bluuebunny/crossref_metadata_2025 huggingface dataset:
A review of techniques and results obtained in one laboratory by an integrated system of methods designed for routine clinical flow cytometric DNA analysis
Authors: Vindeløv Lars L. , Christensen Ib Jarle
Published: August 2005

Establishing flow cytometric DNA analysis as a clinical routine procedure requires adequate and proven guidelines, by which the data can be obtained and interpreted to directly influence management of the individual patient with a specific neoplasm. The present paper is intended as a contribution to such guidelines, of which only fragments are available today.

We have previously described a system of methods, designed for routine flow cytometric DNA analysis. In the present status report our experience, based on approximately 18,000 samples (clinical and experimental) is summarised. Sample acquisition with fine‐needle aspiration, storage at −80°C, internal standardization by chicken (CRBC) and trout red blood cells (TRBC), staining with propidium iodide (PI), and analysis in the flow cytometer is recapitulated, with emphasis on previously unpublished aspects. The method of statistical analysis which has an integrating role is described in some detail. A lack of linearity between channel number and DNA content was determined experimentally, and the coefficient of variation (CV) was found to decrease with increasing channel number. The corrections in the algorithm of deconvolution made necessary by these findings are fundamental for estimating the end results. The zero point adjustment and procedures for changing from one batch of standards to another are described. A systematic approach to interpretation of DNA histograms is attempted and illustrated by data from clinical specimens of malignant lymphoma, breast cancer, small cell lung cancer, cancer of the oral cavity, and bladder cancer. Some problems are still unsolved and visual inspection is required to determine if the quality of the individual histogram is satisfactory. Inspection of the fluorescence/light scatter dot‐plot provides additional information for the recognition of artifacts. The results stress that good quality DNA histograms with as small CVs as possible are important for interpretation of the data. It is essential that statistical methods are employed to extract the key end‐point results. These are the number of subpopulations and their relative representation, and for each subpopulation the DNA index (DI) and the fractions of cells in the cell cycle phases.

For the DNA data to have any rationally based impact on clinical decision making, it must be demonstrated that they have an independent prognostic value. Strategies for final evaluation are discussed. Multicenter trials on fresh material, to accrue quickly the number of patients necessary for firm conclusions, are suggested.
Geometrical constraints on the shape of cultured cells
Authors: Heckman C. A.
Published: August 2005

Previous studies on cultured cells revealed that the values of certain shape descriptors were highly correlated despite the fact that there was little mathematical interdependence (Heckman, CA: In Advances in Cell Culture, Vol. 4, K. Maramorosch, ed, pp 85–156, 1985). The independence of such descriptors was tested by calculating the values of correlations among descriptors for a data set consisting of macroscopic objects. The descriptors were unlinked for this data set, confirming that high correlations reflected specific characteristics of cultured cells. These characteristics were identified by constructing model figures incorporating the geometrical features postulated to be responsible for each correlation. Because the position of the ellipse of concentration depended upon the form in which mass was displaced in figures, the fraction of the figure falling within the area of the ellipse (FINE) and the ratio of the ellipse area to the figure's area (ARAT) became decorrelated from each other in figures with massive projections. Further, correlations between ARAT or FINE and variables measuring cavities and inflections of the perimeter showed a complex dependence on the size, shape, and number of invaginations. Descriptors could become decorrelated due to features present in only a minority of cells of a population. Finally, cell populations that differed with respect to physiological characteristics showed no differences in the value of ARAT:FINE correlation. However, correlations between ARAT and the coefficient of variation of centroid‐to‐perimeter distances were altered. This suggested that correlation coefficients may be themselves powerful descriptors of the shape characteristics of cell populations.
Eight‐Parameter PC‐AT based flow cytometric data system
Authors: Kachel Volker , Messerschmidt Roger , Hummel Peter
Published: August 2005

An 8‐parameter flow cytometric data system is described using an IBM‐at compatible personal computer (PC) and a commercial analog to digital conversion (ADC) board. A dedicated pulse processing interface adapts the flow cytometric pulses to the ADC board and controls the number of parameters to be taken up and the trigger conditions. The trigger thresholds are automatically held at a level immediately above the noise level. For the timing of kinetic measurements a linear voltage ramp of adjustable rise time is available. A built‐in precision voltage source can be used for an overall calibration. The data system is operated by software written in assembly language. Data may be collected and processed in 1–8‐parameter listmode or 1–3‐parameter histogram mode. Functions are available for graphical color displays, numerical integration, multiparameter gating, and printing.
Flow cytometric detection of ornithine decarboxylase activity in epidermal cell subpopulations
Authors: Robertson Fredika M. , Gilmour Susan K. , Beavis Andrew J. , O'Connell Sean M. , Conney Alan H. , Huang Mon‐Tuan , Laskin Jeffrey D. , Hietala Oili A. , O'Brien Thomas G.
Published: August 2005

Using flow cytometry in combination with membrane permeabilization techniques to enhance binding of antibodies with immunoreactive protein within the cytoplasm, we have developed a method to examine the ornithine decarboxylase (ODC) activity present within subpopulations of epidermal cells following acute and chronic exposure to the phorbol ester tumor promoter 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA). The method described has the sensitivity to detect basal levels of ODC as well as increases in ODC at early time points following treatment with TPA and has the additional advantage of allowing subpopulation identification and characterization.
Recovery of RNA from flow‐sorted fixed cells
Authors: Khochbin Saadi , Grunwald Didier , Pabion Michel , Lawrence Jean‐Jacques
Published: August 2005

We describe a method for the preparation of RNA from ethanol‐fixed cells, allowing analysis of the RNA from cells “frozen” in a given physiological state. This technique may have important applications in experiments which require prolonged cell manipulations beforre RNA preparation, such as investigations of cell‐cycle‐regulated gene exprression, which require the preparation of cells for cell‐cycle flow analysis, and even for long‐term cell sorting. It eliminates all the inconveniences associated with the use of fresh cells, and allows cell‐cycle biologists to couple flow cytometry methodology with the advancing techniques of molecular biology.
Flow cytometric measurement of rates of particle uptake from dilute suspensions by a ciliated protozoan
Authors: Lavin Daniel P. , Fredrickson A. G. , Srienc Freidrich
Published: August 2005

Flow cytometry is used to measure rates of ingestion of particles from dilute monodisperse suspensions by the ciliate Tetrahymena pyriformis. The particles used are polystyrene microspheres containing a fluorescent dye. Measurements were made directly, that is, by determining the fluorescence intensities from microspheres ingested by cells in samples collected from the experimental feeding apparatus. The fact that fluorescence intensities from individual cells can be grouped into discrete classes based on the numbers of fluorescent particles associated with the cells makes it possible to calibrate the flow cytometer and convert fluorescence measurements into numbers of particles ingested by average cells. At low particle concentration or high ciliate concentration, ingestion data must be corrected for depletion of particles during the assay, and a method for doing this is described. Experiments at various ciliate concentrations show that ingestion rates are not affected by this concentration. The methods developed should allow measurements of rates of ingestion of particles from concentrated and polydisperse suspensions. For such measurements, nonfluorescent particles together with a fraction of fluorescent tracer particles would be used.
Alterations of growth fraction and DNA content in K562 cells by differentiating agents
Authors: Gorisse Marie‐Claude , Carpentier Yves , Joly Philippe , Comoe Léopold , Desoize Bernard
Published: August 2005

The growth fraction, estimated by the monoclonal antibody Ki‐67 labeling, and DNA content, assessed by ethidium bromide staining, were determined simultaneously in K562 leukemic cells by flow cytometry. A multiparametric analysis enabled the fraction of the cell by flow cytometry. A multiparametric analysis enabled the fraction of the cell population with G1, S, and G2 + M content in Ki‐67‐positive and Ki‐67‐negative cells to be evaluated.

Butyric acid (BUT) was used as positive control. The fraction of Ki‐positive cells decreased with the BUT concentration, while the proportion of cells with G1 DNA content increased only in the Ki‐negative cells.

Adriamycin, aclacinomycin A, and fagaronine induced differentiation, as assessed by benzidine staining and glycophorin A expression. These drugs decreased the fraction of Ki‐positive cells by more than 50% for both anthracyclines and by 25% for fagaronine. Following treatment, Ki‐negative cells displayed a G1, but also a G2 and a S DNA content in different proportions, indicating that induction of quiescent cells by differentiating agents is not a uniform process and is worthy of interest.
Detection of cells resistant to alkylating agents by flow cytometric analysis of DNA damage
Authors: Frankfurt Oskar S. , Seckinger Daniel , Sugarbaker Everett V.
Published: August 2005

DNA damage was measured by flow cytometric analysis of cells sensitive and resistant to alkylating agents. Human ovarian carcinoma cell line A2780 and a subline which is 7 times more resistant to L‐phenylalanine mustard (L‐PAM) were treated with the drug, fixed, and stained with monoclonal antibody (MOAB) F7‐26 which detects single‐stranded regions in alkylated DNA. Mean fluorescent intensity was measured on a flow cytometer. Cells were heated before staining to amplify single‐strandedness in alkylated DNA. Significantly larger amount of MOAB was bound to DNA in sensitive than in resistant cells. Fluorescence increased by 80 channels per νg L‐PAM insensitive cells and only by 17 channels in resistant cells. Sensitive and resistant cells were treated with L‐PAM, mixed in different proportions, and stained with MOAB. Populations of sensitive and resistant cells were clearly separated on fluorescence histograms by more than a decade difference in fluorescence intensity. Presence of 2–5% resistant cells was detected among sensitive cells as a separate cell subset.

We conclude that staining with MOAB F7‐26 can be used as an indicator of cell sensitivity or resistance to alkylating agents. Detection of minor subsets of resistant cells in heterogeneous populations by FCM analysis may be useful for monitoring emerging drug resistance.
Flow cytometric evaluation of nitro blue tetrazolium (NBT) reduction in human polymorphonuclear leukocytes
Authors: Fattorossi Andrea , Nisini Roberto , Le Moli Soccorsa , De Petrillo Guido , D'Amelio Raffaele
Published: August 2005

Oxidative metabolic burst of activated human polymorphonuclear leukocytes (PMN) is most commonly investigated in clinical practice by evaluating nitroblue tetrazolium (NBT) reduction at the single cell level. Reduced NBT precipitates where the redox reaction has taken place and can be visualized as PMN‐associated dark blue granules of formazan in light microscopy. Although widely used and not technically demanding, this method remains subjective and labor intensive, especially when large numbers of samples need to be investigated. We developed a new flow cytometry technique in which PMN membrane was rendered fluorescent by a short incubation with fluorescein‐conjugated Concanavalin A. PMN were then incubated with NBT and increasing doses of a suitable stimulus, such as phorbol myristate acetate (PMA). Formazan has a distinct peak of absorption at 520 nm that represents the peak of emission of fluorescein. As a consequence, formazan quenches the PMN‐associated fluorescence. Data show that a dose‐dependent reduction of fluorescence can be obtained using graded amounts of PMA in normal PMN cultures. PMN‐associated fluorescence remains unchanged in control patients with chronic granulomatous (CGD) disease, a disorder characterized by a selective impairment of PMN oxidative metabolism. Electronic cell size increases upon PMA incubation in normal PMN, irrespective of the presence of NBT. Conversely, forward light scatter intensity decreases in the presence, but not in the absence, of NBT indicating that the phenomenon is due to the capacity of formazan to absorb/scatter the incident light. The present method for easily detecting NBT reducing activity at single cell level by flow cytometry makes use of commonly available, inexpensive reagents and standard instrumentation. It could become a useful test for clinical purposes.
Personal computer interface and software for the ZONAX microscope attachment controller
Authors: Rigney David R. , Velyvis John H. , Wei Jeanne Y.
Published: August 2005

We describe the hardware and software of a general‐purpose interface that permits a personal computer (PC) running MS‐DOS to control cytometric devices, e.g., a scanning stage, shutters, and focus motor attached to a microscope. This note explains how to use the interface to convert the ZONAX microscope attachment controller from 8080‐based hardware control to PC‐based control.
Improved method for measuring intracellular Ca++ with fluo‐3
Authors: Rijkers Ger T. , Justement Louis B. , Griffioen Arjan W. , Cambier John C.
Published: August 2005

The accuracy of flow cytometric measurement of intracellular calcium with fluo‐3 is compromised by variation in basal fluorescence intensity due to heterogeneity in dye uptake or compartmentalization. We have loaded cells simultaneously with fluo‐3 and SNARF‐1. When SNARF‐1 fluorescence is collected at approximately 600 nm, its intensity does notchange upon cell activation. Furthermore, fluo‐3 and SNARF‐1 fluorescence signals exhibit a linear relationship. The ratio of fluo‐3 to SNARF‐1 eliminates a significant proportion of variation in fluorescence intensity caused by variation in fluo‐3 uptake and thus can be used as a sensitive parameter for measuring changes in [Ca2+]i.
Nuclear texture parameters as discriminant factors in cell cycle and drug sensitivity studies
Authors: Colomb Evelyne , Dussert Christophe , Martin Pierre‐Marie
Published: August 2005

The authors have recently shown that cell cycle characteristics of in situ cell populations can be determined using the SAMBA 200 cell image processor by computing 15 densitometric and texture parameters on each Feulgen‐stained nucleus and multiparametric analysis of data. The present paper displays the importance of chromatin pattern assessment and detection of conformational changes in DNA structure, based on nine nuclear texture parameters measured from the grey level cooccurrence and the run‐length section matrices.

Reference files were constructed by merging respective reference files (G0/ G1, S, G2 and M) of MDA AG and MCF‐7, two mammary epithelial cell lines presenting different morphological aspects and hormone responses, these files were found to be valid in the reclassification of any mammary epithelial cell in culture with a diploid or near diploid pattern.

Moreover, the authors demonstrate that chromatin texture changes, following direct interaction of chemotherapeutic drugs with DNA, may be assessed owing to nuclear texture parameters. Consecutive to daunomycin addition (0.5 μg/ml) and concomitant to the appearance of nuclear morphological alterations in MDA AG sensitive cells as viewed by microscopic observation, discriminant factorial analysis showed progressively increasing erroneous reclassification from 15 to 72 h of treatment. These experimental results were exploited with a kinetic mathematical model to quantify the daunomycin blocking effect: 20% in S phase and 80% in G2 phase. Interestingly, no textural change was observed on MDA A1 anthracycline resistant cells, indicating that these texture parameters may permit distinction of drug sensitive cells.

This methodology (1) can be applied to test in vitro resistance‐reversal molecules, (2) may be extended to other therapeutic agents giving rise to conformational changes in DNA structure, and (3) can be applied to cytopunctions or imprints of tumor biopsies with diploid‐like DNA content to follow evolution of drug sensitivity or resistance during course of therapy.
Modalities of synthesis of Ki67 antigen during the stimulation of lymphocytes
Authors: Lopez F. , Belloc F. , Lacombe F. , Dumain P. , Reiffers J. , Bernard P. , Boisseau M. R.
Published: August 2005

The antibody Ki67 is currently used to evaluate the proliferative fraction of solid tumors and some hematological malignancies. We have used phytohemagglutinin (PHA)‐stimulated peripheral blood lymphocytes as a model to study the entry of quiescent cells into cell cycle and to follow their progress to the next cycle. Flow cytometric analysis of lymphocyte samples stained with the antibody Ki67 and a DNA marker has allowed us to follow the expression of Ki67 antigen (Ki67Ag) as a function of the position of the cells in the cell cycle. The use of drugs blocking the stimulated lymphocytes in different phases of the cell cycle permitted us to demonstrate that Ki67Ag expression started from the beginning of the first S phase. The level of Ki67Ag increased during S phase until mitosis, when its expression was maximal. After division, the cells in G1 phase showed a decrease in Ki67Ag expression (possibly corresponding to degradation) until they reentered S phase, when the level of Ki67Ag increased again. The results confirm that the expression of Ki67Ag is related to the proliferative state of the cells and suggest that it may be used to determine the proliferative cell fraction in hematopoietic tissues.
Aberrant Ki‐67 expression in normal bone marrow revealed by multiparameter flow cytometric analysis
Authors: Van Bockstaele Dirk R. , Lan Jar , Snoeck Hans‐W. , Korthout Marcel L. , De Bock Robrecht F. , Peetermans Marc E.
Published: August 2005

Ki‐67 is a commercially available monoclonal antibody that reacts with a nuclear antigen detectable in proliferating cells only. Since its first description, it has been widely used as a “universal” proliferation marker and few groups have questioned the validity of the initially described reactivity, although this was tested only on very restricted experimental models. We wanted to check its reactivity on normal bone marrow (BM) samples using a multiparameter flow cytometric analysis. Although we were able to reproduce the findings of Ki‐67 positivity on cultured and stimulated cells, we could not detect any convincing Ki‐67 positivity on nuclei of normal BM samples. These samples all had a noticeable proliferating compartment as evidenced by their DNA content. These data are in contrast with the data we obtained starting from stressed marrows and marrows cultured in the presence of hematopoietic growth factors, where we found a marked Ki‐67 positivity.

This discrepancy suggests that bone marrow cells, growing and proliferating under steady‐state conditions and guided by natural control mechanisms, may lose their Ki‐67 expression upon exiting the progenitor compartment and entering the differentiating compartment.
Introduction of antibody into viable cells using electroporation
Authors: Berglund Deborah L. , Starkey Jean R.
Published: August 2005

Conditions for labelling an intracellular antigen, p21ras, using electroporation to introduce a fluorescent antibody, are described. Following labelling, cells were evaluated for p21ras associated fluorescence by flow cytometry. Electroporation, sorting, and cell handling parameters were varied to determine optimal conditions for cell viability. Cells were best held in serum containing growth medium both before and after electroporation, while antibody introduction during the electroporation phase was most efficient when carried out in a balanced saline solution. For maximum efficiency of antibody internalization, the antibody needed to be present during electroporation, and medium needed to be replaced several times in the first few hours after electroporation to ensure good cell survival.
DNA histogram debris theory and compensation
Authors: Bagwell C. Bruce , Mayo Sara W. , Whetstone Sherry D. , Hitchcox Shelly A. , Baker David R. , Herbert Donald J. , Weaver Donald L. , Jones Michael A. , Lovett Edmund J.
Published: August 2005

In this paper we describe a theory of DNA histogram debris generation and compensation that can be applied to paraffin‐embedded frozen tissue preparations. The theory predicts the distribution of fragments generated from single and multiple random sectioning of threedimensional ellipsoids representing nuclei. The fragment distribution is assumed to be a major component of the underlying debris in DNA histograms. A comparison of S‐phase fractions (SPF) from matched tissue prepared by frozen and formalin‐fixed paraffin‐embedded DNA methods demonstrates the usefulness of the theory.
Ratiometric measurement of intracellular pH in cultured human keratinocytes using carboxy‐SNARF‐1 and flow cytometry
Authors: Van Erp Piet E. J. , Jansen Monique J. J. M. , De Jongh Gijs J. , Boezeman Jan B. M. , Schalkwijk Joost
Published: August 2005

In this study we describe a method to measure intracellular pH in cultured human keratinocytes using flow cytometry. Keratinocytes pose a technical problem because the population is heterogeneous with respect to size and metabolic activity (nonspecific esterase activity), resulting in variability in dye uptake. In order to compensate for this, dyes were selected that change colour with pH. The ratio of fluorescence intensities at two wavelengths was recorded and used as a measure of intracellular pH by reference to the pH in the presence of the proton ionophore nigericin. However, methods published till now do not routinely combine the ratiometric technique and excitation with an argon ion laser set at 488 nm. Therefore we have tested the recently developed pH‐sensitive dye carboxyseminaphthorhodafluor‐1 (SNARF‐1) as a possible candidate for flow cytometric pH measurements and compared it with 2′,7′‐bis(carboxyethyl)‐5,6‐carboxyfluorescein (BCECF) and 2,3‐dicyanohydroquinone (DCH) with respect to emission spectra, resolution, range, and stability of cellular fluorescence. SNARF‐1 had a practical and stable excitation wavelength of 488 nm rather than UV, it offered the possibility of ratio‐metric measurements on the basis of a real emission shift, and had superior resolution for the pH range 7–8. With SNARF‐1 we found that keratinocytes cultured under low serum conditions (0.2%) contain a higher proportion of cells with relatively low intracellular pH compared to high serum cultures (6%). Furthermore, pH changes were followed by changes in relative DNA content. These findings suggest that intracellular pH can be an early functional proliferation marker for human keratinocytes.
Use of a photolabeling technique to identify nonviable cells in fixed homologous or heterologous cell populations
Authors: Riedy M. C. , Muirhead K. A. , Jensen C. P. , Stewart C. C.
Published: August 2005

Flow cytometric determination of viable versus nonviable cells in fixed samples can be accomplished by utilizing the irreversible binding of photoactivated ethidium monoazide (EMA). EMA is a positively charged molecule which is excluded by cells with intact membranes (viable cells), included by cells with damaged membranes, and can be photochemically crosslinked to nucleic acids using visible light. EMA fluorescence can be excited using a standard argon laser operating at 488 nm and is able to be distinguished from fluorescein and phycoerythrin, Fixation is important when analyzing cells from a potentially infectious origin. EMA is photochemically crosslinked and therefore unable to leak out of cells when removed from the extracellular media, unlike propidium iodide (PI) or other viability stains, which were heretofore commonly used. We demonstrate the usefulness of EMA in combination with fluoresceinated and phycoerythrin labeled monoclonal antibodies in immunophenotyping. The photoaffinity labeling technique allows for a quick and efficient means of identifying nonviable cells which cannot be distinguished on the basis of light‐scattering properties.
DNA slit‐scan flow cytometry of bladder irrigation specimens and the importance of recognizing urothelial cells
Authors: Wheeless Leon L. , Reeder Jay E. , O'Connell Mary J. , Robinson Roy D. , Cosgriff Janice M. , Fradet Yves , Frank Irwin N. , Cockett Abraham T. K.
Published: August 2005

DNA slit‐scan flow cytometry was used to analyze 150 bladder irrigation specimens from 83 patients. Specimens were categorized into groups based on cystoscopy, histology, and cytopathology. Cells were stained for DNA with propidium iodide using a whole cell protocol. Nonspecific fluorescence in the cytoplasm of some urothelial cells together with differential DNA staining of cell types in certain specimens was noted. DNA frequency distributions were analyzed using a semi‐automated technique. Data were gated using slit‐scan morphological features to remove cellular debris, multiple nuclei, and cells exhibiting nonspecific cytoplasmic fluorescence. Specimens were classified abnormal if they were aneuploid or had a hyperdiploid fraction (HDF) greater than 8%. The sensitivity to abnormality was 89% for grade 3 transitional cell carcinoma (TCC), 70% for grade 2 TCC, and 67% for grade 1 TCC. Specificity was 61%. Specimen data were then reprocessed using slit‐scan morphological features to enrich for urothelial cells. The urothelial cells were identified by the ratio of nuclear diameter to cell diameter. This method was found to be in good agreement with immunofluorescent labeling of urothelial cells using the urothelium‐seleetive T16 monoclonal antibody. The sensitivity to abnormality remained 89% for grade 3 TCC and 70% for grade 2 TCC, but fell to 52% for grade 1 TCC. Specificity for the urothelial cell enriched data increased to 77%. Reprocessing of data to enrich for urothelial elements resulted in 16 fewer specimens with an aneuploid DNA distribution and 2 fewer specimens with increased HDF. These data illustrate the necessity for recognition of urothelial cells in the flow analysis of unfixed bladder irrigation specimens stained with propidium iodide using a whole cell technique. Differential staining of blood and urothelial cells can mimic aneuploidy or contribute to an increased hyperdiploid fraction. These may result in the specimen being classified as abnormal if they are not recognized as such.
Detection and analysis by dual‐laser flow cytometry of bacteriophage T4 DNA inside Escherichia coli
Authors: Sanders Cynthia A. , Yajko David M. , Nassos Patricia S. , Hyun William C. , Fulwyler Mack J. , Hadley W. Keith
Published: August 2005

Bacteriophage T4 DNA was detected and analyzed inside E. coli by dual‐laser flow cytometry using a dye combination of Hoechst 33258 (H33258) and chromomycin A3 (CA3) which bind to A‐T‐ and G‐C‐rich regions of DNA, respectively. An exponentially‐growing culture of E. coli ATCC 11303 was infected with T4 bacteriophage at a 1:1 multiplicity of infection. Samples were taken immediately and at 5 min intervals and placed on ice to interrupt viral replication. The samples were then centrifuged, ethanol‐fixed, stained with H33258 and CA3, and analyzed by flow cytometry. Twenty‐five minutes post‐infection, a population of cells which contained T4 DNA began to appear on both a bivariate contour plot and a frequency histogram plot of the data. By 35 min, T4 DNA‐containing cells could be distinguished from E. coli cells containing little or no T4 DNA. The ratio of CA3:H33258 fluorescence was then used to calculate the % G + C value for T4 DNA inside E. coli. A value of 35.6 ± 0.2% was obtained, which agrees with % G + C values determined by traditional methods. These results demonstrate that dual‐laser flow cytometry can be used to study viral DNA inside the bacterial host.
Comparison of microscopic imaging strategies for evaluating immunocytochemical (PAP) steroid receptor heterogeneity
Authors: Sklarew R. J. , Bodmer S. C. , Pertschuk L. P.
Published: August 2005

Receptogram analysis was compared with three other imaging strategies for immunocytochemical assay of estrogen receptors. These included nuclear‐specific methods for analysis of nuclear integrated optical density (IOD) or mean optical denstiy (MOD) histograms, and field‐specific methods, where the pixel optical density (POD) histogram was evaluated for the composite nuclear phase. Measurements in culture and in breast cancer cryosections were treated separately to isolate geometric considerations. In culture receptograms the modality of IOD and MOD histograms and their bivariate contour maps revealed one, two, or more subpopulations with discrete receptor content and concentration. However, when the field of nuclei was imaged as a whole, regardless of the number of subpopulations, POD histograms showed two minima, defining three intranuclear phases. This was due to mottling and variegation of intranuclear chromatin and nucleolar immunostaining and not to differences between nuclei. These limitations were also revealed in breast cancer sections. In POD histograms, % unstained pixels did not provide a reliable estimate of % receptor negative nuclei, as determined by their enumeration. In sections, correction of IOD for nuclear volume variability was essential to suppress artifactual peaks not representing differences in receptor content. This was achieved by multiplying nuclear IOD by the spherical nuclear radius (S) of individual slab sections. Peaks of IOD(S) then reflected receptor content on a true ratio scale. Only receptogram analysis, which incorporates these strategies, permitted objective evaluation of receptor heterogeneity at the level of tumor subpopulations.
Evaluation of flow cytometric methods for determining population potential doubling times using cultured cells
Authors: Terry Nicholas H. A. , White R. Allen , Meistrich Marvin L. , Calkins Dennise P.
Published: August 2005

Various methods have been proposed for determining the potential doubling times (Tpot) of mammalian cell populations by using flow cytometric techniques after labeling the cells with bromodeoxyuridine (BrdUrd). We show here that, in a well‐defined in vitro system where multiple time measurements are possible, all the methods give similar results that are close to the true population doubling time. Of ultimate interest, however, is the accuracy of determination of Tpot from a single time point. In this paper we compare the accuracy and precision of the methods in making such determinations at different times after labeling.

The relative movement (RM) of BrdUrd‐labeled cells that have not divided at the time of assay allows for computation of the length of S phase (Ts). The precision of estimation of Ts was enhanced when a quantity, ν (a function of the fraction of BrdUrd‐labeled divided and the fraction of BrdUrd‐labeled undivided cells), was used to estimate the initial intercept of RM. Furthermore, calculation of Tpot from the formula, Tpot = ln(2) Ts/v, gave values closest to the observed population doubling time. It is suggested that the use of RM with v be the analytical method of choice for the calculation of Tpot from single time‐point observations, preferably made at times between the length of the G2 and M phases (TG2M) and Ts.
Mimosine reversibly arrests cell cycle progression at the G1–S phase border
Authors: Watson Paul A. , Hanauske‐Abel Hartmut H. , Flint Alan , Lalande Marc
Published: August 2005

It has previously been demonstrated that the compound mimosine inhibits cell cycle traverse in late G1 phase prior to the onset of DNA synthesis (Hoffman BD, Hanauske‐Abel HM, Flint A, Lalande M: Cytometry 12:26–32, 1991; Lalande M:Exp Cell Res 186:332–339, 1990). These results were obtained by using flow cytopmetric analysis of DNA content to compare the effects of mimosine on cell cycle traverse with those of aphidicolin, an inhibitor of DNA polymerase α activity. We have now measured the incorporation of bromodeoxyuridine into lymphoblastoid cells by flow cytometry to determine precisely where the two inhibitors act relative to the initiation of DNA synthesis. It is demonstrated here that mimosine arrests cell cycle progression at the G1–S phase border. The onset of DNA replication occurs within 15 min of releasing the cells from the mimosine block. In contrast, treatment with aphidicolin results in the accumulation of cells in early S phase. These results indicate that mimosine is a suitable compound for affecting the synchronous release of cells from G1 into S phase and for analyzing the biochemical events associated with this cell cycle phase transition.
Genetic stock assessment of spawning Arctic cisco (Coregonus autumnalis) populations by flow cytometric determination of DNA content
Authors: Lockwood Samuel F. , Bickham John W.
Published: August 2005

Intraspecific variation in cellular DNA content was measured in five Coregonus autumnalis spawning populations from the Mackenzie River drainage, Canada, using flow cytometry. The rivers assayed were the Peel, Arctic Red, Mountain, Carcajou, and Liard rivers. DNA content was determined from whole blood preparations of fish from all rivers except the Carcajou, for which kidney tissue was used. DNA content measurements of kidney and blood preparations of the same fish from the Mountain River revealed statistically indistinguishable results. Mosaicism was found in blood preparations from the Peel, Arctic Red, Mountain, and Liard rivers, but was not observed in kidney tissue preparations from the Mountain or Carcajou rivers. The Liard River sample had significantly elevated mean DNA content relative to the other four samples; all other samples were statistically indistinguishable. Significant differences in mean DNA content among spawning stocks of a single species reinforces the need for adequate sample sizes of both individuals and populations when reporting “C” values for a particular species.
Sorting minor subpopulations of cells: Use of fluorescence as the triggering signal
Authors: McCoy J. Philip , Chambers William H. , Lakomy Robert , Campbell Joseph A. , Stewart Carleton C.
Published: August 2005

Flow cytometric cell sorting is commonly used to obtain purified subpopulations of cells for use in in vitro and in vivo assays. This can be time‐consuming if the subpopulations of interest represent very low percentages of the cell suspension under study. Often the desired subpopulations are identified by two‐color immunofluorescence staining. Generally, cell sorting is performed with a flow cytometer configured to trigger on light scatter signals, then sort windows are set based upon the signals from both fluorescent markers. We demonstrate that triggering the cytometer with the fluorescence signal from antibody staining common to both of the desired subpopulations, then sorting the subpopulations based upon staining of a second marker, substantially increases the speed of cell sorting vis‐à‐vis traditional methods. This is because undesired events are not analysed, allowing an increase in the throughput rate. While desired subpopulations of cells can be obtained by this method, undesired (i.e., nonstaining) cell “contaminants” increase and may require a second sort. The combined time for the initial enrichment sort and a second sort can be less than sorting once using standard methodology. Alternatively, the degree of contamination may be controlled by adjusting the concentration of the cell suspension and by the sample flow rate.
Use of diploid and triploid trout erythrocytes as internal standards in flow cytometry
Authors: Pierrez José , Ronot Xavier
Published: August 2005

DNA content determination requires the use of standards. Vindelov has shown the need to use two standards. Chicken and trout erythrocytes are commonly used, but they are not ideal standards. On the one hand, their DNA contents rarely frame the studied sample DNA content, and, on the other hand, as their base compositions are different in terms of A+T/G+C, their relative indices change according to the stains used. Use of triploid trout erythrocytes instead of chicken erythrocytes allows elimination of these two drawbacks; however, diploid trout must be differentiated from triploid trout. The present paper shows that an anatomic malformation is found with the triploid trout and so justifies the use of paired diploid and triploid trout as standards to measure nuclear DNA content.
A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification
Authors: Schmid Ingrid , Uittenbogaart Christel H. , Giorgi Janis V.
Published: August 2005

A method was developed for gentle fixation of mammalian cells and permeabilization of their membranes. The method is useful for staining of intracellular antigens or quantification of DNA content simultaneously with cell surface staining. Cells are treated for 1 h at 4°C with 0.25% buffered paraformaldehyde then for 15 min at 37°C with 0.2% Tween 20 detergent in PBS. The procedure permits excellent staining of intracellular proteins, very low coefficients of variation (CV) on the G0G1‐peak of DNA distributions, and preservation of the integrity of cell surface antigens. The low vs. 90° angle light scatter profile of cell clusters is maintained thereby allowing discrimination of different cell populations including human peripheral blood lymphocytes and monocytes for gating and analytic purposes. The method was successfully used on a variety of other cell types, including human thymocytes, murine thymocytes and spleen cells, and several leukemic cell lines. Dual‐color surface antigen staining combined with DNA staining with 7‐amino‐actinomycin D (7‐AAD) on peripheral blood mononuclear cells (PBMC) cultured with tetanus toxoid allowed the determination of the cell subset that was preferentially stimulated. Staining for internal antigens was done on CCRF‐CEM for expression of CD3ϵ and on NALM‐6 for expression of μ. The technique we developed gave bright and specific staining of internal antigens in the examples presented here. It is particularly suited for correlations of internal antigen staining with DNA staining and/or surface immunofluorescence.
3‐D image analysis system and megakaryocyte quantitation
Authors: Zhang Y. J.
Published: August 2005

A quasi‐automatic computer image analysis system has been developed for 3‐D reconstruction of stained serial sections and implemented on an IBAS system. Some new automatic image analysis techniques have been designed and incorporated into the system. For image segmentation, a transition region determination based thresholding method is introduced. Neither histogram calculation nor empirical parameters are needed in the automatic threshold selection. A two step 3‐D reconstruction procedure—symbolic and pictorial reconstructions—is designed to improve the flexibility and the computational capability of the system. The global level registration and local level registration are separated. The former consists of establishing the relationship among a large numbers of profile pairs dispersed in adjacent sections. A pattern matching method based on pattern recognition principles is devised to exploit the information about the statistical character of mismatch caused by deformation of sections and about the relationship of nearby objects. For the latter, an equivalent elliptical approximation method based on the physical theory of the rotation of rigid bodies is proposed. The system has been used for 3‐D reconstruction and quantitation of megakaryocytes in human bone marrow tissue. Features about individual 3‐D megakaryocyte cell and the spatial distribution of megakaryocytes are determined. The latter is a new to megakaryocyte quantitation and is not possible by using conventional stereologic techniques. These experimental results have demonstrated the ability of the system to perform quantitative analysis.
Dicentric chromosome frequency analysis using slit‐scan flow cytometry
Authors: Lucas Joe N. , Mullikin James C. , Gray Joe W.
Published: August 2005

Slit‐scan flow cytometry (SSFCM) was used to quantify the frequency of dicentric chromosomes in human lymphoblastoid cells following γ irradiation. In this study, cultured human cells were irradiated with 0, 0.25, 0.5, 1.0, and 2.0 Gy of 0.66 MeV γ‐rays, cultured for an additional 11 h, and treated for 5 h with colcemid. Chromosomes were then isolated, stained with propidium iodide, and analyzed using SSFCM for total fluorescence and slit‐scan profile. The frequency of chromosomes having DNA contents greater than once and less than twice the DNA content of the number 1 chromosome and producing trimodal profiles was determined at each dose. This frequency was used as an estimate of the relative dicentric chromosome frequency at that dose. The estimated dicentric chromosome frequency per cell, f(D), increased with dose, D, in a linear‐quadratic manner according to the relation
Nuclease‐induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide
Authors: Prosperi Ennio , Giangarè Maria Chiara , Bottiroli Giovanni
Published: August 2005

A flow cytometric analysis of DNA structural changes induced by cleavage with nucleases was performed on isolated HeLa nuclei by assessing changes in stainability with the DNA‐specific fluorochrome propidium iodide (PI). After mild digestion with DNAse I, micrococcal nuclease, or with the single‐strandspecific S1 and Neurospora crassa nucleases, fluorescence intensity of nuclei stained with PI increased by about 15—30% above the value of undigested control samples. No significant modifications were observed with the restriction enymzes Eco RI, Alu I, and Not I. The DNAse I‐induced increase in fluorescence intensity was also observed with the non‐intercalating dye Hoechst 33258, but not with mithramycin. Nuclease‐induced fluorescence intensity changes as determined with PI were found to be dependent on the dye concentration. A constant increase (about 20%) was measured at dye/DNA‐P ratios > 0.11. Below this value (2 μg/ml PI), the fluorescence intensity of digested samples was 15—30% lower than that of undigested controls. This behaviour towards intercalating dyes is similar to that of the relaxed (nicked) vs. the supercoiled (intact) form of circular DNA. These results suggest that conformation‐ but not sequence‐specific nucleases induce a relaxation of DNA supercoils.
Flow cytometric monitoring of glutathione content and anthracycline retention in tumor cells
Authors: Nair Swagata , Singh Shivendra V. , Krishan Awtar
Published: August 2005

We have used an enzymatic (spectrophotometric) and a flow cytometric (GSH‐MBCL) method to compare the glutathione (GSH) content of doxorubicin sensitive (P388) and resistant (P388/R‐84) murine leukemic and human lung cancer cells. The flow cytometric analysis revealed that GSH‐MBCL conjugate formation was dependent on glutathione‐S‐transferase (GST) activity. The human solid tumor cell lines exhibited extensive heterogeneity, high GSH content, and GST activity. In contrast to the enzymatic method, the flow cytometric method did not accurately reflect the 95% reduction in GSH content of cells treated for 24 h with 100 μM BSO. Possible reaction of MBCL with other sulfhydryl groups (other than GSH) in BSO‐treated cells may be responsible for this discordance. We have also shown the feasibility of using dual parameter flow cytometry to monitor cellular anthracycline (daunorubicin) retention and GSH‐MBCL conjugate fluorescence in human tumor cells. These two parameters, which measure drug retention and cellular detoxification, are believed to be the important determinants of chemoresistance in tumor cells.
Concomitant delineation of surface Ig, B‐cell differentiation antigens, and HLADR on lymphoid proliferations using three‐color immunocytometry
Authors: Segal Glenn H. , Edinger Mark G. , Owen Matthew , McNealis Michael , Lopez Phillip , Perkins Alvin , Linden Michael D. , Fishleder Andrew J. , Stoler Mark H. , Tubbs Raymond R.
Published: August 2005

Accurate and consistent enumeration of B‐cell subpopulations in lymphoid tissue was achieved through multiparameter three‐color immunofluorescence and flow cytometric analysis (FCM). Phycoerythrin (PE)‐anti‐CD19 (Leu12) and biotinylated anti‐HLADr/streptavidin‐Duochrome® (PE/Texas Red), used in conjunction with polyclonal fluorescein isothiocyanate (FITC) conjugated anti‐surface immunoglobulin (SIg) antibodies, effectively separated non‐specific binding and background fluorescence from true B‐cell surface FITC immunofluorescence, while concomitantly analyzing for HLADr and CD19 phenotypic expression/deletion. Autofluorescence was measured to establish a fluorescence threshold. A second control measured non‐specific binding of isotypic control mouse Ig and non‐immune rabbit IgG. Cell suspensions from 128 samples of various lymphoid proliferations were studied. In 116 of the 128 samples, kappa/lambda ratios determined by flow cytometry correlated well with immunocytology results obtained using cytospins from the same cell suspension and with histopathologically established diagnosis. Clonality and lineage as defined immunotypically by flow cytometry was concordant with genotypic results in 64 of the 67 cases evaluated. SIg, HLADr, and CD19 deletions were demonstrated by flow cytometry in 8, 4, and 1 case(s), respectively. Discordance was usually attributable to selective loss of large neoplastic cells in flow cytometry specimens or absent expression of SIg by some cytoplasmic Ig (CIg+) lymphomas.
The effect of improvements in cytometer sensitivity on the detection of CD5‐positive B cells with dim fluorescence
Authors: Givan Alice L. , Shenton B. K. , Culvert Jane E.
Published: August 2005

When antigen density on the surface of a cell population is low and variable, the percentage of that population determined to express the antigen (i.e., to be positively stained) depends directly on the sensitivity of the flow cytometer for resolving particles which are dimly fluorescent from those which are unstained. In this study, the sensitivity of a commercial flow cytometer has been improved by changes in the photomultiplier tube, the fluorescence filter, and the amount of stray light entering the fluorescence channel. In a model system with human lymphocytes, modifications to these factors increased the percent of the B‐lymphocyte population found to express the CD5 antigen.