bluuebunny/crossref_metadata_2025 huggingface dataset:
Automated absorption image cytometry of electromagnetically exposed frog erythrocytes
Authors: Chiabrera A. , Viviani R. , Parodi G. , Vernazza G. , Hinsenkamp M. , Pilla A. A. , Ryaby J. , Beltrame F. , Grattarola M. , Nicolini C.
Published: August 2005

Frog erythrocytes dedifferentiate in vitro when exposed to a suitable electromagnetic field in a chemically potentiated microenvironment. These electrochemical stimuli are able to induce a chromatin decondensation, as previously shown by measuring the chromatin accessible sites for acridine orange intercalation by means of flow cytometry (acridine orange green fluorescence). Automated absorption image cytometry of Feulgen stained smeared erythrocytes has been performed to further elucidate the above processes. After measuring or computing the area A of the nucleus, which is related to its volume, the nucleic integrated optical density D, which is related to the DNA content, the average optical density DA = D/A, which is related to chromatin conformation, and the accessible chromatin sites SA = D2/3 A1/3, the following results have been obtained: (a) electromagnetically exposed cells progress to stages which correspond to values of D equal to those of controls, in the potentiating solution, whereas A increases, so that DA is smaller in exposed erythrocytes than in control ones, confirming that chromatin decondensation is occurring as an early dedifferentiation step. (b) By using the electromagnetic signal that is most effective in promoting dedifferentiation, erythrocytes further progress toward more advanced stages, which correspond to larger values of both D and A than in controls, i.e., to larger DNA content. (c) In all cases, the histograms of SA are in agreement with those previously obtained by flow microfluorometry of chromatin conformation (acridine orange green fluorescence). Finally, flow microfluorometric measurements of acridine orange red fluorescence give an increase of RNA content for case (a), as compared with controls, and for case (b) as compared with (a). These results point out that frog erythrocytes can be electromagnetically reactivated, resuming both RNA and DNA syntheses after initial chromatin decondensation.
A model for the computer analysis of synchronous DNA distributions obtained by flow cytometry
Authors: Fox Michael H.
Published: August 2005

A new model for obtaining the distributions of cells in the cell cycle by computer analysis of flow cytometry data is proposed. The model is able to analyze distributions from both asynchronous and synchronous cell populations. A culture of HeLa cells is pulse‐labeled with tritiated thymidine at various times after sychronization by the mitotic selection method, then analyzed by flow cytometry and autoradiography. The results show good agreement between the percentage S‐phase cells determined by the model and by autoradiography.
Selection of viable cells with known DNA content
Authors: Hamori Eugene , Arndt‐Jovin Donna J. , Grimwade Brian G. , Jovin Thomas M.
Published: August 2005

Cells doubly stained with Hoechst 33342 (H‐33342) for DNA content and fluorescein diacetate for viability can be selected on the basis of both criteria using a single UV laser flow sorter. The selection is made possible due to resonance energy transfer occurring between the H‐33342 and fluorescein fluorophores. Both a static fluorescence microscope and a dual laser flow sorter were used to demonstrate that energy transfer occurs in the doubly stained cells and that the sensitized emission in conjunction with the DNA emission can be used to select populations of cells with known DNA content and viability. The results indicate that fluorescein liberated by cellular esterases is freely accessible to the nucleus.
Percentage of cells in the S phase of the cell cycle in human lymphoma determined by flow cytometry. Correlation with labeling index and patient survival
Authors: Braylan Raul C. , Diamond Lawrence W. , Powell Marie L. , Harty‐Golder Barbara
Published: August 2005

The percentage of cells with S phase DNA content (S cells) was determined by flow cytometry in cell suspensions obtained from 27 patients with non‐Hodgkin's lymphomas. DNA was measured in ethanol‐fixed cells stained with propidium iodide after RNase treatment. Cells from 17 samples were also exposed to [3H]‐thymidine and labeling indices were determined. An excellent linear correlation was observed between the percentage of S cells calculated by flow cytometry and the labeling index (r = 0.95). To determine the relationship between the percentage of S cells and the clinical behaviour of the tumors, the survival of the 27 patients was analyzed. None of the patients was undergoing antineoplastic therapy at the time of the study. The patients were separated into 2 groups according to the percentage of S cells calculated by flow cytometry: those with less than 5% S cells and those with higher than 5% S cells. Clinical follow‐up ranged from 5 to 20 months. While 7 of 12 patients with tumors containing more than 5% S cells have died of lymphoma (median survival, 9 months), there has been only one death from lymphoma among 15 patients with tumors containing less than 5% S cells. The difference in survival between these two groups is statistically significant (P = 0.01).
Flow cytometric determination of esterase and phosphatase activities and kinetics in hematopoietic cells with fluorogenic substrates
Authors: Malin‐Berdel J. , Valet G.
Published: August 2005

Viable and formaldehyde fixed rat bone marrow or spleen cells and rat or human blood leukocytes were incubated with fluorescein (fluorescein‐diacetate, ‐dibutyrate and ‐laurate) or umbelliferone (4‐methylumbelliferyl acetate and ‐phosphate) fluorogenic substrates for subsequent flow cytometric determination of cell fluorescence and cell volume. Formaldehyde‐fixed cells preserved between 14 and 20% of the enzyme activity of the unfixed cells and the number of cell clusters for fixed and unfixed cells was the same. The esterase substrates revealed one cell cluster for spleen cells, two for bone marrow cells and four for peripheral blood leukocytes. Phosphatase activity was only associated with a cell cluster of large cells. The time course of substrate cleavage was linear during the first 10 min of incubation. Later on a plateau was reached. Specific enzyme activities were calculated on a single cell level from the simultaneous cell volume and cell fluorescence measurement. Two enzyme activites could be measured simultaneously by using a substrate mixture of umbelliferone acetate and fluorescein acetate.
Preparation of tissues for DNA flow cytometric analysis
Authors: Thornthwaite Jerry T. , Sugarbaker Everett V. , Temple Wally J.
Published: August 2005

A method for measuring DNA in tissue cells by flow cytometry utilizing a one step combination nuclear isolation‐DNA fluorochrome staining procedure is described. A variety of cells and tissues, both in vivo and in vitro, was used to illustrate the universal nature of this technique. These included murine bone marrow, liver testicle, sarcoma brain tumor, rat pancreatic islets, human peripheral blood, colon mucosa, colon cancer, sarcoma and brain tumor tissues. A special nuclear isolation medium, which contained either of the DNA fluorochromes, 4′,6‐diamidino‐2 phenylindole‐2 HCl or propidium iodide, was utilized successfully to isolate single suspensions of DNA fluorochrome stained nuclei in a rapid (5–10 min), consistent manner from a variety of tissues and cells. Multiple sampling of the same tissue or comparison between whole tissues and their single cell isolates showed that a representative sample was being obtained.
Nozzle design for the generation of plane liquid surfaces
Authors: Eisert Wolfgang G. , Dennenloehr Manfred
Published: August 2005

Measurements besides total fluorescence intensity of more elaborate optical techniques such as light scattering, extinction as well as fluorescence polarization and slit scan profiles have been reported in various cell sorter design studies. However the cylindrical liquid stream in air, common to many research system, limits the optical properties of the system. We report the successful application of three new types of nozzles, all having in common the fact that the main optical axes intersect plane liquid surfaces. This has been achieved on the basis of a double sheath arrangement, which we have reported previously. The nozzles generate fluid streams with either rectangular, square or triangular cross‐sections at the location of the optical axis. The latter facilitates the alignment of two beams with different wavelengths on the same optical axis within the liquid. Square or rectangular cross‐sections improve light scattering and extinction measurements and may well be better suited to more complicated geometrical analysis of cells and particles in flow streams.
Centrifugal separation of cells in sputum specimens from patients with adenocarcinoma
Authors: Pressman Norman J. , Albright Craig D. , Frost John K.
Published: August 2005

A Ficoll density gradient centrifugation technique was used to separate fixed cells from the sputa of patients with bronchogenic adenocarcinoma. The selective enrichment of leukocytes, squamous or cancerous cells was achieved while retaining the diagnostically significant cell morphologic features. This technique is shown to typically eliminate most of the leukocytes (96.9%), macrophages (86.7%), squamous cells (70.0%) and necrotic debris from specimen fractions containing the majority of cancerous cells. The maximum purity of cancer cells (14.6%) occurs in high density (ρ=1.135 g/ml) gradient fractions and results in an average 12.2‐fold enrichment for cancer cells in contrast to their relative frequency of occurrence in unprocessed specimens. Differential cell count analyses of serial density centrifugation fractions show that this technique produces comparable enrichment rates for material from bronchogenic adenocarcinoma and squamous cell carcinoma.
Identification of mitogen responding lymphocytes by fluorescence polarization
Authors: Udkoff Ranon , Chan Sandra , Norman Amos
Published: August 2005

Measurements of the intensity and polarization distributions of fluorescein fluorescence in human lymphocyte populations show changes within minutes after exposure of the cells to the mitogens phytohemagglutinin and Concanavalin A. The distributions of polarization, which before mitogen exposure show essentially a single peak, after exposure develop a second peak at a lower value of polarization. Simultaneously, the intensity distributions show a shift to higher levels of intensity. These shifts can be modeled on the basis of a subpopulation of lymphocytes responding to mitogen. Interpretation of the results is complicated by many factors that may influence the shape of the polarization distributions. In particular we show that the measurements are sensitive to the concentrations of phytohemagglutinin, Concanavalin A, Ca++, K+, fluorescein diacetate, the incubation time and the specific donor. Nevertheless, fluorescein fluorescence measurements provide a rapid and sensitive method for studying the physiology of lymphocytes and for identifying responding lymphocyte subpopulations.
Rapid analysis of drug effects on the cell cycle
Authors: Darzynkiewicz Zbigniew , Traganos Frank , Xue Shaobai , Staiano‐Coico Lisa , Melamed Myron R.
Published: August 2005

Using a flow cytometric technique to analyse DNA content and chromatin structure simultaneously, the following parameters of cell cycle progression were estimated in control and drug‐treated L1210 cell cultures: (a) the kinetics of cell exit from the G1 phase; (b) the probability of cell exit from the indeterminate portion of the G1 phase, measured as the half‐time of cell residence in that state; (c) the duration of the deterministic portion of G1 phase; (d) the rates of cell transit through selected “windows” in S phase; (e) the rate of cell entrance into mitosis; (f) the mean duration of the cell cycle (Tc). These parameters are obtained in a single stathmokinetic experiment from measurements of individual samples withdrawn at 30 min‐1 hr intervals from Vinblasatine‐treated cultures. In the same experiment mitotic indices are obtained with high statistical accuracy, and may be used to determine the terminal point of drug action. In addition to cell cycle analysis the method makes it possible to detect drug‐induced changes in nuclear chromatin that are manifested by varying sensitivity of DNA in situ to denaturation by acid. Such changes were found to be associated with defective chromatin condensation, altered histone modifications or intercalation of the drugs into DNA. Using this technique the effects of sodium n‐butyrate and two new antitumor drugs on L1210 cells were investigated.
Texture of white blood cells expressed by the counting densitogram,
Authors: Bins M. , Landeweerd G. H. , Gelsema E. S. , van Montfort L. H. , Halie M. R.
Published: August 2005

The counting of particles and holes in white blood cells during thresholding at different gray levels results in a histogram, the counting densitogram, which contains information about granularity. Parameters describing the shape of this histogram appear to be sufficiently characteristic for the five normal white blood cell classes to allow a 84% correct discrimination between them. This method to quantitate granularity could be valuable for the analysis of cell texture and therefore, when used in combination with geometrical and density histogram parameters, could contribute to the results of morphometric discrimination between other than normal white blood cells.
Modular computer programs for flow cytometry and sorting: The LACEL system
Authors: Salzman G. C. , Wilkins S. F. , Whitfill J. A.
Published: August 2005

A computer‐based data acquisition, display and processing system for flow cytometers has been developed. The Los Alamos Cell Analysis (LACEL) programs and electronic hardware provide the capability to acquire list mode or histogram data for up to eight parameters and control bidirectional sorting based on up to four parameters for each direction. The programs described in this paper also enable the display of single parameter and bivariate histograms and the graphical manipulation of the list mode data. The electronic hardware is described in a companion paper.
Modular electronics for flow cytometry and sorting: The LACEL system
Authors: Hiebert R. D. , Jett J. H. , Salzman G. C.
Published: August 2005

LACEL is a newly developed, fast, general‐purpose data acquisition and processing system for flow cytometric applications. The system's modular electronics allows flexibility in system configurations. The system can process as many as eight input analog parameters and can transfer 16‐bit words between the user's electronics and the computer with standard input/output interfaces. The system's 8‐fold coincidence logic capability can be set to operate with the noncoincidental timing that may occur in multiparameter flow measurements. As many as four parameters can be used to establish amplitude and timing criteria for each of two sorting directions. Two experiments can be on line with the computer at one time.
Analysis of chromosomes from human peripheral lymphocytes by flow cytometry,
Authors: Matsson Per , Rydberg Björn
Published: August 2005

A method of preparation and flow cytometric analysis of chromosomes from human peripheral lymphocytes is described. The procedure allows a resolution coefficient of variation better than 3% using propidium iodide staining and a commercially available flow cytometer.
Measurement of DNA synthesis by flow cytometry
Authors: Noguchi Philip D. , Johnson Joyce B. , Browne Walter
Published: August 2005

Cells were stained for DNA content with Hoechst 33258 combined with propidium iodide and analyzed on an epi‐fluorescence flow cytometer. Scatterplots of red versus blue fluorescence of control cells showed a straight line indicating that both dyes were measuring proportional amounts of cellular DNA. Scatterplots of cells incubated with the thymidine analog bromodeoxyuridine, however, showed a displacement from the control straight line that increased with longer exposure time to bromodeoxyuridine. These results suggest that bromodeoxyuridine incorporation in DNA can be detected by flow cytometry.
The look‐up table: A classifier for cell sorters
Authors: Koper Ger J. M. , Christiaanse Johan G. M.
Published: August 2005

Currently used classifiers for flow cell sorters are described and the look‐up table is introduced as such. An experimental setup of a look‐up table classifier added to a commercially available cell sorter, under control of a microcomputer is described. The contents of the look‐up table are obtained from contrours of regions of interest. These regions are defined by the user who designates the areas on a video color display of the bivariate flow data. Since most cell clusters form elliptic or ovoid shapes in a multi‐dimensional feature space this method enables a better matching of window shapes to cluster outlines, compared to conventional sort decision circuitry.
The fluorescence intensity of propidium iodide bound to DNA depends on the concentration of sodium chloride
Authors: Martens Anton C. M. , van den Engh Gerrit Jan , Hagenbeek Anton
Published: August 2005

While performing DNA analysis with propidium iodide using the FACS‐II cell sorter, it was noted that the fluorescence intensity was not constant during the measurements. When different sheath and sample fluids were used, the fluorescence intensity of a given cell population increased or decreased during the measurement, depending on the concentration of sodium chloride in the fluids. By selecting the appropriate sheath and sample fluid combination, the drift in fluorescence intensity can be avoided.
Flow cytofluorometric analysis of the nuclear division cycle of Physarum polycephalum plasmodia
Authors: Murphy Robert F. , Daban Joan‐Ramon , Cantor Charles R.
Published: August 2005

The nuclear cycle kinetics of Physarum polycephalum plasmodia were examined using flow cytofluorometry. The dyes Hoechst 33342 and propidium iodide were used to stain the DNA of isolated nuclei. In asynchronously growing microplasmodia, S phase consists of 13–15% of the nuclear division cycle time. Nuclei isolated from individual macroplasmodia, which have previously been demonstrated to divide in synchrony, were shown to be less synchronized during late S phase than during mitosis. The results obtained demonstrate the feasibility of flow cytometric measurement of the properties of nuclei isolated from a single cell.
Cell cycle analysis by combining the 5‐bromodeoxyuridine/33258 hoechst technique with DNA‐specific ethidium bromide staining
Authors: Böhmer Ralph‐M. , Ellwart Joachim
Published: August 2005

Cells were grown in 5‐bromodeoxyuridine(BrdUrd)‐containing medium, harvested and stained with a combination of 33258 Hoechst and ethidium bromide for analysis in a two‐parametrical flow cytometer. Both fluorescences were excited by UV laser light, and ethidium bromide was additionally excited by energy transfer from the Hoechst dye. The separation of the two fluorescences proved to be excellent, the projected histograms showing a quality comparable to the quality obtained by single dye staining. The ethidium bromide fluorescence gives information as to where the cell is located within the cycle of DNA replication, while the BrdUrd‐quenched Hoechst fluorescence gives information as to where in the cycle the cell was located at the beginning of BrdUrd incorporation. In this way information is obtained concerning the distance a cell traveled through the cycle during the BrdUrd incubation time. This method may become a powerful tool in many investigations dealing with cell cycle perturbations in culture.
Simplified method for DNA and protein staining of human hematopoietic cell samples
Authors: Crissman Harry A. , Van Egmond Jan , Holdrinet Robert S. , Pennings Arie , Haanen Clemens
Published: August 2005

A rapid reproducible method yielding high resolution analysis of DNA and protein in human hematopoietic cell samples has been developed by modification of the propidium iodide and fluorescein isothiocyanate procedure. Cell staining involves sequential addition of each reagent (RNase, fluorescein isothiocyanate and propidium iodide) to ethanol‐fixed cells and requires no centrifugation steps. Stained cells are analyzed in the reagent solutions. Analysis of bone marrow samples from multiple myeloma patients showed mixed normal and aneuploid populations with a major portion of the aneuploid cells having a significantly higher protein content. This approach permitted differential cell cycle analysis of normal and the aneuploid populations.
Flow cytometric estimation of DNA and RNA content in intact cells stained with hoechst 33342 and pyronin Y
Authors: Shapiro Howard M.
Published: August 2005

The addition of RNA content estimation to flow cytometric measurement of DNA content provides valuable information concerning cells' transitions between quiescent and proliferative states. Equilibrium staining methods employing acridine orange have been used for DNA/RNA content measurement but are difficult to apply to intact cells and impractical for use in conjunction with fluorescent antibodies or ligands for demonstration of cell surface structures. I have used a combination of Hoechst 33342 (HO342) and pyronin Y (PY) to stain intact cells for DNA/RNA content estimation with a dual source flow cytometer using UV and blue‐green or green excitation, measuring HO342 fluorescence at 430–470 nm and PY fluorescence at 590–650 nm. Results obtained with cultured cells and stimulated lymphocytes are in good agreement with those obtained using acridine orange for DNA/RNA staining; about half of the PY fluorescence can be removed from ethanol‐fixed cells stained with HO342 and PY by RNAse digestion. The HO342/PY method can be combined with fluorescein immuno‐fluorescence for detection of cell surface markers. HO342 can be combined with other tricyclic heteroaromatic dyes for DNA/RNA estimation; the combination of HO342 and oxazine 1 can be excited in a dual source instrument using a mercury arc lamp and a helium‐neon laser. The staining procedure is simple; cells in medium are incubated with 5 μM HO342 at 37°C for 45 min, 5 μM PY (or oxazine 1) is then added and cells are analyzed without washing after an additional 45 min incubation. Suitability of these dye combinations for vital cell staining and sorting remains to be determined.
Measurement of the distribution of time intervals between cell passages in flow cytometry as a method for the evaluation of sample preparation procedures
Authors: Lindmo Tore , Fundingsrud Kåre
Published: August 2005

It is expected that the time sequence of cells passing through the sensing region of a flow cytometer should follow the statistics of a Poisson process. Thus, measurement of time intervals between passages of fluorescent microspheres and cells stained with mithramycin demonstrated the negative exponential distribution characteristic of such a process. However, analysis of cells stained with fluorescein‐iso‐thiocyanate showed marked deviations from a negative exponential distribution of time intervals. The distribution had an additional component due to time intervals with mean value about 60 times shorter than the population mean, and in certain cases more than 50% of the cells were registered with such short time intervals. This percentage could, however, be reduced to acceptable levels by modifications of the sample preparation procedure. Measurement of the distribution of time intervals between cell passages is proposed as a method for the evaluation of possible improvements in sample preparation procedures.
Mathematical evaluation of two parameter flow cytometric histograms
Authors: Scholz Karl‐U.
Published: August 2005

A general procedure was developed to evaluate two‐parameter flow cytometric data. These data usually show overlapping distributions representing different subpopulations. To calculate the fractions of cells in the various subpopulations, the maximum likelihood method is used, assuming an unknown number of Gaussian distributions with constant coefficient of variation in the histogram. A test on simulated data shows that Gaussians can be separated clearly by the procedure if the distance between their mean values is larger than twice the maximum standard deviation. The analysis of different measurements of double‐stained (DNA/protein) cultured cclls out of the same pool yield the same fractions in the phases of the cell cycle with acceptable error rates. To simulate samples with different fractions in the phases of the cell cycle, defined mixtures of proliferating and resting cells of the same type were prepared and measured for DNA and cell size. The calculated fractions are consistent with the expected variation of G1, S and G2 + M fractions depending on the mixture ratio.
The effect of hexamethylene‐bis‐acetamide on multiparameter analysis of friend leukemia cells
Authors: Mishal Z. , Fourcade A. , Tapiero H.
Published: August 2005

Friend leukemia cells were induced to differentiate by hexamethylene‐bis‐acetamide (HMBA). The relationship between cell growth cell volume distribution, fluorescence intensity and fluorescence anisotropy of the membrane probe 1,6 diphenyl 1,3,5 hexatriene (DPH) was analyzed in differentiated and undifferentiated cells with the aid of the fluorescence activated cell sorter (FACS II). The induction of a differentiated state was associated with a decrease in cell volume and an increase of fluorescence anisotropy. The inhibition of this induction by dexamethazone prevented the decrease of cell volume without affecting the increase of fluorescence anisotropy. We conclude therefore that the high degree of anisotropy is not correlated to the differentiated state nor with the volume of the cells.
G2‐delay after irradiation with α‐particles as studied in synchronized cultures and by the bromodeoxyuridine‐33258H technique
Authors: Hieber L. , Beck H.‐P. , Lücke‐Huhle C.
Published: August 2005

Division delay of mouse L‐929 fibroblasts after α‐irradiation is due to a pronounced lengthening of their G2‐phase. Experiments on synchronously and asynchronously growing cultures revealed a cell cycle phasedependent sensitivity of this effect: Cells irradiated in G2 or at the Gl/S border suffered a longer G2‐delay than cells irradiated at mid‐ or late‐S. Progression through G2 was nearly normal at doses up to 0.3 Gy if cells were exposed during Gl phase.
DNA content and kinetic characteristics of non‐Hodgkin's lymphoma: Determined by flow cytometry and autoradiography
Authors: Costa Aurora , Mazzini Giuliano , Bino Giacinta Del , Silvestrini Rosella
Published: August 2005

The determination of DNA content and [3H]thymidine labeling index was carried out on malignant lymph nodes from 74 patients with non‐Hodgkin's lymphoma. Analysis of cellular DNA content was performed using propidium iodide as DNA‐specific fluorescence dye. The ploidy was expressed as the DNA ratio between the relative DNA content of the human lymphoid G0/1 cells to that of chicken red blood cells. Forty‐five of the 74 non‐Hodgkin's lymphomas (61%) were aneuploid populations and the majority of these (91%) showed a hyperdiploid DNA content. A higher frequency of aneuploidy (72%) was observed in tumors with unfavorable histology than in those with a favorable histology (55%). Moreover, among aneuploid lyrophomas heterogeneous populations were observed in 24% of the cases. The evaluation of flow cytometric data using Fried's deconvolution procedure showed no statistically different frequency of G0/1, S and G2 + M cells between the two groups of tumors with favorable and unfavorable histology. On the contrary, a statistically different frequency of G0/1 and S cells was observed between the two groups of tumors with low and high labeling indices (P < 0.01). A correlation was found between autoradiographic and flow cytometric determination of S phase cells (P < 0.001).
High resolution DNA content measurements of mammalian sperm
Authors: Pinkel D. , Lake S. , Gledhill B. L. , Van Dilla M. A. , Stephenson D. , Watchmaker G.
Published: August 2005

The high condensation and flat shape of the mammalian sperm nucleus present unique difficulties to flow cytometric measurement of DNA content. Chromatin compactness makes quantitative fluorescent staining for DNA difficult and causes a high index of refraction. The refractive index makes optical measurements sensitive to sperm head orientation. We demonstrate that the optical problems can be overcome using the commercial ICP22 epiillumination flow cytometer (Ortho Instruments, Westwood, MA) or a specially built cell orientating flow cytometer (OFCM). The design and operation of the OFCM are described. Measurements of the angular dependence of fluorescence from acriflavine stained rabbit sperm show that it is capable of orienting flat sperm with a tolerance of ± 7 degrees. Differences in the angular dependence for the similarly shaped bull and rabbit sperm allow discrimination of these cells. We show that DNA staining with 4‐6 diamidino‐2‐phenylindole (DAPI) or an ethidium bromide mithramycin combination allows resolution of the X and Y populations in mouse sperm. They have also been successful with sperm from the bull, ram, rabbit, and boar. Reliable results with human sperm are not obtained. The accuracy of the staining and measurement techniques are verified by the correct determination of the relative DNA content of these two populations in sperm from normal mice and those with the Cattanach [7 to X] translocation. Among the potential uses of these techniques are measurement of DNA content errors induced in sperm due to mutagen exposure, and assessment of the fractions of X and Y sperm in semen that may have one population artifically enriched.
An epiilluminator/detector unit permitting arc lamp illumination for fluorescence activated cell sorters
Authors: Koper Ger J. M. , Bonnet Jan , Christiaanse Johan G. M. , Ploem Johan S.
Published: August 2005

The application of arc lamps to flow cytometers is discussed and epiillumination for jet‐in‐air cell sorters is introduced. An epiilluminator/detector unit equipped with a mercury arc lamp constructed for a commercially available cell sorter is described. Experiments in which laser and mercury arc lamp illumination were compared show that the signal‐to‐noise ratio for the arc lamp illumination is predominantly limited by shot noise from constant light backgrounds due to reflected excitation light and ambient light. Arc lamp illumination can be used for the sorting of highly fluorescent objects such as cells stained for DNA by for example: ethidium bromide, propidium iodide, or the Hoechst dyes. The simultaneous employment of mercury arc and laser light sources as an inexpensive dual wavelength system is discussed.
EMOSS: An epiillumination microscope objective slit‐scan flow system
Authors: Weller Laura A. , Wheeless Leon L.
Published: August 2005

An epiillumination microscope objective slit‐scan flow system has been fabricated utilizing two dimensional slit scanning with hydrodynamic sample stream focussing. Low resolution (4 μm) analysis of cellular fluorescence is facilitated by the definition of a stabilized flow plane through hydrodynamic focussing. Coincidence of the region of stabilized flow with the focal plane of the microscope objective will allow for the collection and subsequent imaging of fluorescence from cells oriented along this plane. Two orthogonal slit‐scan contours are generated as a cell traverses the excitation region. It is hoped that the need for a three dimensional system will be precluded by preferential orientation of the cells in the region of stabilized flow. Cellular fluorescence is collected by a high numerical aperture epiillumination optical system and imaged onto two orthogonal slits. Two photomultiplier tubes are used to detect fluorescence.

It is anticipated that the epiilumination microscope objective slit‐scan flow system will be used with a variety of fluorescent stains and markers, as well as extended to the research of light scattered by cells. (Steen, H. B., Cytometry 1:26–31, 1980).
A hematology data‐logging terminal
Authors: Paquette Gerard A. , Sparks Thomas E.
Published: August 2005

Intended as a link between a central analysis facility and remote microscope analyzers at international sites, a data‐logging terminal has been developed which exploits basic analyzer control and data acquisition capabilities. The terminal depends on the analyzer's ability to assimilate, interpret, and respond to external commands transferred through its standard communications interface. Within the limits of the analyzer's high‐level control language, the data‐logging terminal is able to direct microscope operation and data acquisition as well as to initiate algorithms. Internal instrument algorithms can then establish and maintain focus, acquire specified objects, direct various scene segmentation sequences, and execute feature extractions. The data‐logging terminal can thus command the analyzer to find scenes of interest and extract data from them. User access to a limited instrument operating interface is provided through its keyboard and alphanumeric display. The terminal can also request and receive image, feature, or count data over the communications interface. Such data are recorded, together with identifiers entered through the keyboard, onto diskettes which may be transferred by mail or telecommunications for processing with the image analysis and pattern recognition techniques available at the central analysis facility. Anticipated applications include evaluation of sample‐preparation techniques, correlation of image data with sampling conditions, training of special classifiers, and analysis of instrument performance.
Effects of hoechst 33342 on survival and growth of two tumor cell lines and on hematopoietically normal bone marrow cells
Authors: Fried Jerrold , Doblin Jeffrey , Takamoto Shigeru , Perez Amaury , Hansen Herbert , Clarkson Bayard
Published: August 2005

Our objective in this investigation was to determine whether Hoechst 33342, which is widely recognized as a DNA specific fluorochrome for living cells, is in fact nontoxic and kinetically nonperturbing at dye concentrations required to achieve acceptable DNA distributions. Three cell types were tested: HeLa S‐3; SK‐DHL2, a human lymphoma cell line; and hematopoietically normal human bone marrow cells. In the third system, only the cloning efficiencies were determined. Results differed considerably for the different cell types. While HeLa cells yielded excellent DNA distributions and were almost completely resistant to the cytotoxic and cytokinetic effects of the dye, SK‐DHL2 cells were highly sensitive to the fluorochrome even at dye concentrations which produced very poor DNA distributions. Human bone marrow cells were intermediate in their stainability and toxicity response, and acceptable DNA distributions could be obtained at the nontoxic dye concentration of 2.5 μM. Clearly, different cell types differ considerably with respect to their cytotoxic and kinetic responses to Hoechst 33342. In some cases it may not be possible to ensure adequate staining of the cells for flow cytometry without significantly altering their viability and/or proliferative behavior.
Comparative kinetics of poly (6‐thioinosinic acid) and 6‐mercaptopurine treated L1210 cells
Authors: Tomasovic Stephen P. , Higashikubo Ryuji , Cohen Arthur M. , Broom Arthur D.
Published: August 2005

Poly(6‐thioinosinic acid), poly(s6I), a synthetic polynucleotide shown to inhibit L1210 ascites growth in vivo, was compared with 6‐mercaptopurine (6‐MP) for their effects on cellular kinetics of L1210 cells in vitro. These kinetic perturbations were monitored by changes in growth rates, cell volumes and cell‐cycle distributions. At equimolar exposure doses (based on molar concentration of purine bases), 0.1 mM poly(s6I) was more effective than 6‐MP in reducing growth rate and total cell number. A 20‐fold increase in the 6‐MP exposure dose (to 2 mM) was required to reduce total cell number similar to 0.1 mM poly(s6I). Concomitant determination of Coulter volume distributions of treated cell populations indicated 0.1 mM poly(s6I) increased the mean population volume more than either concentration of 6‐MP. Cell‐cycle distributions obtained by flow cytometry showed that, simultaneously with the increase in mean population volume, poly(s6I) exposure caused a decrease of cells in G1 and S and a pronounced accumulation of cells in G2. Treatment with 6‐MP caused similar cell‐cycle redistributions but always less than for poly(s6I). Flow sorting of these redistributed populations indicated the increase in mean population volume during treatment with 0.1 mM poly(s6I) was almost equally due to an increased percentage of G2 cells and to an increased volume of cells with the G2 DNA content. However, with 0.1 mM 6‐MP, increased volume of G2 cells was predominant. Although poly(s6I) was synthesized in an attempt to develop more effective delivery forms of biologically active compounds like 6‐MP and it has close structural similarities to 6‐MP, its mechanism of action is unknown.
A polycarbonate filter technique for collection of sorted cells
Authors: Folstad L. , Look M. , Pallavicini M.
Published: August 2005

We describe the application of polycarbonate filters to the collection of cells sorted by flow cytometry. Cells are sorted directly onto polycarbonate filters and transferred to microscope slides which are then processed to dissolve the filter and remove pore outlines. The technique results in cell preparations which retain cellular morphology and have high cell recovery of 82–100%.
Is the central dogma of flow cytometry true: That fluorescence intensity is proportional to cellular dye content?
Authors: Kerker M. , Van Dilla M. A. , Brunsting A. , Kratohvil J. P. , Hsu P. , Wang D. S. , Gray J. W. , Langlois R. G.
Published: August 2005

Measurements and theoretical calculations of fluorescent emission from four samples of polystyrene microspheres (diameter 0.92, 1.63, 1.90 and 4.18 m̈m) containing the same fluorescent dye show a general dependence upon particle size, emission angle, and polarization conditions. However, for the excitation and detection conditions used in flow cytometry, the relative fluorescent intensities measured for the four particle sizes are proportional to the dye content to +10% accuracy, independent of particle size. Accordingly, the central dogma of flow cytometry ‘that fluorescence is proportional to cellular dye content’ is valid to this accuracy for these solid, highly refractive polymer particles. Most mammalian cells are much less refractive, therefore, should conform more closely to the central dogma.
Rapid, one step staining procedures for analysis of cellular DNA and protein by single and dual laser flow cytometry
Authors: Crissman H. A. , Steinkamp J. A.
Published: August 2005

Detailed, simplified techniques are described for simultaneous staining and analysis of DNA and protein in a number of mammalian cell types. Cell staining involves the addition of appropriate dye mixtures to unfixed or ethanol‐fixed cells and subsequent analysis of cell populations in the staining reagents generally within 10 to 20 minutes. The approach is novel in that no centrifugation steps are involved during the staining procedure, thus, eliminating cell clumping and cell loss and making the procedures appropriate for samples containing limited numbers of cells.

For single wavelength analysis, staining of DNA and protein in ethanol‐fixed cells was accomplished with a dye solution containing propidium iodide, fluorescein isothiocyanate and RNase. After 20 minutes at room temperature cells were analyzed using the 488 nanometer (nm) laser excitation line. For dual laser analysis the following dye combinations were employed without RNase: mithramycin‐rhodamine 640, mithramycin‐substituted rhodamine isothiocyanate, Hoechst 33342‐rhodamine 640 and Hoechst 33342‐rhodamine isothiocyanate. Unfixed cells were also stained with the Hoechst 33342‐rhodamine 640 dye combination. Mithramycin was excited at 457.9 nm, Hoechst 33342 at 333–363 nm, and the rhodamine dyes at 568 nm.

Cell types analyzed included Chinese hamster ovary cells, cultured mouse colon 26 cells, mouse embryo forelimb bud cells, and rat cell samples obtained by lung lavage.
Flow cytometry of human testicular tissue
Authors: Pfitzer Peter , Gilbert Peter , Rölz Gero , Vyska Karel
Published: August 2005

Flow cytometry of testicular tissue from autopsies showed differences in the proportion of substained spermatozoa (1 CA), spermatids (1 CB), diploid 2C nuclei and premeiotic spermatozoa in the 4 C class. The evaluation of the histograms was performed with computer assistance. Healthy accident cases or those delivered in forensic medicine furnished the control values. Patients with hepatic cirrhosis showed a nearly complete loss of the haploid class, while cases with fatty degeneration of the liver demonstrated a reduction. Circulatory insufficiency in cases of acute shock resulted in an increase in the 4C values of meiotic prophase I and a decrease of spermatids. Spermatozoa were normal in these cases. After prolonged shock a nearly complete breakdown of spermatogenesis was observed. In cases of atherosclerosis an irregular degree of reduction was seen. Cancer patients showed a reduction of spermatogenesis in correlation to therapy.
Cell cycle operation during batch growth of fission yeast populations
Authors: Agar D. W. , Bailey J. E.
Published: August 2005

Batch cultivation provides a continuous sequence of different environments useful for studying responses of cell cycle controls. Flow cytometry measurements have been made of the frequency functions for protein, RNA, and DNA at different times during batch growth of the fission yeast Schizosaccharomyces pombe. The mean cellular protein and RNA contents and their variances tend to increase with increasing population specific growth rates. Analysis of the midexponential phase DNA frequency function data indicates that DNA synthesis occupies 12% of the total cell cycle time and is completed at the same time as cell separation. Coordination of DNA synthesis and cell separation is less precise when population growth rate is low in late lag and early stationary phases.
The effect of some β‐lactam antibiotics on escherichia coli studied by flow cytometry
Authors: Martinez Octavio V. , Gratzner Howard G. , Malinin Theodore I. , Ingram Marylou
Published: August 2005

The effects of three beta‐lactam antibiotics on Escherichia coli were studied by means of flow cytometry. Since these agents block bacterial cell wall synthesis in such manner as to prevent septal formation without appreciably affecting nucleic acid synthesis, the resulting cell elongation caused by these agents can be assessed by nucleic acid fluorescent staining. It was shown by this technique that the somatic effects of cefazolin, cefamandole and moxalactam were related both to the antibiotic concentration and time of exposure to the drugs and were observable within 30 minutes of the initial exposure of the cultures to these agents. These results demonstrate that fluorescent cytometry can provide accurate assessment of the effects of compounds that inhibit cell wall formation. This technology could be a useful tool for comparing antibiotic somatic effects on bacteria and for rapidly and reliably determining their sensitivity and resistance to these agents.
Conversion of linear histogram flow cytometry data to a logarithmic display
Authors: Brunk C. F. , Bohman R. E. , Brunk C. A.
Published: August 2005

A routine is described that readily allows the rescaling of linear histographic data to a corresponding logarithmic histogram. This procedure significantly improves data display, particularly where a wide range in the measured parameter is encountered. The logarithmic scale displays peaks with band widths more proportional to their respective coefficients of variation than is the case in a linear display. Rescaling several linear histograms to a common logarithmic scale allows the combination of these linear data even though the linear ranges are different. This routine is presented as a program written in BASIC for execution on a microcomputer.
The analysis and interpretation of DNA distributions measured by flow cytometry
Authors: Dean Phillip N. , Gray Joe W. , Dolbeare Frank A.
Published: August 2005

A principal use of flow cytometers is for the measurement of fluorescence distributions of cells stained with DNA specific dyes. A large amount of effort has been and is being expended currently in the analysis of these distributions for the fractions of cells in the G1, S, and G2 + M phases. Several methods of analysis have been proposed and are being used; new methods continue to be introduced. Many, if not most, of these methods differ only in the mathematical function used to represent the phases of the cell cycle and represent attempts to fit exactly distributions with known phase fractions or unusual shapes. In this paper we show that these refinements probably are not necessary because of cell staining and sampling variability. This hypothesis was tested by measuring fluorescence distributions for Chinese hamster ovary and KHT mouse sarcoma cells stained with Hoechst‐33258, chromomycin A3, propidium iodide, and acriflavine. Our results show that: (a) single measurements can result in phase fraction estimates that are in error by as much as 40% for G2 + M phase and 15–20% for G1 and S phases; (b) different dyes can yield phase fraction estimates that differ by as much as 40% due to differences in DNA specificity; (c) the shapes of fluorescence distributions and their interpretation are very dependent on the dye being used and on its binding mechanism.
Flow cytometric analysis of G1 exit kinetics in asynchronous L1210 cell cultures with the constant transition probability model
Authors: Sharpless T. K. , Schlesinger F. H.
Published: August 2005

Following addition of mitotic inhibitors to asynchronous cultures of mouse lymphoid cell line L1210, the rate of cell exit from G1 phase accelerates during the first 2–3 hr then declines with apparent first order kinetics. The simplest mathematical model that calls for biphasic G1 exit kinetics is the two‐state model with constant transition probability. It posits an exponential distribution of residence times in the first state, which is wholly contained in the G1 phase, and a fixed duration, Tb, for the rest of the cycle.

We derive the distribution of expected times‐to‐division predicted by the model for a population in exponential steady state growth, and integrate this to obtain the predicted G1 exit curve for the blocked population as a function of two parameters, Tb and the population doubling time, Td.

The experimental exit curves were fractions of the population remaining below an early S phase DNA content threshold, as a function of time after adding Colcemid, vincristine or vinblastine to exponentially growing cultures. Doubling times were determined from the rate of accumulation of blocked cells in G2 + M. An iterative method was then used to obtain a least‐squares fit of Tb and an observational parameter, Tt, representing the average time‐to‐division of cells at the chosen DNA threshold.

The fitted G1 exit curves match the data well, showing that the constant transition probability model is a useful mathematical description of the G1 kinetics of these cells. The method described is a convenient way to estimate the parameters of this model for asynchronous, exponentially growing populations.