cultures of normal rat tissues and the increase in proliferative activity can be reversed by the addition of 10−6 M retinoic acid but not by retinyl acetate. In a human mammary tumor cell line (MCF‐7), the cell kinetic activity was significantly depressed by antiestrogens (i.e., tamoxifen) as determined by both flow cytometry and tritiated thymidine uptake studies. Finally, a human tumor model system was developed with the MCF‐7 cell line in estrogenized nude mice. Flow cytometry of the resulting tumor indicates a tetraploid tumor similar to the cell culture. Cell kinetic analysis of these nude mice tumor cells demonstrates the higher proliferative activity of the estrogenized animals compared to the tamoxifen‐treated animals.

In conclusion, the use of flow cytometry to rapidly measure cell kinetic activity of mammary tissue has been demonstrated. The effects of estrogen or estrogen and progesterone hormones seem to increase the proliferative activity, while antiestrogens decrease this activity. Findings are observed in both in vivo and in vitro conditions or normal tissues, mammary cancer tissue, and cultured cells.
Automated analysis and survival selection of anchorage‐dependent cells under normal growth conditions
Authors: Schindler Melvin , Allen Margaret L. , Olinger Max R. , Holland John F.
Published: August 2005

An instrument is described that can automatically analyze and select for a subpopulation of anchorage‐dependent cells in tissue culture. Cells that label with fluorescently tagged antibodies or demonstrate structural variations are saved from exposure to a destructive high‐intensity argon laser beam. The surviving population may then be cloned. The cell selection may occur in a tissue culture plate or in a microflow incubator which is designed to maintain a constant flow of media at 37°C across cells growing on a glass coverslip. This incubator sits on an inverted microscope which focuses the laser beam to a diameter as small as 1 μm. A high‐speed computer‐controlled two‐dimensional stage moves the cells past the beam for analysis, the results of which determine the fate of each cell: whether it is to be destroyed by radiant energy or selected for survival and subsequent proliferation. Another selection strategy performed by the instrument involves growing the cells on a thin, blackened polyester film which can be cut by the argon laser beam. Cells selected for cloning are then circumscribed. The heat of cutting welds the circumscribed film to a plastic coverslip surface or tissue culture chamber bottom. Nonselected cells may be removed by pulling the unattached polyester sheet from the attachment surface. The selected cells remain on polyester film disks welded to the plastic. Selections may be done automatically under computer control or manually by operator direction of stage movements.

This instrument extends the art of automated cell selection and analysis to normal cell lines that must maintain cell‐substratum contact (anchorage dependence) for differentiated cell function, e.g., neurons, fibroblasts, or kidney cells.
RNA content and chromatin structure of CHO cells arrested in metaphase by colcemid
Authors: Melamed Joseph , Darzynkiewicz Zbigniew
Published: August 2005

To evaluate the stability of cells arrested in metaphase, cell viability, RNA content, and chromatin structure (the latter probed by the DNA in situ sensitivity to acid‐induced denaturation) were studied in uniform‐age mitotic CHO cell populations maintained either at 37°C (in the presence of Colcemid) or at 0–4°C for up to 6 h. No significant changes in cell viability and RNA content were seen throughout the experiment for both groups of cells. The sensitivity of DNA in situ to denaturation was significantly increased during the initial 40 min of cell arrest in mitosis. However, no further chromatin changes for up to 6 h were evident regardless of whether cells were kept at 37°C with Colcemid or at 0–4°C in its absence. The data indicate that neither significant deterioration of metaphase cells nor progressive chromatin changes are expected during stathmokinesis experiments in vitro or during the metaphase cell arrest in cytogenetic studies lasting up to 6 h. Also, no RNA turnover can be detected in mitotic cells during this time interval.
Tuning meter can replace oscilloscope for flow cytometer alignment
Authors: Hovorka George
Published: August 2005

The differentiator and pulse averaging circuit described in this technical note can be used in place of the oscilloscope normally required to align a flow cytometer.
Evaluation of a new blood cell counter with sheath flow system
Authors: Tatsumi Noriyuki , Tsuda Izumi , Fujimoto Keiji , Matsumoto Hideaki
Published: August 2005

A blood cell counter with a sheath flow system has been developed to eliminate the drawback of the Coulter type blood cell counter, namely, signal distortion caused by the cells passing through the electric aperture gate. With the new system, signal distortion was low and a near‐normal distribution curve for erythrocytes and resin particles was obtained. The counter has a computer program for determining the red cell distribution width, which represents an actual size difference at 20% of the relative frequency of the distribution curve. This is independent of mean cell volume, and is considered to be of clinical importance. We examined the values in 2,300 healthy subjects and patients with various hematological disorders.
A dual laser analysis of the migration of XRITC‐labeled, FITC‐labeled, and double‐labeled lymphocytes in sheep
Authors: Abernethy Nevin J. , Chin Warren , Lyons Helen , Hay John B.
Published: August 2005

Substituted rhodamine isothiocyanate (XRITC) has been used to study lymphocyte migration in sheep. After being labeled in vitro with XRITC, lymphocytes appeared in the efferent lymph of single lymph nodes with the same kinetics as cells labeled with fluorescein isothiocyanate (FITC). The recovery of intravenously injected XRITC‐labeled cells was followed in lymph for several days. The kinetics and recoveries were compared with data obtained using FITC, chromium‐51, and indium‐111. XRITC was found to be a suitable label and, using dual laser (argon and krypton) flow cytometry, it could be analyzed simultaneously with FITC. In addition, it was possible to relabel FITC‐stained cells with XRITC after they were recovered in lymph. The migratory characteristics of such doublelabeled cells were not different from singlelabeled cells.
Cell kinetic effects of incorporated 3H‐thymidine on proliferating human lymphocytes: Flow cytometric analysis using the DNA/nuclear protein method
Authors: Pollack A. , Moulis H. , Greenstein D. B. , Block N. L. , Irvin G. L.
Published: August 2005

Phytohemagglutinin‐stimulated human peripheral blood lymphocytes incorporating high concentrations of 3H‐thymidine accumulate in G2 and show a consequent reduction in the number of cells entering M (division delay). The simultaneous flow cytometric analysis of DNA content (propidium iodide fluorescence) and nuclear protein content (fluorescein isothiocyanate fluorescence) allows for the accurate quantitation of these events; G2 and M are separated in the bivariate distributions. A good correlation was observed between mitotic indices, quantitated by manually counting mitotic cells, and integration of the M area in DNA/nuclear protein histograms. Moreover, significant differences in G2 nuclear protein levels were found between untreated and 3H‐thymidine‐treated lymphocytes. In order to characterize this effect, G2 was empirically divided into low nuclear protein (G2A) and high nuclear protein (G2B) compartments. 3H‐thymidine caused an initial accumulation of lymphocytes in G2A, followed within 3–6 h by a gradual movement of some cells into G2B, with a subsequent accumulation of cells in G2B. The results suggest that the distribution of cells in G2 (G2A and G2B), the average nuclear protein content of G2B cells, and the proportion of cells in M are parameters that when used in combination provide a unique description of radiobiological effects.
Anthracycline resistance and consequences of the in situ–in vitro transfer
Authors: Šonka J. , Stöhr M. , Vogt‐Schaden M. , Volm M.
Published: August 2005

Adriamycin‐resistant and normal cells of the sarcoma 180 of the mouse undergo qualitatively different deflections from the in situ state when prepared for an experiment. Resistant cells perform a fast reactive decline in the proliferative activity. They are capable of quiescence as defined by the time needed for the induction of the proliferation. Sensitive cells seem to be unable to quiesce and are only slowed down. These facts must be taken into account in interpretation of similar results. Differences in experiments need not necessarily imply differences in situ. Such in vitro appearing differences between sensitive and adriamycin‐resistant cells of the murine sarcoma 180 include the retention of the mitochondria‐specific stain rhodamine 123 and the uptake of anthracyclines, both being reduced in resistant cells.

After labeling sensitive cells with thymidine in vivo and sorting them according to their rhodamine 123‐derived fluorescence, the label was only found in the major, highly fluorescing fraction. A small low‐fluorescing fraction remained unlabeled. We were able to demonstrate similar results with labeled anthracyclines applied to both the sensitive and the resistant cells in a short period between the removal of the cells from the ascites and the cells sorting. The adriamycin resistance seems to be joined with the ability of the cells to reduce their proliferative activity following changes to unfavorable conditions in vitro. Quiescent cells of the resistant line demonstrate the “anthracyclin pump.” Substances which are known to increase the sensitivity of anthracycline‐resistant cells (TWEEN, verapamil) also shift the cells from low to high rhodamine 123‐fluorescence.
Flow cytometry of human colorectal tumors: Nuclear isolation by detergent technique
Authors: Petersen Steffen E.
Published: August 2005

A simple one‐step technique for detergent isolation and DNA staining of nuclei from mouse colon and from human colorectal tumors was investigated. Nuclear yield increased with treatment time, up to 24 h. There were only minor differences when detergent concentrations from 0 to 0.6% were used. The lowest (0.03%) concentration was most effective. No loss of nuclei was effected by cell lysis and no selectivity was observed for isolation of certain cell‐cycle phases or ploidy classes from heterogeneous tumors. The nuclei were stable in the stain‐detergent solution for 24 h, but lymphocytes were sensitive to the possible proteolytic activity of one of the two commercial RNase preparations. Of the total number of parenchymal nuclei in mouse liver, as estimated by a stereological method, approximately 60% were isolated by the procedure (approximately 0.9 × 108 nuclei/g tissue). From mouse colon the average nuclear yield was 1.8 × 108/g, and from human colorectal tumors 0.9 × 108/g (ranges 0.3–1.9 × 108). Microscopic examination of undissolved tissue fragments from the preparation of tumors and mouse colon showed a high selectivity for isolation of epithelial and neoplastic nuclei, leaving the stroma with its nuclei almost intact.
A monoclonal antibody reactive with 5‐bromo‐2‐deoxyuridine that does not require DNA denaturation
Authors: Gonchoroff Nick J. , Greipp Philip R. , Kyle Robert A. , Katzmann Jerry A.
Published: August 2005

We describe a mouse monoclonal antibody (BU‐1) reactive with 5‐bromo‐2‐deoxyuridine (BrdUrd). The antibody is different from previously described BrdUrd monoclonal antibodies in that BU‐1 does not require pretreatment of cells with strong DNA denaturants in order for the antibody to react with BrdUrd incorporated in the DNA. The antibody can be used in immunocytochemical and indirect immunofluorescent assays and can be used to identify cells that have incorporated BrdUrd. Double staining with BU‐1 antibody and propidium iodide has been used to confirm S‐phase measurements with the BU‐1 antibody. Immunocytochemical stains using the BU‐1 antibody do not destroy cell morphology and allow cell identification to be performed simultaneously with S‐phase measurements. Flow Cytometer two‐color fluorescence analysis allows the simultaneous identification of cell surface or cytoplasmic markers and S‐phase quantitation. The BU‐1 antibody should broaden the application of cell kinetic measurements to individual elements of cell populations that are heterogeneous with respect to morphology, surface marker, and other biological features.
Simultaneous analysis of cell surface antigens, bromodeoxyuridine incorporation and DNA content
Authors: Houck David W. , Loken Michael R.
Published: August 2005

A method has been developed for correlating the presence of cell surface markers with bromodeoxyuridine (BrdUrd) uptake. We have found that paraformaldehyde fixation will maintain immunofluorescent cell surface staining through acid denaturation that is necessary for anti‐BrdUrd reactivity to single‐stranded DNA. In addition, the forward angle light scattering was maintained, eventhough the cells had been exposed to 2 N HCl and detergent. The protocol was tested on three model systems: mouse thymus, a human cell line (CCRF‐SB), and peripheral blood leukocytes. It was found that there was no specific loss of lymphocyte subsets.
Multivariate analysis and list mode processing of murine hemopoietic subpopulations for cytokinetic studies
Authors: Pallavicini M. G. , Summers L. J. , Giroud F. J. , Dean P. N. , Gray I. W.
Published: August 2005

Multivariate analyses and list mode data processing were used to obtain cytokinetic information on flow cytometrically distinct hemopoietic subpopulations. In one application, viable, unfixed hemopoietic subpopulations were discriminated on the basis of cyanine dye fluorescence and orthogonal light scatter; Hoechst dye fluorescence was measured to determine the proliferative status of the subpopulations. In another application, ethanol‐fixed mouse bone marrow cells were triply stained with Hoechst dye, rhodamine‐conjugated wheat germ agglutinin (WGA), and a fluorescein‐labeled monoclonal antibody against bromodeoxyuridine. In both of these studies, flow cytometric data for all three variables were acquired in list mode fashion, stored on magnetic tape, and processed by list mode software on a computer based multivariable pulse‐height analyzer. In the first application, subpopulations distinguished by cyanine dye intensity and light scatter appeared to be more related to cell lineage and cell size than proliferative status. In the second application, WGA affinity discriminated two subpopulations in mouse bone marrow S‐phase cells in each subpopulation that actively incorporated bromodeoxyuridine (BrdUrd). List mode data processing obviates the need for routine electronic sorting of cells and thus facilitates characterization of discriminated subpopulations. In this regard, it is particularly useful for the study of flow cytometrically distinct, low frequency subpopulations.
Mapping of the pattern of DNA replication in polytene chromosomes from Chironomus thummi using monoclonal anti‐bromodeoxyuridine antibodies
Authors: Allison Linda , Arndt‐Jovin Donna J. , Gratzner Howard , Ternynck Thérèse , Robert‐Nicoud Michel
Published: August 2005

We present results from a nonautoradiographic study of DNA replication in polytene chromosomes from dipteran larvae. Monoclonal antibodies with specificity for 5‐bromodeoxyuridine (BrdUrd) were used to localize by indirect immunofluorescence the sites of BrdUrd incorporation and to follow the dynamics of DNA synthesis in salivary gland cells of 4th instar Chironomus thummi larvae. This technique presents numerous advantages over autoradiographic procedures and allows mapping of DNA synthesis patterns at the level of resolution of one chromosomal band. Several replication patterns were observed, classified according to characteristic features, and tentatively assigned to specific periods of the S‐phase. In early S‐phase, DNA synthesis is first detectable in puffs and inter‐bands, later in bands. Most chromosomalbands appear to initiate DNA synthesis synchronously; however, in bands within centromeric and heterochromatic regions the start of synthesis is delayed. At mid S‐phase, all the bands show uniform staining. Subsequent staining patterns are increasingly differential with the bands displaying characteristic fluorescence intensities. As replication progresses through the late S‐phase period, the chromosomes show a decreasing number of fluorescent bands. The last bands to terminate replication are located in centromeric and heterochromatic DNA‐rich regions and a few bands of low DNA content in region IIAa‐c.
The use of FITC‐conjugated gated monoclonal antibodies for determination of S‐phase cells with fluorescence microscopy
Authors: Gunduz Nurten
Published: August 2005

A method is modified to determine the DNA synthesizing cells in primary human breast tumors and cells with idiopathic thrombocytopenic purpura (ITP) with FITC‐conjugated monoclonal antibody against bromodeoxyuridine (FITC‐M‐anti‐BrdUrd) and fluorescence microscopy. The DNA synthesizing cells were also determined from a portion of the same tissues by classical tritiated thymidine labeling (3HdThd) and autoradiography. The results from bromodeoxyuridine labeling index (BrdUrd‐LI) and tritiated thymidine labeling index (3HdThd‐LI) obtained from the same tissues were compared. The mean BrdUrd‐LI for breast tumor was 5.4 ± 1.0% and the mean 3HdThd‐LI was 5.5 ± 1.1%. Similarly, the labeling indexes obtained from mononuclear leukocytes of healthy donors had means of 0.5 ± 0.1% and 0.6 ± 0.1% for BrdUrd‐LI and 3HdThd‐LI, respectively. The change in the proliferation rate of mononuclear leukocyte population in the samples obtained from patients with ITP could be observed by both methods. The mean BrdUrd‐LI of mononuclear leukocytes for this hematological disorder was 5.4 ± 0.9% and the mean 3HdThd‐LI was 6.1 ± 0.8%. These results suggest that this relatively simple technique offers an alternative method for determining the DNA synthesizing cells in a given cell population.
Direct and indirect flow cytomyetric enumeration of 6‐thioguanine‐resistant human peripheral blood lymphocytes
Authors: Matsson P. , Amnéus H. , Djupsjöbacka M. , Eriksson B. , Eriksson L. , Fellner‐Feldegg H. , Zetterberg G.
Published: August 2005

Methods based on flow cytometry and sorting, auto radiography, and cloning were used to evaluate the potential for the enumeration of 6‐thioguanine‐resistant human peripheral blood lymphocytes assumed to be deficient with respect to the enzyme hypoxanthineguanine‐phosphoribosyl‐transferase. Flow cytometric sorting of proliferating cells in the late S‐and the G2‐stages by means of DNA content, as measured by propidium iodide fluorescence, enabled an enrichment of variant cells to about 99%. The main source of false events was contaminating doublets of G0/G1 cells appearing in the sorting region. Doublet discrimination measured as the difference between pulse height and area (Ortho‐50) accomplished no further improvement. A combination of propidium iodide fluorescence and bromodeoxyuridine incorporation, measured by fluorescent anti‐bromodeoxyuridine‐DNA antibodies, allowed flow cytometric enrichment to about 99.99% of variant cells.

By sorting of 3H‐thymidine‐labeled cell nuclei from the late S‐and the G2‐phases and subsequent autoradiographic evaluation, partly resistant variants could be discriminated; variant frequencies of the same magnitude as for the cell cloning methods were obtained.
Detection of cytosine arabinoside resistant cells at low frequency using the bromodeoxyuridine/DNA assay
Authors: Waldman Frederic M. , Dolbeare Frank , Gray Joe W.
Published: August 2005

This report describes a rapid and sensitive procedure for detection of cytosine arabinoside‐ (Ara‐C) resistant mouse leukemia cells (L1210) in a predominantly Ara‐C‐sensitive population. L1210 cell lines sensitive or resistant to Ara‐C were grown and treated with Ara‐Cin vitroorin vivo. Ara‐C‐resistant cells were detected as those cells with S‐phase DNA content retaining the ability to incorporate bromodeoxyuridine (BrdUrd) after treatment with Ara‐C. The BrdUrd incorporation ability of the S‐phase cells was assessed by simultaneous flow cytometric measurement of cellular DNA content and amount of incorporated BrdUrd. The proportion of Ara‐C‐resistant cells was accurately estimated at frequencies approaching 10−3.
Cytopress: Automated slide preparation of cytologic material from suspension
Authors: Oud Peter S. , Haag Ditty J. , Zahniser David J. , Ramaekers Frans C. S. , Huysmans Anita C. L. M. , Veldhizen José A. M. , Verheyen René H. M. , Verrijp Kiek , Broers Jos L. V. , Herman Chester J. , Vooijs G. Peter
Published: August 2005

This paper describes a new automated system to prepare slides of cytological material from suspension. The system collects material on a filter tape by filtration and transfers it to glass slides by means of pressure‐fixation. Using cervical cells as a model, results show that a well‐defined cell number is evenly deposited over a standardized area, while a small number of cells is retained on the tape and a negligible number lost in the filtrate. Contamination is very small. Application of the system to other cytological material (fine needle aspirations, monolayer and cell suspension cultures, agar cultures, and isolated nuclei) is shown. In general, more than one slide can be made from one sample. Several histological staining procedures as well as immunofluorescence labeling protocols can be applied to the preparations obtained in this way. This system thus introduces a method that will standardize specimen preparation, is quick, saves operator time, and can be used for both diagnostic and research applications.
Changes in accessibility of DNA to various fluorochromes during spermatogenesis
Authors: Evenson D. , Darzynkiewicz Z. , Jost L. , Janca F. , Ballachey B.
Published: August 2005

Accessibility of mouse testicular and vas deferens (vas) sperm cell DNA to acridine orange, propidium iodide, ellipticine, Hoechst 33342, mithramycin, chromomycin A3, 4′6‐dimidino‐2‐phenylindole (DAPI), and 7‐amino‐actinomycin D (7‐amino‐AMD) was determined by flow cytometry. Permealized cells were either stained directly or after pretreatment with 0.06 N HCL. For histone‐contanining tetraploid, diploid, and round spermatid cells, HCI extraction of nuclear proteins caused an approximately sixfold increase of 7‐amino‐AMD stainability but had no signicant effect on DAPI stainability. For these same cell types, the stainability with other intercalating (acridine orange, propidium iodide, ellipticine) and externally binding (Hoechst 33342, mithramycin, chromomycin A3) dyes was increased by 1.6‐ to 4.0‐fold after HCI treatment. In sharp contrast, HCI treatment of vas sperm did not increase the staining level of 7‐amino‐AMD, DAPI, or propidium iodide but did increase the staining level for the other intercalating dyes (1.3‐ to 1.5‐fold) and external dyes (1.3‐ to 1.9‐fold). Elongated spermatids that contain a mixture of protein types including histones, transition proteins, and protamines demonstrated the greatest variability of staining with respect to type of stain and effect of acid extraction of proteins. In general, for nearly all dyes, the round spermatids had an increased level and tetraploid cells had a decreased level of stainability relative to the same unit DNA content of diploid cells. The observed differential staining is discussed in the context of chromatin alterations related to the unique events of meiosis and protein displacement and replacement during sperm differentiation.
Interactive multi‐window integration of two‐parameter flow cytometric data fields
Authors: Kachel Volker
Published: August 2005

Integration is necessary to determine the particle content of regions of interest of flow cytometric two‐parameter fields. The improved program of the Cytomic 12 analyzer (1) offers: window trace integration for relatively simple window structures. The field of interest is surrounded by an integration trace (window). Eight independent windows can be stored and successively evaluated. It also offers painted field integration for complicated window structures. The pointer or a small window is interactively moved over the structures to be integrated like the brush of a painter. The “painted field” defines the window to be integrated. Window sets and painted fields can be stored on a floppy disk. Painted fields can be added and may also serve as look up tables for sorting.
Programming for the DNA analysis of FCM data on an IBM microcomputer
Authors: Pavlik Edward J. , Flanigan Robert C. , Bartmas Jonathan , Van Nagell John R. , Donaldson Elvis S. , Hanson Michael B. , Gallion Holly , Kenady Daniel E.
Published: August 2005

The analysis of data generated on a flow cytometer (FCM) is often performed on a computer obtained especially for dedicated use with the flow cytometer. This computer component can be expensive and also presents the FCM user with the added burden of mastering specialized programming language or of accepting the secret analytical processes of protected proprietary program routines. We believe that the evolution of more accurate and efficient FCM analyses that have the power to consider complex signal distributions can be assisted by the availability of analysis programs written in languages common to many users. DNA analysis routines written for a relatively inexpensive micro‐computer (IBM PC/XT) in Basic and Pascal are described here. The routines can automatically process multiple FCM data files and can provide high‐resolution graphic hardcopy. A foreground/background utilization is also described that allows the computer to be available for other uses in the laboratory.
Flow cytometry of DNA content using oxazine 750 or related laser dyes with 633 nm excitation
Authors: Shapiro Howard M. , Stephens Sandra
Published: August 2005

The laser dyes oxazine 750 (OX750), LD700, and rhodamine 800 (R800) can be used in an instrument employing a low‐power heliumneon laser source for flow cytometry of DNA content in ethanol‐fixed or detergent‐permeabilized cells. Cells in near‐isotonic medium are stained with 10–30 μM dye, and fluorescence excited at 633 nm. The dyes do not appear to stain RNA, and the intensity of DNA staining is not changed when 2 μMHoechst 33342 is added to cells simultaneously with redexcited dye. The effects on fluorescence of addition of DNA to LD700 or R800 in aqueous solution are strongly influenced by the base composition of the DNA; binding mechanisms remain to be determined.
Analysis of protein incorporation of radioactive isotopes in the chinese hamster ovary cell cycle by electronic sorting and gel microelectrophoresis
Authors: Pipkin James L. , Anson Jeanne F. , Hinson William G. , Schol Henry , Burns E. Robert , Casciano Daniel A.
Published: August 2005

The patterns of [3H]‐leucine and [32P]‐phosphate incorporation of proteins extracted with varying molarities of sodium chloride were analyzed from nuclei physically sorted from six fluorescence windows after propidium iodine staining of the G0 + G1 and G2 + M phases of the Chinese hamster ovary (CHO) cell cycle. Eight hundred nanograms of protein were used in each electrophoretic analysis obtained from 200,000 nuclei, a portion of the sample, from each window. Autoradiography was performed in a two‐dimensional polyacrylamide gel ultra‐microelectrophoresis apparatus (UMEA) designed and fabricated in this laboratory. There was a net reduction and/or loss of [3H]‐leucine‐ and [32P]‐phosphate‐labeled protein regions from the autoradiographs occurring primarily in the G2 + M phase. Two phosphorylated proteins that were stage specific were observed in partitions of the G2 + M phase. The use of isolated proteins and the coelectrophoresis of these markers demonstrated the similarity in mobility of a number of proteins seen in the autoradiographs of proteins extracted with high and low salt molarities and implied they are synonymous. Coelectrophoresis indicated that a substantial number of high molecular weight proteins that decreased or disappeared at late stages of G2 + M and early mitosis were composed, in part, of nucleolar proteins.
Quantitation and sorting of vitally stained natural killer cell‐target cell conjugates by dual beam flow cytometry
Authors: Storkus Walter J. , Balber Andrew E. , Dawson Jeffrey R.
Published: August 2005

We have detected formation of stable associations, or conjugates, between fluorescein diacetate‐ (FDA) stained human natural killer (NK) cells and Hoechst 33342‐ (HO342) stained tumor cells by dual laser flow cytometry. Conjugates in mixtures of effectors and targets emitted both green (FDA) and blue (HO342) fluorescence. This was confirmed by cell sorting. More than 90% of the conjugates included one target and one effector cell. Conjugate formation frequency was temperature independent between 4 and 37°C, optimized by 10 min, and stable for 1 hr. Enrichment of effector populations for cells mediating lysis of standard NK targets and for cells reacting with OKM1, Leu‐7, and Leu‐11b monoclonal antibodies also enriched conjugate‐forming cells. Lysis of either OKM1+, Leu‐11b+ effector subpopulations with antibody and complement eliminated, but treatment with these antibodies alone had no effect on, conjugate formation. Effector pretreatment with Leu‐4 or 3A1 and complement increased the frequency of conjugation slightly. Flow‐determined frequencies of NK‐conjugate formation with 14 target cell lines correlated well with data derived from standard microscopic assays. However, the flow method was more rapid, could be used when target and effector were of comparable size, and permitted isolation of conjugates by sorting.
Physical discrimination between human T‐lymphocyte subpopulations by means of light scattering, revealing two populations of T8‐positive cells
Authors: Terstappen L. W. M. M. , De Grooth B. G. , Nolten G. M. J. , Ten Napel C. H. H. , Van Berkel W. , Greve J.
Published: August 2005

Light‐scattering properties of human T‐lymphocyte subpopulations selected by immunofluorescence were studied. Based on differences in orthogonal light scattering, two subpopulations of T8‐positive cells can be distinguished. The first population (T8a) has the same orthogonal light‐scattering properties as T4‐positive cells, whereas the orthogonal light scattering of the second population (T8b) was about 70% larger. Orthogonal light scattering of Leu7‐positive lymphocytes resembles that of the T8b population. We have studied the occurrence of the subpopulation in healthy individuals and we discuss their possible functional identification. Light‐scattering properties of lymphocyte subpopulations in two patients with B‐cell chronic lymphatic leukemia suggest that this observation is of clinical interest.
Malignancy index based on flow cytometry and histology for renal cell carcinomas and its correlation to prognosis
Authors: Baisch H. , Otto U. , Klöppel G.
Published: August 2005

Fifty‐five human renal cell carcinomas, removed by nephrectomy, were classified using flow cytometry and histology. The parameters obtained by flow cytometry were DNA index, fractions of cells in the phases, and fractions of tumor cells versus normal cells. From histology, the routine classification of tumor structure and morphology were obtained, and a nuclear grading according to Arner et al. (Acta Chir Scand [Suppl] 346:1–12, 1965) was determined. All parameters including tumor stage according to Robson et al. (J Urol 101:297–301, 1969) were subjected to discriminant analysis in order to define a malignancy index. The patients were divided into two groups with good and poor prognosis (low and high risk). Those patients who were free of multiple metastases in 2 years and more after nephrectomy were assigned to the group “good prognosis”; those who developed multiple metastases for 2 years represented the “poor prognosis” group. The malignancy index determined by discriminant analysis yielded 91% correct predictions of prognosis.
A statistical analysis of prescreening alarms in a population of normal and abnormal gynecologic specimens
Authors: Wheeless L. L. , Robinson R. D. , Cox C. , Berkan T. K. , Reeder J. E.
Published: August 2005

A multidimensional slit‐scan flow system has been developed to serve as an automated prescreening instrument for gynecological cytology. Specimens are classified abnormal based on the number of cells having elevated nuclear fluorescence (alarms). An alarm region in a bivariate histogram of nuclear fluorescence versus nuclear‐to‐cell‐diameter ratio is defined. Alarm region probability arrays are calculated to estimate the probability that an alarm falling in a particular bin of the alarm region is either from a normal or an abnormal specimen. From these arrays, a weighted alarm index is generated. In addition, summary indices are derived that measure how the distribution of alarms in each specimen compares with the average distributions for the normal and abnormal specimen populations. These indices together with current features are evaluated with respect to their utility in specimen classification using a nonparametric classification technique known as recursive partitioning. Resulting classification trees are presented that suggest information in the distribution of alarms in the bivariate histogram. In addition, they validate the features and rules currently used for specimen classification. Recursive partitioning appears to be useful for multivariate classification and is seen as a promising technique for other applications.
Flow cytometric analysis of hairy cell leukemia using right‐angle light scatter
Authors: Van Bockstaele Dirk R. , Berneman Zwi N. , Peetermans Marc E.
Published: August 2005

We report on an easy and reliable method for the enumeration of the typical cells present in the blood, bone marrow, and spleen of patients with hairy cell leukemia. Samples of five patients were analyzed. In three patients, the hairy cells were very accurately followed during treatment with alpha‐interferon, using right‐angle light scatter to differentiate them from other leukocytes present in total blood. Excellent correlation was obtained with the microscopic hairy cell enumeration. Additional verification by cell sorting and immunofluorescence studies confirmed these findings.
Binding of Hoechst 33258 to chromatin in situ
Authors: Stokke Trond , Steen Harald B.
Published: August 2005

The binding of Hoechst 33258 to rat thymocytes, human lymphocytes, and NHIK 3025 tissue culture cells was studied by measuring the fluorescence and light scattering of the cells as functions of dye concentration using flow cytometry. The results indicated that there were two different modes of binding of Hoechst 33258 to chromatin in situ at physiological pH. Type 1 binding, which dominated at total dye/phosphate ratios below 0.1 (0.15, M), was characterized by a binding constant of the order 107 M−1 and fluorescence with high quantum yield. Further binding of the dye resulted in a reduced blue/green fluorescence ratio, indicating that secondary sites were occupied. Binding at secondary sites above a certain density (0.1 ≤ bound dye/phosphate ≤0.2) induced strong quenching of fluorescence and precipitation of chromatin. Precipitation was quantitated by measuring the large‐angle (≥ 15°) light scattering of the cells above 400 nm, i.e., outside the Hoechst 33258/DNA absorption spectrum, as a function of dye concentration. In contrast, the light scattering at 365 nm, i.e., within the absorption spectrum of Hoechst 33258/DNA, was independent of the total dye/phosphate ratio. The coefficient of variation of the light‐scattering (≥ 400 nm) histograms decreased with Hoechst 33258 concentration. Type 2 binding to histone‐depleted chromatin was cooperative (Hill‐coefficient ≈ 2) and the apparent binding constant was 2–3 · 105 M−1 as determined from quenching and precipitation data. The final stoichiometry in the dye/chromatin complex calculated from quenching data was q = 0.70 (bound dye/phosphate ratio), while light‐scattering data yielded q = 1.35. The dye‐induced precipitation of intact chromatin of human lymphocytes was not cooperative (Hill‐coefficient ≈ 1.0). The precipitation of DNase 1 digested chromatin, however, was described by two‐component kinetics. The components had Hill‐coefficients of 1.0 and 2.4, respectively; the appearance of the latter was interpreted as reflecting precipitation of relaxed chromatin. Histone‐depletion increased the number of Hoechst 33258 type 1 binding sites in chromatin by about 30%, while the dye‐induced increase in light scattering decreased by about 40%.
Characterization of f‐Met‐Leu‐Phe‐stimulated fluid pinocytosis in human polymorphonuclear leukocytes by flow cytometry
Authors: Davis Bruce H. , McCabe Elizabeth , Langweiler Marc
Published: August 2005

N‐formylated chemotactic peptide stimulation of human neutrophils initiates a number of cellular processes, such as lysosomal enzyme release and superoxide anion production, that are indicative of the events of neutrophil activation during the acute inflammatory response in disease. This study characterizes a newly recognized neutrophil activation event, N‐formylated chemotactic peptide‐stimulated fluid pinocytosis in human neutrophils, using a novel flow cytometric assay for this activity. Fluid pinocytosis was found to be inhibited by acidic pH and low temperature but could be enhanced by cytochalasin B treatment or surface adherence by neutrophils. The activity measured by this new assay of fluid pinocytosis appears to be separate and distinct from lysosomal enzyme release and receptor‐mediated adsorptive endocytosis in neutrophils. The physiologic significance of N‐formylated chemotactic peptide‐stimulated fluid pinocytosis is not known, but a possible relationship to neutrophil locomotion is discussed.
Measurement of myeloid maturation by flow cytochemistry in HL‐60 leukemia: Esterase is inducible, myeloperoxidase is not
Authors: Ross Dennis W.
Published: August 2005

The phenomenon of leukemic cell maturation requires a measurment of myeloid maturation to understand the process and to exploit it as a means of therapy for leukemia. The HL‐60 leukemic cell line was used as a model of induced leukemic cell maturation in order to develop a method of quantitating granulocytic and monocytic maturation in response to drug therapy. An automated flow cytochemistry system (Hemalog‐D) was employed to measure mean cell volume, myelo‐peroxidase (MPO), and nonspecific esterase (NSE). For granulocytic maturation induced by vitamin A or DMSO, MPO and cell volume decreased by 50%, maintaining a constant mean cellular MPO concentration throughout maturation from promyelocyte to neutro‐phil‐like forms. For monocytic maturation induced by low‐dose ARA‐c, the mean NSE increased substantially, while cell volume remained constant. Unlike MPO concentration, NSE was truly inducible and thus a useful quantitative measure of maturation caused by low‐dose ARA‐c. Flow cytochemistry and cytofluorometry may be developed to allow for quantitative monitoring of therapeutic trials of induced maturation in human leuke‐mias. However, this will require adapting these techniques to the complexity of human leukemias in vivo, and the necessity of handling heterogeneous populations encountered in bone marrow samples.
Modification of a laser‐based flow cytometer for high‐resolution DNA analysis of mammalian spermatozoa
Authors: Johnson L. A. , Pinkel D.
Published: August 2005

Modification of a Coulter EPICS V orthogonal laser‐based flow cytometer/cell sorter allows resolution of X and Y mammalian sperm populations based on DNA content. The modification consists of beveling the sample injection tube situated in the flow chamber, adding a second fluorescence detector directly forward along the laser beam axis, and routing the collected fluorescence through an optical fiber bundle to one of the existing photomultiplier tubes. The X and Y chromosome‐bearing spermatozoa from the bull, boar, and ram can be resolved using this system.
G2 arrest, binucleation, and single‐parameter DNA flow cytometric analysis
Authors: Ronot Xavier , Hecquet Christiane , Larno Suzanne , Hainque Bernard , Adolphe Monique
Published: August 2005

One important facet of flow cytometry involves the effects of pharmacological agents on cell cycle progression. Comparative G2 fraction perturbations were examined: effects of sodium butyrate on articular chondrocytes, effects of an antineoplastic agent (SOAz) and an antirheumatic drug (D‐penicillamine) on HeLa cells. Even though DNA flow cytometric analysis detects preferentially an induction of G2 arrest, the mode of action of these agents on the cell cycle is different. Sodium butyrate and D‐penicillamine lead to an increase of binucleate cells due to cytokinesis perturbation. Because of similar fluorescence intensity, distinguishing G2 from binucleate GO/1 cells is not easily possible using DNA content measurement and reflects a failure of flow cytometry in the detection of binucleate cells. Rapid cell cycle analysis of single cells should contribute greatly to the study of pharmacological interactions, but DNA flow cytometric measurements obtained from cultured cells exposed to certain agents must be cautiously interpreted because those may interact on cytokinesis and induce artefacts in histogram interpretation.
Accuracy and reliability of flow cytometric DNA analysis using a simple, one‐step ethidium bromide staining protocol
Authors: Petersen S. E.
Published: August 2005

Sources of variation and error were investigated for a simple flow cytometric analysis of DNA content of detergent‐isoiated nuclei stained with ethidium bromide. Using the ploidy classes of mouse liver nuclei, deviations from linearity were assessed for three different instruments. In more extreme settings, the maximum deviations for a FACS instrument were up to 6 to 9%, but in general deviations were around 1% or lower for all instruments. As biological DNA standards, human peripheral lymphocytes and trout erythrocytes appeared to be suitable and easy to store frozen. The erythrocytes had dyebinding characteristics similar to those of human lymphocytes and a 20% lower fluorescence, thus being well suited as an internal standard, as was demonstrated in tumor ploidy analyses performed with varied tissue concentration. Staining homogeneity was improved when staining time was extended to 24 h, at which time male and female lymphocytes were completely separated with an average difference in DNA content of 1.9%. A small difference in fluorescence between mitogen‐stimulated and unstimulated lymphocytes was reduced to less than 1% after 24 h of staining. In general, the manipulations of the conditions for the analysis resulted in maximum variations of around 1%, indicating the robustness and reliability of the technique.
The use of an image analyser in human tumour clonogenic assays
Authors: Kahn Edmond , Benard Jean , Di Paola Robert
Published: August 2005

The counting of tumoral colonies growing in semisolid media must be achieved in human tumour clonogenic assays. An image analyser can be advantageously used to overcome the limit of the eye when performing this task. Colonies growing from human cancer cell lines were analysed. Quantitative results on the cytotoxic effect of chemotherapeutic agents were obtained that could not be reached by means of the eye.
DNA and nuclear protein measurement in isolated nuclei of human endometrium
Authors: Oud Peter S. , Reubsaet‐Veldhuizen José A. M. , Henderik Jos B. J. , Pahlplatz Martin M. M. , Hermkens Huub G. , Tas Johan , James Jan , Peter Vooijs G.
Published: August 2005

Feulgen‐DNA and nuclear light green‐protein measurements have been performed in isolated nuclei of normal (nonmalignant) and malignant human endometrial homogenates.

The DNA content of the GO/G1 fraction of malignant endometrium showed much overlap with that of normal endometrium, or was slightly increased. Two of the 18 carcinomas were clearly aneuploid. No correlation was found between the histological grade and the DNA content. The tumors of clinical stage II and higher all had a higher DNA content than that of normal endometrium.

The percentage of cells present in the proliferative fraction was higher in proliferative endometrium than in secretory and postmenopausal atrophic endometrium. For malignant endometrium, percentages were found comparable to that of normal endometrium or higher. No correlation was found with the histological grade. Tumors of stage II and higher had intermediate values compared to those of carcinomas below stage II.

The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. However, for postmenopausal women, most values of the carcinomas exceeded that of normal, atrophic, endometrium. Within the tumor population, no correlation was found with the histological grade. Higher values were found with tumors of clinical stage II and higher.
DNA and nuclear protein measurement in columnar epithelial cells of human endometrium
Authors: Oud Peter S. , Reubsaet‐Veldhuizen José A. M. , Beck Hans L. M. , Pahlplatz Martin M. M. , Hesselmans Gerard H. F. M. , Hermkens Huub G. , Tas Johan , James Jan , Peter Vooijs G.
Published: August 2005

Propidium iodide DNA flow cytometry, Feulgen‐DNA, and nuclear light green protein scanning cytometry were performed in columnar epithelial cells of normal, nonmalignant human endometrium and endometrial adenocarcinomas. Columnar cells were identified by immunohistochemical staining for cytokeratin 18, an intermediate filament protein specifically present in columnar cell epithelium. DNA measurements derived from flow and scanning cytometry showed comparable results. The DNA content of the G0/G1 fraction of the adenocarcinomas had a considerable overlap with that of normal endometrium, with that of the carcinomas shifted toward higher values. For the carcinomas, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas. Most of the clinical stage I tumors showed a DNA content in the normal diploid region. Three of the four carcinomas of clinical stage II and higher had an increased DNA content.

For the carcinomas, the percentage of cells in the proliferative fraction, as determined from scanning cytometric derived DNA histograms, was comparable to that of normal endometrium, or higher. No correlation was found with the histological grade. Tumors of clinical stage II and higher had intermediate values compared to carcinomas of lower stages.

The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. Within the tumor population, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas, and clinical stage.

Based on the aforementioned parameters, no discrimination could be obtained between normal and malignant endometrium. However, when the DNA content of the G0/G1 fraction was combined with the coefficient of variation of the nuclear protein/DNA ratio, a clear discrimination could be obtained with only two false‐positive cases.