bluuebunny/crossref_metadata_2025 huggingface dataset:
A new flow cytometric pulse height analyzer offering microprocessor controlled data acquisition and statistical analysis
Authors: Kachel V. , Schneider H. , Schedler K.
Published: August 2005

The instrument described is capable of storing up to 112 one parameter histograms (128 channels) or 16 one parameter and 3 two parameter histograms (64 × 64 channels). A low cost 10 Mhz oscilloscope displays the graphical and alphanumerical data. During data acquisition, the original pulses, an analogue rate meter and the growing histogram are displayed simultaneously on the screen. The instrument may be completely operated by an 18 element keyboard. The data processor allows calculation of integrals, mean values, standard deviations and coefficients of variation of operator selectable regions of the acquired histograms. Gaussian distributions determined by the statistical parameters may be compared directly with the measured distributions. Distribution curves can be normalized, shifted, added, subtracted or compared with artificial distributions which are generated by superimposition of distinct Gaussian curves. Exponentially fitted background curves can be subtracted from the measured histograms. A text line at the top of the screen displays the numerical values and explanatory remarks. The two parameter histograms may be rotated, observed from the sides or back and projected either on the x‐or y‐axes. The projections or selected lines or columns can be transferred to the one parameter range and treated statistically. Sequences of one parameter distributions may be transferred to the two parameter range and displayed as pseudo‐two‐parameter histograms.
The effect of isoproterenol on nuclear protein synthesis in electrostatically sorted rat hepatocytes
Authors: Pipkin J. L. , Hinson W. G. , Hudson J. L. , Martin J. L. , North B. M. , Pack L. D.
Published: August 2005

Rat hepatocyte nuclei were electrostatically sorted and collected from specific regions of the cell cycle. Proteins were extracted from homogeneous populations of nuclei with cetyltrimethylammonium bromide and chromatographed by one‐ and two‐dimensional acid‐urea polyacrylamide gel electrophoresis. Small quantitative differences were observed in amido black stained two‐dimensional patterns of the nuclear proteins of the G0 + G1 and G2 + M populations. However, comparison of the two‐dimensional autoradiographs of 3H‐leucine labeled nuclear proteins in these populations revealed dramatic differences. After isoproterenol administration, animals showed enhanced labeling of certain nuclear proteins in G2 + M populations, but no noticeable effect in protein labeling was observed in the G0 + G1 populations. Four days after the administration of isoproterenol, fluorescent histograms exhibited increased amounts of DNA in the G2 + M populations. Comparison of the total amino acid composition of two proteins from control animals revealed the distinct chemical contrast of these proteins. Both proteins possessed higher ratios of acidic to basic residues than anticipated for basic proteins. These procedures and findings should prove useful for further understanding of mechanisms of toxicant effects upon nuclear protein metabolism.
High resolution optics combined with high spatial reproducibility in flow
Authors: Eisert W. G.
Published: August 2005

Accurate sizing in flow using optical methods generally requires high resolution optics and specially designed flow systems. Flow systems developed by this group have following features: (a) double sheath configuration for optical index match, (b) no curved optical surface in the sensing area, (c) gradual hydrodynamic focusing over a long distance to minimize mechanical shearing, (d) precision spatial positioning of cells by reducing suspension fluid diameter to a cell diameter or less, (e) total thickness between outer surfaces of the flow chamber at the viewing area of 1.5 mm or less.

Cells intersect a laser light beam focussed to circular as well as elliptical cross‐sections of 1μ or less in diameter. Cellular extinction is monitored during transit through the beam. Cell length is derived from the time for flight measurement and corrected for absolute values by continuous velocity reference using a second laser beam intersecting the cell stream at a predetermined distance. This second spot may be circular or elliptical, of a different polarization and/or frequency.

Simultaneous fluorescence intensity and diameter measurements were performed on test particles using different optical geometries. The influence of the particle structure on fluorescence measurements is demonstrated where high resolution sizing is required at the same time.
A morphometric expression of differentiation in fine‐needle biopsies of breast cancer
Authors: Stenkvist Björn , Bengtsson Ewert , Eriksson Olle , Jarkrans Torsten , Nordin Bo
Published: August 2005

An automated method of particle sizing in fine‐needle biopsies of human breast carcinomas is described. The average size of clusters of cells aspirated from breast carcinomas was strongly correlated with cytologic differentiation of the tumors and also with the recurrence rate 3 years after mastectomy so that smaller cluster size was associated with poor differentiation and a poor prognostic outcome. It thus seems possible to express in quantitative terms the degree of differentiation of breast carcinomas.
Evaluation of selected aerosol‐control measures on flow sorters
Authors: Merrill John T.
Published: August 2005

Flow sorters produce microdroplets as part of their normal operation, and if these microdroplets escape into the room, they are potentially hazardous. We have tested several aerosol‐control measures on a commercial flow sorter. To accomplish this, T‐4 phages were introduced into the sorter's liquid jet through the sample injection tube, and culture plates containing lawns of T‐4‐sensitive Escherichia coli bacteria were exposed around the sorter for each operational configuration. After the exposure, the plates were incubated and then scored for plaques. A single phage‐containing microdroplet landing on a plate was sufficient to cause a plaque of lysed bacteria to form. The number of plaques was thus an indicator of how much aerosol was released for each configuration. Aerosols were controlled most effectively by catching the central, undeflected stream in a vacuum‐exhausted tube; this technique, coupled with the manufacturer's vacuum‐exhaustion of the air around the sorting location, produced no plaques. Several failure modes were tested, including having the central stream hit the outside of the catch‐tube or deflection plates, and loss of the manufacturer's vacuum‐exhaustion system. Many flow sorters allow the undeflected stream to splash into a flask or beaker, an approach that produces the most plaques if instrument‐failure modes are excluded. The simple addition of a catch‐tube removed this major contributor to the aerosol production.
A flow system for partial automation of plating efficiency tests
Authors: Macevicz S. , Dean P. , Peters D. , Pinkel D. , Gray J.
Published: August 2005

A flow system that sterilely deposits a known number of cells into a collection vessel has been constructed and evaluated. In this device, the cells from a single‐cell suspension flow through a nozzle that constrains them to the center of the emerging jet. The cells in the jet are illuminated by a 0.5‐mW helium‐neon laser; they are detected, sized and counted on the basis of the light scattered as they pass through the laser beam. The device has been used for plating efficiency tests in a tissue culture laboratory. We found that it saved considerable labor by eliminating the dilution step from the standard plating efficiency procedure. The biologic effect of machine plating was determined by comparing plating efficiencies of KHT, CHO‐T5, and CHO‐AA8 cells established by hand plating with those established by machine plating. Results indicate that machine plating reduces the plating efficiency of the CHO‐T5 cells while the plating efficiencies of the CHO‐AA8 and KHT cells are unaffected.
Cytophotometric analysis of lytically and persistently infected tissue culture cells with measles virus
Authors: Harms H. , Aus H. M. , ter Meulen V.
Published: August 2005

Lytically and persistently infected VERO and LUB cells were cytophotometrically characterized with the aid of computer analysis. Images scanned at 260 and 280 nm were processed by computer algorithms. With this approach, infected cells could be segmented and differentiated from uninfected cells. Lytically and persistently infected cells could be distinguished by distinct differences in nuclear and cytoplasmic optical densities. These findings are supported by biologic data based on the analysis of virus‐specific proteins and nucleic acids. The applied computer aided cytophotometry provides a new approach in the study of virus‐cell interaction.
Automated selection of metaphase cells by quality,
Authors: van den Berg H. T. C. M. , de France H. F. , Habbema J. D. F. , Raatgever J. W.
Published: August 2005

The performance of metaphase‐finding systems could be improved if they were able to determine the quality of the cells detected. This paper discusses the extent to which this may be realized by the introduction of a metaphase‐quality parameter. Data obtained from 300 cells were statistically analyzed. Seventeen features were measured and nine visual properties were determined for each cell. Discriminant analysis and regression analysis were used to extract those features and visual properties which contribute to assessment of metaphase quality. Rather low correlations were found between the selected measured features and visual properties. A quality‐parameter based on a linear combination of cluster projections, areas and perimeters was found to account for 64% of the variation between visual and measured quality indicators. In addition, an increase in the predictive value for finding usable metaphases from 28–68% was achieved.
Cell cycle dependency of tumor promoter on murine erythroleukemic cells,
Authors: Zucker Robert M.
Published: August 2005

The tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) affected the cell cycle of murine erythroleukemic cells. In logarithmically growing cells a transient G1 block lasting approximately 6 hr developed after the addition of TPA. To investigate this cell cycle effect of TPA, cells were synchronized either by a double thymidine block or by centrifugal elutriation and were analyzed by flow cytometry and electronic nuclear volume. Using double thymidine blocked synchronized cells or elutriated separated fractions containing cells other than G1, a transient G1 block occurred as the cells entered early G1. The G1 elutriated cell population was blocked in the G1 period by TPA before cell cycle traverse. This transient block in G1 was not observed after the next cell cycle traverse. Addition of TPA to butyric acid or dimethylsulfoxide induced‐cells resulted in a transient G1 block in addition to inhibition of differentiation. TPA was more inhibiting on dimethylsulfoxide‐induced cells than butyric acid‐cells.
Flow cytometric electronic direct current volume and radiofrequency impedance measurements of single cells and particles
Authors: Hoffman R. A. , Johnson T. S. , Britt W. B.
Published: August 2005

A flow‐system instrument is described that uses direct current and radiofrequency (rf) currents to detect simultaneously the low and high frequency impedance changes produced by biologic cells or particles suspended in saline traversing through a 93‐μm diameter sensing orifice. For nonmembranous particles and plastic microspheres both the Coulter direct current volume and rf‐parameter signals are proportional to particle volume. Cells having an intact plasma membrane produce rf‐impedance changes dependent additionally on the electrical properties of the plasma membrane and intracellular structures. Thus, biologically different cells having the same Coulter direct current volume can be distinguished if they differ in their electrical rf impedance properties.
An immunofluorescence method for monitoring DNA synthesis by flow cytometry
Authors: Gratzner H. G. , Leif R. C.
Published: August 2005

An immunofluorescence method for monitoring DNA synthesis in single cells has been developed for flow cytometry. With antiserum which is specific for 5‐bromodeoxyuridine (BrdUrd) and a second fluorescent label, BrdUrd‐incorporation pulses of 30 min are detectable. The fluorescence intensity of the incorporated BrdUrd, as determined by immunofluorescence, is related to the amount of BrdUrd incorporated, as shown by isotopic methods and cell sorting. Thus, the technique may be applicable to determining rates of replication per cell. Multiple samples of as few as 1 × 105 cells can be fixed, hydrolyzed and treated with the anti‐BrdUrd antiserum. Nuclear‐bound IgG is localized by fluorescein‐labeled avidin‐D. Since the technique uses whole cells, other parameters such as light scatter and DNA content can be simultaneously monitored so that cohorts of “labeled” cells can be followed through the cell cycle.
Clinical application of flow cytometry: A review
Authors: Laerum Ole Didrik , Farsund Tore
Published: August 2005

A survey of the use of flow cytometry for clinical purposes is given. In the last decade the main clinical application of this technique has been measurements of cellular DNA content for estimation of cell cycle distribution and ploidy studies. A large body of data is now available on the presence of aneuploidy in different malignant diseases. By measurements with high resolution, the demonstration of abnormal cellular DNA content in several types of tumors can be of definite diagnostic value when combined with conventional diagnostic procedures. The prognostic significance of different types of DNA aberrations is so far not established. Attempts to monitor cancer treatment by studying altered cell cycle distribution have not been successful, although some applications are of potential value. The main reasons for this are the complexity of tumor tissue as well as difficulties with interpretation of altered cell cycle distribution caused by drug combinations. For further progress in this field more emphasis on other cell constituents than DNA measured by flow cytometry is desirable, either as single or as multiparameter measurements.
Dual laser beam focussing for flow cytometry through a single crossed cylindrical lens pair
Authors: Watson James V.
Published: August 2005

Two laser beams (argon 488 nm blue, and krypton UV lines) have been focussed either to the same point or to two different points in a vertical plane in a multiparameter flow cytometer. The technique exploits the focal length shortening of the longer wavelength beam that occurs with spherical and astigmatic lens aberrations. Ethidium bromidestained DNA is excited by both the argon 488 nm and krypton UV lines and the excitation intensities of both beams were set to give the same red fluorescence pulse height from ethidium bromide‐stained nuclei. DNA histograms were obtained for sequential focussing of the two beams and these were virtually identical irrespective of which beam was the first excitor and irrespective of which beam the histogram was recorded from. Coincident focussing of the two exciting beams resulted in a histogram of double the emission intensity.
Flow cytometric analysis of the uptake of hoechst 33342 dye by human lymphocytes
Authors: Szabó Gábor , Kiss Attila , Damjanovich Sándor
Published: August 2005

Human peripheral blood lymphocytes have been shown to resist staining with the DNA binding fluorochrome Hoechst 33342 by the cellular membrane. The rate of uptake of the dye is strongly temperature‐dependent with minimal uptake rate below 16°C. The activation energy of dye transport was found to be 135 kJ/mol above 20°C and about 20 kJ/mol below 16°C. Metabolic inhibitors accelerated, instead of inhibiting, the transport of the dye. Dead cells have been shown to stain promptly in contrast with the gradually staining viable cells. The uptake process in the vital staining conditions is suggested to involve a carrier mediated mechanism. Application of Hoechst 33342 as a fluorescent indicator of viability is proposed.
Flow fluorometric study of DNA content in nonproliferative Euglena gracilis cells and during proliferation
Authors: Bonaly J. , Mestre J. C.
Published: August 2005

Ethanol‐fixed Euglena gracilis cells have been analyzed by flow microfluorometry during the lag, logarithmic and stationary phases. The histogram of a plateau stage culture reveals, as expected, an unimodal distribution, but the peak is at a lower fluorescence intensity as compared to G1 logarithmic cells. The fluorescence intensity drops as the cells enter the stationary stage. Ultimately the decrease represents a change of about 25%. When cells recover from the plateau stage, the fluorescence intensity increases during the lag phase, and climbs to the level found in a Gl logarithmic population. The reason for the decrease in the fluorescence intensity during the stationary stage may be due to a possible loss of DNA or to a decrease in the number of chromatin‐binding sites for intercalating ethidium bromide.
Flow cytometric analysis of cellular endogenous fluorescence simultaneously with emission from exogenous fluorochromes, light scatter and absorption
Authors: Thorell Bo
Published: August 2005

A cytofluorimeter capable of simultaneous 4‐parameter analysis (i.e., endogenous fluorescence (NAD(P)H), exogenous fluorescent dyes, absorption and small angle scatter) is described. Analyses have been performed on isolated rat liver cells, white blood cells, bone marrow, thymus cells and yeast under different metabolic states. This allows correlations between cell function and concentration of cell substances, size, refractive indices, etc. Kinetic measurements are also possible.
The application of flow cytometry for the quantification of the response of experimental tumors to irradiation
Authors: Beck H.‐P. , Brammer I. , Zywietz F. , Jung H.
Published: August 2005

R1H rhabdomyosarcomas of the rat were irradiated with 15 Gy of x‐rays and the depopulation and repopulation of the tumor was determined by means of flow cytometry, quantitative histology and colony assays. The results show that flow cytometry yields important information about the cell population kinetics of irradiated tumors, but misleading results are obtained if flow cytometry is not combined with the determination of the numerical density of cells.
Expression of tumor‐associated antigens and kinetic profile of two in vitro human melanoma cell lines
Authors: Badaracco G. , Corsi A. , Maisto A. , Natali P. G. , Starace G. , Zupi G.
Published: August 2005

The growth characteristics and the antigenic profile of two human melanoma established cell lines in vitro are reported. The growth curves of the two cell lines show some peculiar features which are also detected in their DNA distribution content. In the late stages of growth, in fact, the cell DNA content resumes the initial distribution, suggesting a partial recovery of the cell proliferating potential. When the two cell lines were tested by indirect immunofluorescence with monoclonal antibodies to human melanoma associated antigens, a rather constant expression of these epithopes was observed.
Cell cycle kinetics of irradiated synchronous and asynchronous tumor cells with DNA distribution analysis and BrdUrd‐Hoechst 33258‐technique
Authors: Nüsse Michael
Published: August 2005

Radiation‐induced progression delay in S‐phase and G2‐block, both depending on time of irradiation in the cell cycle, were measured in synchronized Ehrlich ascites tumor cells in vitro using flow cytometric analysis of DNA content in single cells. Similar results could be obtained by the BrdUrd‐Hoechst 33258 technique after irradiating asynchronous cells. BrdUrd replaces thymidine in newly synthesized DNA which is not stainable by the thymidine‐specific dye Hoechst 33258. The temporal development of the fluorescence distributions after addition of BrdUrd to the medium has been measured in the flow cytometer. In asynchronous cells the mean durations of the cell cycle phases and their perturbations after irradiation could be calculated, and the data agreed with the results in synchronized cells. Division delay after irradiation consists of a small progression delay in S‐phase and a block in G2‐phase. Both effects depend on time of irradiation during the cell cycle with delay in S‐phase increasing in irradiated late S‐cells and G2‐block increasing in irradiated G2‐cells. Data from both types of experiments were used with a simple model describing the fractions of cells in the cell cycle phases after irradiation had been applied.
The effect of abnormal cell proportion on specimen classifier performance
Authors: Castleman Kenneth R. , White Benjamin S.
Published: August 2005

In two previous papers we developed formulas relating the performance (error rates) of a two‐class specimen classifier to the performance of a preceding two‐class classifier and the number of cells examined (K. R. Castleman and B. S. White, Analytical and Quantitative Cytology 2:117, 1980 and K. R. Castleman and B. S. White, Cytometry 1:156, 1980.). This analysis assumed a certain proportion (p) of abnormal cells on an abnormal specimen. In this paper we examine what happens when a system designed assuming one value of p is presented with a positive specimen having a different abnormal cell proportion. We show that the specimen false negative error rate increases dramatically as p decreases below the design value, and conversely. This suggests that the specimen classification performance of a particular system should be quoted only with reference to the abnormal cell proportion of the specimens used for testing.
Proliferation kinetics of perturbed cell populations determined by the bromodeoxyuridine‐33258 technique: Radiotoxic effects of incorporated [3H]thymidine
Authors: Beck Hans‐Peter
Published: August 2005

A simple analytical method is described for the evaluation of flow cytometric data of perturbed cell populations obtained by applying the BrdUrd‐33258 Hoechst technique. This procedure allows for determining the growth curve, the efflux from and the influx into G2 + M as well as the transit characteristics of cell cohorts through the G2 + M phase. As an example of the application of the method, the radiotoxic effects of incorporated 3H‐thymidine on the proliferation of L‐929 cells in vitro are described.
Enrichment of viable lymphocytes in defined cycle phases by sorting on the basis of pulse width of axial light extinction
Authors: Monroe John G. , Havran Wendy L. , Cambier John C.
Published: August 2005

A correlation between cell size and cycle state is demonstrated for mitogen stimulated murine lymphocytes. Cell cycle analysis was performed using multiparameter cytofluorometric analysis of acridine orange stained cells. Cell size was determined by measuring “time of flight” of axial light extinction. Cells with diameters of 5.0–6.0 μm are shown to be predominately in G0. Among stimulated cells, those 6.0–7.0 μm in diameter are predominately early and late G1 (80%) with some S phase cells. Cells in G2 + M were found to predominate among lymphocytes with diameters of 7.5–12 μm. This approach provides a method for isolation of lymphocytes in specific phases of the cell cycle which are suitable for subsequent functional studies.
Flow cytometric analysis of RNA content in different cell populations using pyronin Y and methyl green
Authors: Pollack Alan , Prudhomme David L. , Greenstein David B. , Irvin George L. , Claflin Alice J. , Block Norman L.
Published: August 2005

Pyronin Y (PY) was used, in flow cytometric (FCM) systems, to estimate the RNA content per cell in formalin fixed EL4 leukosis tumor cells, enzyme dispersed R3327‐G rat prostatic adenocarcinoma cells, mouse spleen cells stimulated with concanavalin A, and human peripheral blood lymphocytes stimulated with phytohemagglutinin. Preincubation of the cells with methyl green (MG) blocked PY binding to DNA such that the intracellular fluorescence from MG‐PY was due primarily to its binding to RNA. Treatment of the cells with ribonuclease resulted in a 3‐ to 5‐fold reduction in the fluorescence intensity of intracellular MG‐PY.

Mitogen stimulation of either mouse or human lymphocytes resulted in an increase in DNA (propidium iodide fluorescence) and RNA (MG‐PY fluorescence) content per cell over resting levels. Further, the changes in stimulated human lymphocyte DNA and RNA contents following 24, 48, and 72 hr of cell culture were monitored. The results showed that RNA levels were significantly increased prior to that of DNA. Also, the effects of different cell cycle phase specific blocking agents on lymphocyte cell cycle traverse were investigated. We found that: (a) actinomycin D inhibited the increases in cellular RNA and DNA; (b) hydroxyurea inhibited the increases in cellular DNA whereas increases in cellular RNA were only slightly reduced; (c) tritiated thymidine caused an accumulation of cells having high DNA and RNA contents; and (d) Colcemid promoted an accumulation of cells having high DNA contents while causing a reduction of cells having high RNA contents. These results were nearly identical to reports by other investigators using the metachromatic dye acridine orange to quantitate RNA per cell. Thus, the MG‐PY technique described is indicated to provide a stable and accurate measure of RNA content per cell.
Detection of transformed cells using a fluorescent probe: The molecular basis for the differential reaction of fluorescamine with normal and transformed cells
Authors: Parry Gordon , Blenis John , Hawkes Susan P.
Published: August 2005

Normal and transformed fibroblasts can be discriminated by a flow cytometry assay on the basis of their differential reaction with fluorescamine. The cause of altered reactivity of transformed cells with this fluorescent probe has been investigated by a detailed analysis of its reaction with chicken embryo fibroblasts transformed by a temperature sensitive mutant of Rous sarcoma virus. The subcellular distribution of fluorescent adducts characterized by cell fractionation and gel electrophoresis procedures supports the hypothesis that transformed cells possess a surface barrier which decreases the accessibility of fluorescamine to reactive macromolecules. The barrier has been identified as being composed at least partly of hyaluronic acid, because of the ability of purified and specific hyaluronidase (from Streptomyces hyalurolyticus) to modulate the response of transformed cells to fluorescamine. Enzyme treatment of transformed cells prior to reaction with fluorescamine causes them to resemble nontransformed cells both in the nature of components labeled and in their fluorescence intensity.

It is suggested that fluorescamine monitors an altered surface hyaluronic acid composition which occurs upon transformation. Its significance is discussed in terms of the known physical properties of the molecule and the finding that it is an early event in the process of transformation.
A follow‐up study of the progression or spontaneous regression of leukemia in mouse strains
Authors: Szöllösi J. , Ember I. , Trón L. , Kertai P.
Published: August 2005

C3H/He‐mg and AKR mice were injected intraperitoneally with lymphocytes prepared from the spleen of (C3H/He‐mg X AKR)F1 hybrid mice bearing Gross‐virus leukemia. Lymphocyte suspensions were prepared from the spleen, mesenteric lymph node and thymus of mice at different days after the injection and analyzed for the ratio of leukemic to normal lymphocytes with a flow cytometer. The size and the fluorescein content of the cells incubated with fluorescein diacetate served for the distinction between normal and leukemic lymphocytes. After injection with leukemic lymphocytes, lethal leukemia developed in the AKR strain, whereas the C3H/He‐mg strain was characterized by a spontaneous regression after a transient development of leukemia. The percentage of leukemic lymphocytes was the highest in the spleen and it was the lowest in the thymus regardless of the stage of the disease. In the thymus of C3H/He‐mg mice the leukemic lymphocytes could be detected only during the crisis period.
Comparison of two flow cytometric assays for cellular RNA—acridine orange and propidium iodide
Authors: Wallen C. Anne , Higashikubo Ryuji , Dethlefsen Lyle A.
Published: August 2005

Two flow cytometric assays for cellular RNA, two‐step acridine orange (TSAO) and propidium iodide (PI), were compared with each other and with ultraviolet (uv) spectrophotometry of RNA to determine their ability to quantitate cellular RNA and to differentiate between proliferating and quiescent (Q) cells. The model system used for these comparisons was cells from unfed cultures of two mouse mammary tumor lines designated 66 and 67. The growth kinetics of unfed 67 and 66 cells were characterized by a decrease in cellular RNA (a factor of 2) with the decrease occurring throughout the entire population of cells as they entered plateau phase. The time course of the RNA decrease as monitored by the PI assay closely corresponded to that observed by uv spectrophotometric measurements. The TSAO assay, however, agreed with the uv spectroscopy and PI assays on the extent of the RNA decrease but showed no decrease in RNA until 24 hours after the other assays indicated that a significant decrease had occurred. The plateau cells from 67 and 66 unfed cultures had a greatly lowered RNA content and were predominately (>97%) in the G1 phase of the cell cycle in terms of DNA content. However, when compared with exponentially growing cells, they were a distinct Q population. This distinction could be observed using either the TSAO or the PI assay. The TSAO assay provided better resolution of the Q population and has the added advantage of giving DNA distribution simultaneously. Therefore, both flow cytometric assays are particularly useful in this system; the TSAO for monitoring the Q population and the PI for determination of kinetic changes in total cellular RNA.
Analysis of glycosaminoglycans of flow sorted cells: Incorporation of [35S]sulfate and [3H]glucosamine into glycosaminoglycans of B16‐F10 cells during the cell cycle
Authors: Blair Owen C. , Burger Douglas E. , Sartorelli Alan C.
Published: August 2005

The incorporation of [35S]sulfate and [3H] glucosamine into cetyl pyridinium chloride (CPC) precipitable glycosaminoglycans was determined in B16‐F10 cultured cells sorted with respect to DNA content. Incorporation into surface material was measured indirectly as the difference between the radio‐activity of control and trypsin treated cells. Approximately 80% of the total cellular [35S] sulfate labeled CPC precipitable material, but only 5% of that labeled by [3H]glucosamine, was removed by this mild trypsin treatment. Incorporation of [35S]sulfate into the trypsin removable surface material increased progressively from G1 to S to G2 + M in both long‐term (48 hours) and shortterm (1 hour) labeled cells, while the ratio of surface to total incorporated [35S]sulfate remained relatively constant. Incorporation of [35S]sulfate into total cellular glycosaminoglycans in long‐ and short‐term labeled cells increased as cells progressed from G1 to S to G2 + M; the incorporation of [3H]glucosamine into CPC precipitable material also increased progressively from G1 to S to G2 + M in long‐term labeled cells but was greater during S phase relative to G1 or G2 + M in short‐term labeled cells. The degree of sulfation of glycosaminoglycans as represented by the ratio of [35S]sulfate to [3H]glucosamine of double labeled cells was relatively constant in long‐term labeled cells but was increased during the G1 and G2 + M phases of short‐term labeled cells. Comparison of the degree of sulfation of short‐term with long‐term labeled cells suggests that highly sulfated glycosaminoglycans may be turned over more rapidly during G1 and G2 + M phases of the cell cycle.
Flow sorting of mouse pancreatic B cells by forward and orthogonal light scattering
Authors: Nielsen O. , Larsen J. K. , Christensen I. J. , Lernmark Å.
Published: August 2005

Mouse islet cell suspensions were separated into subpopulations based on forward low angle and orthogonal scattered light. Separated fractions were analyzed for hormone content by radioimmunoassays. The pancreatic B‐cells had the highest orthogonal light scattering, while they overlapped in forward low angle light scattering with other endocrine (glucagon, pancreatic polypeptide and somatostatin containing cells) and nonendocrine cells. Living and paraformaldehyde fixed islet cell suspensions showed similar distributions of combined light scattering.
Determination of linear fluorescence intensities from flow cytometric data accumulated with logarithmic amplifiers
Authors: Muirhead K. A. , Schmitt T. C. , Muirhead A. R.
Published: August 2005

Logarithmic amplifiers are useful in accumulating flow cytometric data with a large dynamic range. However, quantitative comparison of fluorescence intensities for different samples or different subpopulations within a sample is simplified by the conversion of data from log space back to linear space. A method is described in which fluorescent polystyrene spheres of differing intensities are used to construct a calibration curve for the logarithmic intensity scale. This allows calculation of relative linear intensity for each channel of the logarithmically accumulated data and determination of linear fluorescence means and coefficients of variation for comparative purposes. Fluorescent spheres of appropriate intensity may also be used as internal standards to monitor instrument and/or stain stability for samples accumulated using logarithmic amplifiers.
RNA and DNA content of isolated cell nuclei measured by multiparameter flow cytometry
Authors: Piwnicka Magdalena , Darzynkiewicz Zbigniew , Melamed Myron R.
Published: August 2005

Nuclei were isolated from various cell types including Chinese hamster ovary (CHO) and L1210 leukemia cell lines, primary cultures of fibroblasts, nonstimulated and stimulated human lymphocytes and mouse liver cells, by using different isolation techniques. The isolated nuclei were subsequently stained with acridine orange (AO) and their fluorescence was measured by flow cytometry. Various procedures designed to stain DNAversusRNA differentially with AO were tested, and the staining of isolated nuclei was compared with that of whole cells. Control incubations with RNase and DNase were performed to estimate in whole cells and in nuclei the contribution of DNA and RNA to the fluorescence intensity at the respective wavelength bands of maximum emission for DNA (F530) and RNA (F>600).

Depending on the cell type, 10–20% of total cell RNase‐sensitive F>600is localized in the nuclei. The RNase‐resistant portion of F>600of isolated nuclei represents the stainability of DNA. Suppression of cell proliferation in subconfluent cultures results in a decrease in both whole cell and in nuclear RNA content. Nonstimulated lymphocyte nuclei have considerably lower RNA content than nuclei from lymphocytes stimulated by pokeweed mitogen. Two subpopulations of nuclei having the same (2C) DNA content but differing in RNA content, are present in mouse liver; the cells entering S phase originate from the high RNA population.
Replication kinetics of Chinese hamster chromosomes as revealed by bivariate flow karyotyping
Authors: Cremer C. , Gray J. W.
Published: August 2005

In this paper we show that the replication kinetics of individual chromosomes can be estimated by bivariate flow cytometric analysis of chromosomes isolated from 5‐bromo‐2′‐deoxyuridine (BrdUrd) treated cells. To study the timing of replication of chromosomes, Chinese hamster M3–1 cells were grown for different times in BrdUrd containing medium. Colcemid was added during the last 90 minutes of each BrdUrd labeling period. Chromosomes were isolated from the Colcemid blocked mitotic cells and stained with Hoechst 33258 (HO) and chromomycin A3 (CA3). Bivariate HO‐CA3 flow karyotypes were measured in a dual beam flow cytometer. Labeling cells with BrdUrd for 2, 2 1/2, 3, 3 1/2, and 4 hours before chromosome isolation resulted in progressive quenching of the HO fluorescence of chromosomes Y and 10, 11, M2 while the HO fluorescence of all other chromosomes was either not quenched or was only slightly quenched. The CA3 fluorescence of all chromosomes was slightly enhanced by BrdUrd treatment. A quantitative evaluation of the quenching of HO fluorescence suggested that: a) greater than 50% of HO binding DNA of chromosomes 10, 11, M2 is synthesized in the last quarter of S phase; b) the Y chromosome synthesizes more than 50% of HO binding DNA in the last tenth of S phase making the Y chromosome the latest replicating M3‐1 chromosome. All other chromosomes have synthesized greater than 50% of their HO binding DNA before the last third of S phase.
Adaptation of the ektacytometer to automated continuous pO2 changes: Determination of erythrocyte deformability in sickling disorders
Authors: Bessis M. , Feo C. , Jones E. , Nossal M.
Published: August 2005

The ektacytometer is a visco‐diffractometric method to quantitate erythrocyte deformability. A continuous‐flow system has been added to the basic ektacytometer to record automatically cell deformation as ektacytometer index, (EI) as a continuous function of oxygen tension. Samples from normal donors (AA), donors heterozygous for the sickle cell gene (AS), and homozygotes (SS) were studied with this modified ektacytometer. As expected, the normal red cells showed no change in EI upon deoxygenation. At low pO2, the homozygotes were quite rigid, and their EI rose upon reoxygenation. Erythrocytes from AS subjects, when studied under conditions of physiologic pH and osmolarity, showed essentially normal deformability characteristics. When the osmolarity was raised to 310 and the pH lowered to 7.1, however, the AS cells also became rigid at low pO2. The system was also employed to study the effect of potassium cyanate, a known anti‐sickling agent. By increasing hemoglobin‐oxygen affinity, this agent shifted the pO2 versus EI curve to the left; thus, for any given pO2, the sickle cell population was more deformable in the presence of potassium cyanate than in its absence.
A new flow chamber and processing electronics for combined laser and mercury arc illumination in an impulscytophotometer flow cytometer
Authors: Severin E. , Ohnemus B. , Kiegler S.
Published: August 2005

A flow cytometer of the impulscytophotometer (ICP)‐type has been developed for two‐parameter flow cytometry. It uses two different methods of excitation simultaneously: an argon laser beam focused onto the cell stream within a modified flow chamber, and the light of a high pressure mercury (Hg) lamp using Köhler illumination. The two fluorescence signals are collected sequentially by the same photomultiplier tube. The signals are processed on a cell‐by‐cell basis using new hardwired analog signal processing electronics enabling their accumulation and display as two‐parameter frequency distribution histograms.
The significance of DNA measurements for the early detection of bronchial cell atypia
Authors: Ono J. , Auer Gert
Published: August 2005

Investigations in an experimental model system and in clinical material indicate that squamous bronchial carcinoma is regularly preceded by the occurrence of bronchial cells with DNA values exceeding significantly the normal 4c region. The studies also show that cytomorphological methods do not allow the discrimination between milder forms of atypia and nonspecific cellular alterations. We describe a combined cytomorphological and cytochemical diagnostic procedure which facilitates the distinction between atypical squamous cells and nonspecific cellular changes in the bronchial tree.
Simultaneous three color and electronic cell volume analysis with a single uv excitation source
Authors: Haskill Stephen , Becker Susanne , Johnson Tim , Marro Dominic , Nelson Kay , Propst Roy H.
Published: August 2005

We have adapted an Ortho ICP‐22 flow cytometer (Ortho Instruments, Westwood MA) for the simultaneous measurement of three independent fluorochromes and cell volume. This has been accomplished by the addition of a third photomultiplier tube and the development of a new electronic cell volume (ECV) flow cell. Cells are first analyzed as they pass through the 100 U ECV aperture and are then excited approximately 15 μsec later by the 365 nm mercury arc beam reflected by a 400 nm dicroic mirror. Independent blue, green and red signals can be associated by a delay circuit to the ECV signal from the same cell. We have developed this system as an aid in the analysis of tumor cell and macrophage heterogeneity and differentiation. The choice of stain combinations to be used is extremely flexible and permits the anlysis of a wide range of enzyme activities in conjunction with DNA/RNA and phagocytic probes. Data presented indicates the value of this approach in identifying the presence of plasminogen activator‐like activity in both tumor and inflammatory cells within a malignant effusion as well as the quantitative expression of a number of markers of macrophage differentiation. Although the described techniques have been developed on a mercury are instrument, they can be used equally well with cell sorters.
Flow sorting in the study of cell‐cell interaction
Authors: Schaap G. H. , Verkerk A. , Vijg J. , Jongkind J. F.
Published: August 2005

Undifferentiated mouse teratocarcinoma cells were cocultivated with differentiated mouse endoderm cells in order to study the possible induction of teratocarcinoma cell differentiation.

A difference in DNA content between the two cell types was experimentally introduced to enable the reisolation of the teratocarcinoma cells after cocultivation. Pseudotetraploid (2s) endoderm cell lines were produced from pseudodiploid (1s) cells by treatment of these cells with cytochalasin B and flow sorting of tetraploid cells, using Hoechst 33342 as a viable DNA stain, with subsequent cloning of sorted single cells.

In model experiments, where mixtures of 1s teratocarcinoma and 2s endoderm cells were stained with Hoechst 33342, the teratocarcinoma cells could be reisolated with a purity of about 97%.

After a cocultivation period of 24 days viable teratocarcinoma cells could be isolated from the cocultivation mixture with a purity of 95%. Two dimensional analysis of the protein pattern of these cells indicated that cocultivation did not induce a differentiated (endoderm) pattern.

Therefore according to this analysis the teratocarcinoma cells were not induced to differentiate during a 24 day cocultivation period. The method described offers excellent possibilities for studying cell‐cell interaction in vitro.
Isovolumetric sphering of erythrocytes for more accurate and precise cell volume measurement by flow cytometry
Authors: Kim Young Ran , Ornstein Leonard
Published: August 2005

Typical mammalian erythrocytes are biconcave disks. Flow cytometric light‐scattering measurements of red cell volume show large, orientation‐dependent variations in signal size. To eliminate errors caused by shape factors, we sphere erythrocytes isovolumetrically on an Auto‐Analyzer manifold (Technicon Instruments Corp., Tarrytown, N. Y.) using a carefully controlled ratio of concentrations of a salt of long chain sulfated fatty alcohol (e.g.), sodium dodecyl sulfate (SDS) to albumin in neutrally buffered isotonic saline, producing a suspension with at least 2 1/2 hours of stability with respect to cell volume, cell count and retention of hemoglobin. A two‐step manifold modification of this procedure is preferred. It uses the endogeneous serum protein and a first SDS‐saline reagent without added albumin at a low dilution, and further dilution with a second SDS‐saline containing a very low concentration of glutaraldehyde. This bypasses a serious shelf life problem with the albumin‐SDS reagent.

Microhematocrit and Coulter electronic impedance measurements were correlated with light‐scattering measurements on our own flow cytometer system of unsphered, sphered, and sphered and fixed samples. These reveal marked superiority over unsphered samples (e.g., reduced coefficients of variation of cell volume and improved correlation with microhematocrit) of the sphered and of the sphered and lightly fixed cells. The resulting tighter unimodal cell volume distributions also provide the following advantages: (1) The separation of signals of large platelets from that of the smallest red cells is substantially increased permitting better resolution of microcytic red cells in some abnormal bloods, and (2) bimodality of red cell volume distribution in abnormal bloods is more easily resolved.
A new method for cell volume measurement based on volume exclusion of a fluorescent dye
Authors: Gray Martha L. , Hoffman Robert A. , Hansen W. Peter
Published: August 2005

Several methods currently in use for measuring mean corpuscular volume include: centrifuged packed cell volume, electronic impedance, and light scattering methods. Although these techniques are widely used and accepted, there are problems inherent to each method which may produce systematic errors that are difficult to estimate. This paper describes a new flow cytometric method of cell volume determination, based on the principle of volume exclusion, which may overcome the systematic errors of the methods currently in use. This method requires that the cells be suspended in a fluorescent dye which is unable to penetrate the cell membrane. The level of fluorescence which is produced when a narrow stream of the cell suspension is excited by a focused laser beam will remain constant until a cell arrives in the illuminated region thereby causing a decrease in fluorescence which is directly proportional to the cell's volume. The volume exclusion method is shown to give an estimate of mean red cell volume which correlates well with existing methods.
In‐use evaluation of the omnicon automated tumor colony counter
Authors: Herman Chester J. , Pelgrim Olga E. , Kirkels Wim J. , Verheijen Rene , Debruyne Frans M. J. , Kenemans Peter , Vooijs G. Peter
Published: August 2005

The reproducibility and accuracy of the Omnicon (Bausch and Lomb Inc., Rochester, NY) automated tumor colony counter for counting tumor colonies growing in double layer soft agar is evaluated and the reproducibility is compared with manual tumor colony counting. Replicate within day run‐to‐run colony counts of the Omnicon show a median correlation coefficient (r) of >0.985, and day‐to‐day median r of >0.980. In contrast, for manual colony counting, the best intra‐observer reproducibility achieved is a r of 0.943 and the best inter‐observer reproducibility is a r of 0.831. Analysis of results from individual culture plates counted by the Omnicon on 5 separate days shows a median coefficient of variation of 10% with 77% of the culture dishes showing coefficients of variation of colony counts over 5 days of less than 20%. Counting of culture plates during incubation shows that the Omnicon is counting tumor colonies developing after plating of a single cell suspension.
Cells on microspheres: A new technique for flow cytometric analysis of adherent cells
Authors: Bloch Donald B. , Smith Brian R. , Ault Kenneth A.
Published: August 2005

Technical problems have previously prevented the application of fluorescence activated cell sorting to the study of adherent cell populations. We have developed a procedure for attachment of human monocytemacrophages to 14–20 micrometer microspheres. These adherent cells on microspheres retained phagocytic capacity, could be stained for cell surface antigens using indirect immunofluorescence, and could be maintained in long‐term culture. They could be examined and sorted on a fluorescence activated cell sorter while still in the adherent state. This technique facilitates the flow cytometric study of adherent cells and permits the isolation of subpopulations of these cells.