bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantification of an in vitro cell‐cell adhesion assay using interactive laser scanning cytometry
Authors: Newton Sean C. , Millette Clarke F.
Published: August 2005

We are interested in identifying cellcell adhesion molecules on the surface of Sertoli cells that mediate Sertoli cellspermatogenic cell adhesion. Numerous cell‐cell adhesion assays employ microscopic observation, photomicroscopy or radioactive isotopes for quantification. Previously, we developed an in vitro assay for testicular cell interactions. This assay was, however, time consuming using photography for analysis. We have now modified this system using laser cytometry to quantify adherent cells. Rat testicular epithelial cells are cultured for approximately 6 days before labelling with fluorescein diactetate (FDA) to assess confluency by image scanning so that spermatogenic cell binding can be normalized to available epithelial cell surface area. Rat spermatogenic cells are labeled with FDA before addition to epithelial cell monolayers. In some studies, purified spermatogenic cell populations were isolated to determine average cell size. We found that spermatocyte area varied between 225—500 μm2, spermatids were 100—225 μm2 and residual bodies were < 100 μm2. Using these parameters, scanning cytometry allows the differential analysis of adhesion by individual germ cell sub‐classes from mixed cell suspensions, saving time, animals, and major expense. The scanning laser assisted assay is faster, more reproducible and less subjective than earlier cell‐cell adhesion assays using light microscopy or isotopes. This experimental approach should facilitate any cell‐cell adhesion assay in which one cell type is adherent to a substrate.
Three‐dimensional component labeling of digital confocal microscope images enumerates replication centers in BrdUrd labeled fibroblasts
Authors: Baumann Peter H. , Schormann Thorsten , Jovin Thomas M.
Published: August 2005

An algorithm (3Dlabel 1) for labeling three dimensional binary data sets has been developed. Serial optical sections are acquired using a confocal laser scanning microscope. After filtering and thresholding operations, contiguous elements in the three dimensional data sets are identified and labeled. The results are used to calculate the number and size of objects. Additionally, the labeled data are displayed. by applying an algorithm (3Ddisp1) to generate stereo pairs, in which label numbers are color coded and depth is cued by intensity. These procedures have been applied in investigations of the temporal and spatial distribution of replication centers throughout S phase in BrdUrd pulse labeled mouse fibroblasts.
One or multiple samplings for flow cytometric DNA analyses in breast cancer‐prognostic implications?
Authors: Fernö Mårten , Baldetorp Bo , Ewers Sven‐börje , Idvall Ingrid , Olsson Häkan , Sigurdsson Helgi , Killander Dick
Published: August 2005

Flow cytometric assessments of DNA ploidy status and the S‐phase fraction (SPF) have been shown to yield prognostic information in breast cancer. The aim of the present investigation was to elucidate the reproducibility of results with regard to tumor DNA heterogeneity, and to ascertain whether the prognostic value of DNA measurements might be enhanced by analyzing two pieces of a tumor instead of one.

Agreement with regard to ploidy status (diploid versus non‐diploid) was obtained in 90% of cases (71/79) when two adjacent sections of the tumor were investigated, and in 77% of cases (10/13) when four biopsies from different quadrants of the tumor specimen were investigated. The corresponding figures for agreement in SPF (divided into three categories, < 7.0%, 7.0–11.9%, and > 112%) were 75% (59/79; 2‐sample series) and 55% (7/13; 4‐biopsy series).

The main reason for variance in ploidy results was the difficulties in distinguishing near diploid cell populations. Discrepancies in SPF categories could be explained by minor fluctuations in SPF values near the cut‐off levels, or by variance in ploidy status, the fraction of nondiploid nuclei, and background noise due to cell debris.

There was a stepwise increase in recurrence rate (RR) among patients with increasing SPF category (RR:20%, 41%, and 53%). Patients whose SPF categories varied, from low or intermediate in one part of the tumor to high in another, seemed to have a poor prognosis (RR = 57%).

We conclude that the variation in ploidy status and SPF between different parts of the same tumor specimen is largely to be explained by uncertainty in the interpretation of the DNA histograms and by difficulties inherent in the estimation of SPF, rather than by the existence of DNA tumor heterogeneity. Evaluation of a different part of the same tumor specimen may be of value, especially in the case of SPF, as this may yield additional prognostic information. These findings also emphasize the need for good quality control of FCM DNA analysis.
Detection of viral surface antigens on HIV‐2ben infected human tumor cell lines by flow cytometry
Authors: Böhm Dorothea , Nick Sigrid , Voss Gerald , Hunsmann Gerhard
Published: August 2005

The human monocytic cell line U‐937 clone 2 and two T‐cell lines CEM and MOLT‐4 clone 8 were infected with HIV2ben, a recent isolate of HIV‐2. Infection and subsequent antigen expression on the cell surface was monitored by flow cytometry using a rabbit‐anti‐serum against tween‐ether‐treated HIV‐2ben and a fluoresceinisothiocyanate‐conjugated IgG against rabbit‐IgG.

The sensitivity of the three cell lines to infection with HIV‐2ben correlated with the percentages of CD4‐expressing cells but not with the levels of CD4‐expression on the cell. The appearance of viral surface antigens preceeded the formation of syncytia and correlated closely with the infecting virus dose. After 1–2 weeks in culture, 20–85% of the cells of each line expressed viral surface antigens. The variation depended on the cell type and cell culture conditions. The MOLT‐4 clone 8 and the U‐937 clone 2 cells died around 10 or 20 days, respectively, after HIV‐2ben infection. Only HIV‐2ben infected CEM cells grew permanently.

Flow cytometry was an appropriate method to monitor the expression of viral proteins on the cell surface of HIVinfected cell lines. Flow cytometry proved to be more sensitive than determination of RT activity in supernatants of HIV‐infected cells and more precise than light microscopy examinations.
Multivariate flow cytometry of epidermal regeneration provoked by a skin irritant and a tumor promoter
Authors: Kirkhus B. , Glasø M. , Clausen O. P. F.
Published: August 2005

The DNA content and the changes in cellular and nuclear size of isolated regenerating mouse epidermal basal cells were studied after topical application of the skin irritant cantharidin and the tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) to the back skin of hairless mice. The DNA and protein contents of isolated basal cells were stained with propidium iodide and fluorescein isothiocyanate, respectively, and analysed by flow cytometry using the total protein fluorescence as an estimate of cell size and the DNA fluorescence pulse width as an estimate of nuclear size. Transmission electron microscopy was used to identify cells sorted from regions in the bivariate DNA/protein distributions.

The results showed that both chemicals induced an increase in cellular as well as nuclear size of the basal cells. The increase in size was higher in TPA treated than in cantharidin treated animals, anti the bivariate DNA/protein distributions of TPA treated cells differed from those of cantharidin treated cells in that two subpopulations of cycling keratinocytes could be identified. These deviations are probably related to the higher proliferative response observed after TPA treatment and the possibility that proliferative subpopulations in epidermis respond differently to TPA. It may reflect mechanisms providing for a growth advantage of initiated cells, important in tumor promotion. About 8% of the cells in the suspensions from treated animals were non‐cycling non‐keratinocytes, probably infiltrating leukocytes.

The results indicate a strong correlation between rapid regenerative cell cycle progression, i.e., reduced G1 transit time and increased cellular and nuclear size. The increase in cellular size was observed simultaneously in all cell cycle phases, well before the wave of cells entering S phase 16 h after application. This suggests that an increase in size may be essential for the subsequent initiation of DNA synthesis and the reduction in cell cycle time. The results support the hypothesis that the increase in cell size may be caused by genie activation or posttranscriptional processes rather than an increase due to an initial block in cell cycle progression.
Quantitation of human anti‐mouse antibody in serum by flow cytometry
Authors: Labus J. M. , Petersen B. H.
Published: August 2005

Clinical investigations utilizing murine monoclonal antibodies require techniques for the detection of the human anti‐mouse antibody (HAMA) response in patient serum. We report here a flow cytometric assay for the quantitation of HAMA. Commercially available beads conjugated with goat anti‐mouse antibody provide a solid phase matrix for a triple bridge immunoassay. The measurement of fluorescein labeled antibodies by flow cytometry allows accurate quantitation of the HAMA. The assay will detect antibody levels of approximately 1.0 ng/ml. Antibody recovery in serum samples with known amounts of antibody added was greater than 90% at levels ⩾ 10 μg/ml. Serum samples obtained from 41 patients prior to and after single or multidose infusions of KS1/4‐Desacetylvinblastine were analyzed. These results were compared with HAMA titers previously determined by ELISA. With few exceptions, patients with low titers as determined by ELISA demonstrated low HAMA potencies by flow cytometry and those with highest titers the highest potencies. Patients with no detectable HAMA by ELISA were also negative by flour analysis. The results of our studies demonstrate that HAMA levels can be accurately and quantitatively determined by flow cytometry.
Activated and memory T lymphocytes in human milk
Authors: Wirt Daniel P. , Adkins Linda T. , Palkowetz Kimberly H. , Schmalstieg Frank C. , Goldman Armond S.
Published: August 2005

Since activated macrophages and cytokines are found in human milk (HM), a flow cytometry study was conducted to determine whether T cells in HM display phenotypic markers of recent or previous activation. HM was collected during the first 3 d of lactation. The Paint‐a‐Gate program was used to optimize gating on the lymphocyte population. A mean ± 1 SD of 4 ± 3% of total HM leukocytes were lymphocytes and 96 ± 3% were macrophages and granulocytes (N=33 subjects). HM lymphocyte populations were further analyzed in five subjects. T calls (CD3+) represented 83 ± 11% and B cells (CD19+) were 6 ± 4% of HM lymphocytes. The mean CD4/CD8 ratio of T cells in HM was 0.88 (range 0.40–1.25). This ratio was significantly decreased compared to the peripheral blood (PB) of control adults (P < 0.02) and postpartum women (P < 0.02), due mostly to a significant increase in CD8+ CD3+ cells in HM compared to the PB of control adults (P < 0.002) and postpartum women (P < 0.05). T cells bearing markers of recent activation were significantly increased in HM compared to the PB of control adults: 85 ± 7% of CD3+ cells in HM were HLA‐DR+ (controls, 10 ± 4%; P < 0.001), and 15 ± 6% of CD3+ cells in HM were IL‐2R+ (controls, 6 ± 2%; P < 0.001). Subpopulations of CD4+ and CD8+ cells in HM defined by the T200 isoforms CD45RA and CD45RO were markedly altered compared to PB, indicating a striking shift from virginal to antigen‐primed (memory) T cells in HM and suggesting certain functional capacities for these cells. Virtually all CD4+ cells (99.8 ± 0.4%) and 92 ± 5% of CD8+ cells in HM were CD45R0+ (vs. 71 ± 12% and 50 ± 10%, respectively, in the PB of postpartum women; P < 0.001). CD4+ and CDS8+ cells expressing CD45RA were correspondingly markedly reduced (P < 0.001). This phenotypic pattern of T cells in HM may result from T cell activating substances in HM and/or selective homing of T cells to the breast. Conversely, activated T cells in HM may be responsible for cytokines in HM. These activated and memory T cell populations may be transferred to the infant via breastfeeding.
Quantitative and qualitative DNA variations in Anabaena azollae strasb. living in Azolla filiculoides lam.
Authors: Canini Antonella , Caiola Maria Grilli , Ferrucci Luigi
Published: August 2005

Heterocysts and vegetative cells of the filamentous nitrogen‐fixing Anabaena azollae isolated from the apex to the basal leaf cavities of Azolla filiculoides were examined by epifluorescent microscope after fluorochrome staining. Acridine orange (AO), DAPI, and chromomycin fluorochromes were used in order to evidence total DNA content and respectively, A + T and G + C bases.

Measurements of fluorescence intensities were made on photographic prints by the automatic image analysis system Quantimet 970.

Heterocysts contained higher amounts of DNA than did vegetative cells, and their content strongly increased in the basal leaf cavities. The heterocyst DAPI brightness was quite uniform, whereas in vegetative cells DAPI brightness increased from the apex to the basal groups. In vegetative cells from the apex to the median group, the percentage of DAPI brightness was 60–85% with respect to AO brightness, whereas in heterocysts of the same groups DAPI brightness was 40–50% with respect to AO brightness. In the basal group, brightness due to DAPI staining was comparable with those of previous group both in heterocysts and in vegetative cells, whereas chromomycin brightness increased strongly in heterocysts.

These data slow that heterocyst changes its DNA content and composition in the basal leaf cavities, suggesting that its lifetime is not completely over.
Comparison between flow cytometry and image cytometry in ploidy distribution assessments in gynecologic cancer
Authors: Kaern Janne , Wetteland Jarle , Tropé Claes G. , Farrants George W. , Juhng Sang‐Woo , Pettersen Erik O. , Reith Albrecht , Danielsen Håvard E.
Published: August 2005

The DNA content in 37 tumors from 34 women with gynecological cancer was measured by flow cytometry (FCM) and interactive image cytometry (ICM). Agreement was obtained in 81% of cases as regards ploidy levels, but seven tumors (19%) showed different ploidies. Of these, five were classified as diploid by FCM but either aneuploid (three cases) or polyploid (two cases) by IM Two other tumors were aneuploid by ICM but polyploid (one case) and unclassifiable (one case) by FCM. All tumors classified as aneuploid by FCM were also aneuploid by ICM, and all tumors classified diploid by ICM were also diploid by FCM. Of six patients whose tumors were classified as euploid (five diploid and one polyploid) by FCM but classified as aneuploid by ICM, five relapsed, and three of these have died of disease. On the basis of these findings, it is concluded that ICM must be performed in cases classified as diploid by FCM to ensure that small subpopulations of aneuploid tumor cells are not overlooked.
Calibration of a flow cytometric assay of glucose‐6‐phosphate dehydrogenase activity
Authors: Severin E. , Seidler E.
Published: August 2005

The reduction of tetrazolium salts to colored formazans is a reaction which has been exploited both in histo‐ and cytochemistry. Tetrazolium salts forming fluorescent formazans prove suitable for measuring defined cellular dehydrogenase activities in automated processes. This study considers an important aspect of formazan measurement in flow cytometry, namely, calibration. Calibration is performed by correlating the number (and fluorescence intensity) of formazanbearing cells measured by flow cytometry with simultaneously performed biochemical analyses of the same material. The method is demonstrated by an example of glucose‐6‐phosphate dehydrogenase. Using the data of a typical experiment, the enzyme activity is expressed in femtomol of hydrogen transferred per cell during incubation time. Furthermore, through spatially resolved double excitation of formazan and nuclear DAPI fluorescence, an independent analysis of cell cycle and cellular enzymatic activity is established.
Time‐resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization
Authors: Seveus Lahja , Väisälä Mikko , Syrjänen Stina , Sandberg Minna , Kuusisto Ari , Harju Raimo , Salo Juha , Hemmilä Ilkka , Kojola Hannu , Soini Erkki
Published: August 2005

Fluorescent lanthanide chelates with long decay times allow the suppression of the fast decaying autofluorescence in biological specimens. This property makes lanthanide chelates attractive as labels for fluorescence microscopy. As a consequence of the suppression of the background fluorescence the sensitivity can be increased.

We modified a standard epifluorescence microscope for time‐resolved fluorescence imaging by adding a pulsed light source and a chopper in the narrow aperture plane. A cooled CCD‐camera was used for detection and the images were digitally processed.

A fluorescent europium chelate was conjugated to antisera and to streptavidin. These conjugates were used for the localization of tumor associated antigen C242 in the malignant mucosa of human colon, for the localization of type II collagen mRNA in developing human cartilaginary growth plates, and for the detection of HPV type specific gene sequences in the squamous epithelium of human cervix.

The specific slowly decaying fluorescence of the europium label could be effectively separated from the fast decaying background fluorescence. It was possible to use the europium label at the cell and tissue level and the autofluorescence was effectively suppressed in in situ hybridization and immunohistochemical reactions in both frozen and formaldehydefixed, wax‐embedded specimens.
Distribution of non‐heme porphyrin content of individual erythrocytes by fluorescence image cytometry and its application to lead poisoning
Authors: Sassaroli Massimo , Dacosta Rui , Väänänen Heikki , Eisinger Josef
Published: August 2005

The quantitation of the non‐heme porphyrin content of circulating erythrocytes is an important tool in the screening, diagnosis, and management of lead disease, iron deficiency anemia, and certain porphyrias. Useful information about these pathological conditions may be obtained from the distribution of the non‐heme porphyrin content of individual red blood cells. An image‐based cytometry system was developed and used to determine the cellular distributions of zinc protoporphyrin (ZPP) in the erythrocytes of normal and lead‐exposed human subjects. The fluorescence cytometry system described here lends itself to a wide range of statistical studies of cell populations, in which extrinsic fluorescent probes or antibodies may be used instead of porphyrin. Image‐based cytometry appears to offer a number of important advantages over the more conventional flow cytometry systems, including greater sensitivity, applicability to a wider range of cellular parameters, and the ability to retain images of each cell used in the survey for subsequent examination. Pairs of absorption and fluorescence images of many fields of dispersed immobilized red cells were acquired by means of a program which controls the microscope stage, focussing, shutters, filter wheels, and a cooled, slow‐scan CCD camera. The observed marked differences in the ZPP distributions of erythrocytes from donors with chronic and acute lead exposure are discussed in terms of the metabolism of internalized lead.
Comparison of strategies to detect and quantitate uniquely marked cells in intra‐ and inter‐species hemopoietic chimeras
Authors: Pallavicini M. G. , Langlois R. G. , Reitsma M. , Gonzalgo M. , Sudar D. , Montoya T. , Ul. Weier H. , Haendel S.
Published: August 2005

Evaluation of the outcome of successful bone marrow transplantation and indepth studies of transplantation biology rely increasingly upon detection and enumeration of donor hemopoietic cells in the transplanted recipients. The ability to detect and enumerate low levels of donor engraftment in interphase cell subpopulations in hemopaietic chimeras is particularly important for studies of mixed lineage chimerism, early relapse manifestations, and engraftment of subpopulations present at low frequency. We describe and compare the sensitivity and specificity of DNA‐based detection strategies (fluorescence in situ hybridization, in vitro DNA amplification using the polymerase chain reaction) and flow cytometric analysis of cell surface markers to detect cells carrying marker DNA or proteins in syngeneic (mouse‐to‐mouse) and xenogeneic (mouse‐to‐human, monkey, sheep) backgrounds. DNA‐based detection strategies offer advantages of rapid analysis and enumeration of target cell frequencies with detection sensitivities approximating 10−4. The sensitivity of immunofluorescence‐linked flow cytometric‐based detection of nucleated leukocytes approached 10−3, whereas flow cytometric‐based detection of fixed human erythrocytes was feasible at cell frequencies of 10−5. Data described in this manuscript should facilitate selection of appropriate methodologies for assessment of hemopoietic chimerism following transplantation.
Analytical methods for the study of liver cell proliferation
Authors: Gerlyng Per , Stokke Trond , Huitfeldt Henrik S. , Stenersen Tom , Danielsen Håvard E. , Grotmol Tom , Seglen Per O.
Published: August 2005

Various cytometric methods for analysis of regenerating rat liver growth (DNA ploidy distributions, binucleation, and DNA synthesis by in vivo BrdUrd incorporation) were evaluated. The overall hepatocellular growth rate (labeling index), the binucleation rate, and separate indices for mononuclear and binuclear cells could be measured simply by microscope counting of collagenase‐isolated hepatocytes immunostained for BrdUrd. Flow cytometry of cells stained for BrdUrd and DNA provided labeling indices for the various hepatocellular DNA ploidy classes as well as for nonparenchymal cells (identified by their size‐dependent light scatter), but could not distinguish between mononuclear and binuclear hepatocytes. Image cytometry, using fluorescence or Feulgen staining, was inferior to flow cytometry in terms of speed and DNA resolution, but allowed a complete analysis of all hepatocellular DNA ploidy and nuclearity classes. It may therefore be the method of choice, particularly for analysis of liver cell cultures from which single cells are not easily obtained. Fluorescence staining would seem to be preferable to Feulgen staining, since the latter could not be used simultaneously with BrdUrd staining and therefore required a two‐step analysis. A non‐immunological method, based on the ability of incorporated BrdUrd to quench DNA staining by a Hoechst dye, could only be applied to isolated nuclei, thus giving no information about binucleation. The latter method may be useful for analysis of tumors which are difficult to dissociate to intact whole cells.
A statistical analysis of flow cytometric determinations of phagocytosis rates
Authors: Fredrickson A. G. , Hatzis Christos , Srienc Friedrich
Published: August 2005

Uptake of particles by phagocytosing cells is a process that exhibits variability of its rate. This variability is inherent in the mechanism of particle uptake and in the mechanisms that determine the distribution of physiological states within a population of phagocytosing cells. When numbers of particles ingested by cells are determined flow cytometrically an additional measurement variability is superimposed on and interacts with the aforementioned biological variability. In one method of determining population phagocytosis parameters, which involves fitting theoretical equations to experimental time course data on the fractions of cells which have ingested 0, 1, 2, particles, the effects of measurement variability are circumvented, although this usually has the cost of not using all the sample data obtained. However, in a second, simpler, method which is based on determining the time course of the number of particles ingested by an average cell, measurement variability is not circumvented and its effects must be considered. An analysis of the combined effects of biological and measurement variability on the results obtained with the simpler method is presented in this paper. Experimental results for phagocytosis of latex microspheres of uniform size and fluorochrome content by populations of the ciliate Tetrahymena pyriformis show that, for this system, measurement variability is entirely negligible in comparison with biological variability. This conclusion might not apply to other systems, however, and situations which might make measurement variability of some significance are mentioned in the paper. The equations given can be used for the analysis of such situations.
Clinical Applications of Cytometry: 6th Annual Meeting
Authors: Giorgi J. V. , Hurtubise P. E. , Cram L. S. , Parker J. W. , La Via M. F.
Published: August 2005

The Sixth Annual Clinical Applications of Cytometry Meeting was held September 11–14, 1991, in Charleston, SC. Attendance reached a record 470. The meeting provides a forum for interactions among investigators who utilize cytometry as a tool in their clinical immunology, cell biology, hematology, and cancer investigations. Clinical laboratory directors and their technical staff find the meeting of practical value because of the presentation of new applications that they can take home to their own laboratories. The emphasis of the meeting is on advances in the application of cytometry to clinical problems. Often, advances result from new dyes or reagents or improved instrumentation. Sometimes they result from advances in biology that make the studies possible. Occasionally a new way of looking at the same data provides a useful answer. In every case, the effort is to provide a reliable, straightforward way to quantitate biologic information in order to provide improved diagnosis or treatment of human disease.
Quantitation and mapping of integrated human papillomavirus on human metaphase chromosomes using a fluorescence microscope imaging system
Authors: Callahan Daniel E. , Karim Amna , Zheng Gemin , Ts,o Paul O. P. , Lesko Stephen A.
Published: August 2005

Integrated human papillomavirus type 16 (HPV‐16) DNA was directly visualized on metaphase chromosomes in the two human cervical carcinoma cell lines SiHa and CaSki by fluorescence in situ hybridization with a biotinylated DNA probe (7.9 kb). The fluorescence intensities of hybridization signals from single copies and dispersed clusters of integrated HPV‐16 DNA were quantified using a microscope equipped with a cooled‐CCD camera that was interfaced to an image processor and host computer. Hybridization signals were localized on chromosomes using separate, registered images of 4′, 6‐diamidino‐2‐phenylindole (DAPI) or propidium iodide stained metaphase chromosome spreads. In both SiHa and CaSki spreads, a single fluorescein signal was observed on one or both chromatids of chromosome 13, which was identified by simultaneous hybridization with a biotinylated centromere probe specific for chromosomes 13 and 21. Ratios of the distance from 13pter to the HPV‐16 signals to the entire chromosome length were 0.63 ± 0.05 in both SiHa and CaSki cells, indicating the possibility of a common integration domain on chromosome 13. In SiHa cells, no additional signals were observed on other chromosomes. This observation, taken together with literature reports that SiHa cells contain 1 to 2 copies of the HPV‐16 genome in this region of chromosome 13, suggests that each fluorescein signal on chromosome 13 represents one equivalent of the HPV‐16 genome. The total integrated fluorescence intensity in isolated CaSki metaphase chromosome spreads was approximately two orders of magnitude greater than that of a single copy of HPV‐16 DNA in SiHa cells, indicating an increase in HPV‐16 copy number. The relative increase in copy number indicated was similar to that reported in the literature.
Immunofluorescent labeling of centromeres for flow cytometric analysis
Authors: Schmitz Annette , Guilly Marie‐Noëlle , And Barbara Trask , Engh Ger Van Den
Published: August 2005

A procedure to stain the centromeric region of chromosomes for dual beam flow cytometric analysis is described. Serum from a CREST (Scleroderma syndrome) patient presenting a high titer of anticentromeric antibodies was chosen on the basis of specificity of labeling of cells on slides. The high affinity of the antibodies to centromeres and low binding to chromosomal arms allowed the development of an indirect immunofluorescent labeling procedure using isolated and unfixed chromosomes stabilized by Mg++ ions. Discontinuous Ficoll gradients were used to separate chromosomes from unbound antibodies. With this procedure, chromosome clumping and degradation were minimal. The chromosomes were then stained with the DNA dyes Hoechst 33258 and chromomycin A3, before dual beam flow cytometric analysis. Flow karyotypes, with good chromosome peak resolution, were obtained for both human and hamster chromosomes subjected to the immunolabeling procedure. For quantification of FITC fluorescence due to bound antibody, chromosomes were counterstained with Hoechst only. The FITC intensity of antibody‐labeled human and hamster chromosomes were 4–10 and 20 times greater than control chromosomes, respectively. These results suggest that the staining procedure may be suitable for immunolabeling of chromosomes with antibodies recognizing other nuclear proteins and their subsequent quantification by flow cytometry.
A quantitative method for evaluating bivarlate flow cytometric data obtained using monoclonal antibodies to bromodeoxyuridine
Authors: Allen White R. , Terry Nicholas H. A.
Published: August 2005

A method is presented for analyzing data from bivariate analysis of cell populations exposed to bromodeoxyuridine and subsequently examined both for the presence of BrdUrd and for the cellular DNA content. It is shown that certain features may be defined in the bivariate data which are constant independent both of cell type and, within limits, experimental variability. These landmark features include the ratio of red, DNA, fluorescence of G2 + M cells to G1 cells, the ratio of green fluorescence corresponding to the non‐specific binding of unlabeled G2 + M cells to unlabeled G1 cells, and the distribution of green fluorescence in unlabeled cells. The landmarks make it possible to standardize rules for establishing the separation line between‐labeled and unlabeled cells as required in these experiments to obtain estimates of cytokinetic parameters. Values obtained for the DNA synthesis time and the potential doubling time which result from different decision rules for distinguishing labeled from unlabeled are compared in two murine tumor lines. The potential doubling time, but not the DNA synthesis time is shown to depend sensitively on the separation line. Suggestions are presented for analyzing clinical data with this procedure.
Flow cytometric detection of the mitochondrial BCL‐2 protein in normal and neoplastic human lymphoid cells
Authors: Aiello Antonella , Delia Domenico , Borrello Maria Grazia , Biassoni Daniela , Giardini Roberto , Fontanella Enrico , Pezzella Francesco , Pulford Karen , Pierotti Marco , Porta Giuseppe Della
Published: August 2005

The bcl‐2 proto‐oncogene, rearranged and deregulated in B‐cell lymphomas bearing the t(14;18) translocation, encodes an inner mitochondrial membrane protein that blocks apoptotic cell death. We have developed a sensitive immunofluorescence assay for the single‐ and multicolor flow cytometric analysis of bcl‐2 protein in relation to other markers and cell cycle, based on a fixation‐permeation step of cells with paraformaldehyde and Triton X100 and the use of a bcl‐2 specific monoclonal antibody (MoAb). As an application of this method, we have examined the expression of bcl‐2 in normal and neoplastic lymphoid cells. We have found that > 80% of normal Tand B‐cells are bcl‐2 positive; following in vitro mitogen activation, the bcl‐2 reactivity decreased slightly in the former but markedly in latter cells. In both cases the bcl‐2 expression was not restricted to a specific phase of the cell cycle, as evidenced by two‐color analysis. On lymphoblastoid cell lines, the bcl‐2 staining intensity was variable and not necessarily correlated to molecular rearrangements of the bcl‐2 gene. Among fresh B‐cell non‐Hodgkin's lymphomas (B‐NHL), most sporadic Burkitt's cases were bcl‐2 negative. Of four centroblastic‐centrocytic cases with rearrangements of the bcl‐2 gene, only two presented elevated amounts of bcl‐2 protein, indicating that the levels of bcl‐2 are not diagnostic of the translocation. The flow cytometric analysis of bcl‐2 protein allows study and quantification, as the single cell level and in selected cell subsets, of the expression of the bcl‐2 gene and provides an important tool for assessing its role in hematopoietic cell development, proliferation, and neoplastic conversion.
Seric factors influence cell cycle modifications during commitment of murine erythroleukemia cells to differentiation
Authors: Mencherini Fabienne , Khochbin Saadi , Chabanas Agnès , Lawrence Jean‐Jacques
Published: August 2005

Cell cycle modifications are among the early events which take place during the induced differentiation of murine erythroleukemia (MEL) cells; a transient accumulation of the cells in the G1 phase of the cell cycle, followed by a re‐entry of the cells into a proliferation state, has been described. In order to characterize a putative role of serum in such variations, we have studied the modifications of the cell cycle parameters when cells were induced to differentiate in the presence or in the absence of seric factors. We show that, in the absence of exogenous factors brought by serum, the G1 accumulation was enhanced both in amplitude and in duration, but cells were still able to bypass the G1 block and re‐enter into the S phase. These results indicate that the resumption of cell proliferation after the transient block is under synergistic control of seric and endogenous factors, but these later are sufficient to overcome the block. However, MEL cells, were unable to differentiate in the absence of seric factors, as measured by the number of benzidine‐positive cells during induction with hexamethylene‐bisacetamide (HMBA) or butyric acid. This capacity to differentiate was recovered when serum was added back to the culture medium, and the efficiency of recovery was maximal when cells underwent a full round of DNA replication in the presence of serum after the G1 block. The analysis of two molecular markers of cell differentiation conned these results.
Dose effects of LPS on neutrophils‐ in a whole blood flow cytometric assay of phagocytosis and oxidative burst
Authors: Böhmer R. H. , Trinkle L. S. , Staneck J. L.
Published: August 2005

Whole blood phagocytosis (P) and oxidative burst (OB), a rapid and sensitive flow cytometric method for quantifying neutrophil activation, was modified for single laser systems by using propidium iodide labeled Staphylococcus aureus and 2′,7′ dichlorofluorescein diacetate. The purpose of the present study was to characterize this assay with respect to the stimulatory activity of bacterial Upopolysaccharide (LPS) on phagocytosis. Blood from healthy donors was preincubated with log doses of bacterial LPS B (0. 1–1,000 ng/ml) or sterile pyrogen‐free saline at 37°C from 0–120 minutes. LPS increased both P and OB in a dose‐dependent manner (up to 62 and 121%, respectively) at all time points tested, and this effect on P and OB could be detected even with no preincubation. This LPS‐induced phagocytic activity could be blocked by the addition of polymyxin B (10 pμg/ml) during preincubation. The priming effect of LPS was maximal at 45 min. P and OB were inhibited by preincubation with EDTA, at doses > 2 mM (60 and 80% inhibition, respectively). These observations are consistent with the exquisite sensitivity of the neutrophil to endotoxin. This method can evaluate neutrophil response to immunomodulatory and chemotherapeutic agents in a physiological milieu. These findings re‐emphasize the necessity of using pyrogen‐free reagents in any study of neutrophil function.
On the measurement of cytokinetics by continuous labeling with bromodeoxyuridine with applications to chick wing buds
Authors: Allen White R. , Fallon John F. , Savage Mary P.
Published: August 2005

The cytokinetic properties, specifically the phase‐transit times, TG1, TS, and TG2+M, of chick wing bud cells were estimated using data obtained from continuous labeling of stage 20 embryos with bromodeoxyuridine (BrdUrd). The presence of BrdUrd was detected with monoclonal antibodies, and the amount of DNA in the cells was determined with propidium iodide. The fraction of cells in each cell cycle phase, the fraction of labeled cells, and the relative movement, a measure of the mean DNA content, of all labeled cells were evaluated using bivariate flow cytolnetry at successive times following introduction of the label. Equations are presented to describe the fraction of unlabeled cells in G2 + M; which gives a direct estimate of TG2 +M; the fraction of all labeled cells, whiA can then be used to estimate TG1; and, finally, the relative movement, which provides an estimate of TS. Thus, the data measured in these experiments together provide estimates of the progression through the cell cycle of limb mesoderm cells.
Eukaryotic DNA replication is a topographically ordered process
Authors: Humbert Catherine , Usson Yves
Published: August 2005

This paper describes the relationship between the BrdUrd replicating pattern of a cell and its localization within the S phase by means of topographical features and DNA content measurement. The present study follows an objective ranking of the, BrdUrd patterns obtained from a spectral analysis of the BrdUrd images. The pattern ranking was consistent with the DNA content increase throughout the S phase. Five texture groups were arbitrarily set up for the purpose of multivariate analysis. Nine topographical parameters were computed for each BrdUrd‐labelled nucleus. The descriptive quality of these parameters was assessed by means of factorial discriminant analysis. These parameters made it possible to characterize objectively the known pattern distributions of replication sites qualitatively described in the literature. © 1992 Wiley‐Liss, Inc.
Differences between platelet and microparticle glycoprotein IIb/IIIa
Authors: Nomura Shosaku , Suzuki Masahiko , Kido Hirofumi , Yamaguchi Kazuyuki , Fukuroi Tsutomu , Yanabu Mutsumasa , Soga Tetsuji , Nagata Hirokazu , Kokawa Terutoshi , Yasunaga Kojiro
Published: August 2005

Glycoprotein (GP) IIb and IIIa are major constituents of the platelet membrane which are involved in forming the fibrinogen receptor on activated platelets. We used flow cytometry to study the effects of ethylene‐diamine tetraacetic acid (EDTA) on the membrane GPIIb/IIIa complexes of platelets and microparticles, and to study the effects of cations on dissociated GP complexes. Microparticles were detected by both the volume signal and by fluorescence using an FITC‐conjugated anti‐GPIb antibody (NNKY5–5). When platelets were stimulated with ADP, calcium ionophore A23187, or thrombin, fibrinogen binding to the platelet surface increased markedly. However, fibrinogen binding to microparticles showed little increase in response to such agonists. Microparticle GPIIb/IIIa complexes were dissociated by incubation with EFTA at 37°C but did not reassociate after treatment with divalent cations (Ca2+, Mg2+, and Mn2+) in contrast to platelet GPIIb/IIIa complexes. These results suggest that some interaction of GPIIb/IIIa and linked structures like the platelet cytoskeleton may be involved in the reassociation of dissociated GPIIb and GPIIIa, perhaps explaining the failure of reassociation of microparticle GPIIb/IIIa (i.e., the fibrinogen binding to microparticles). © 1992 Wiley‐Liss, Inc.
Evaluation of a method for detection of cells with reduced drug retention in solid tumours
Authors: Carpentier Y. , Gorisse M. C. , Desoize B.
Published: August 2005

A method for detection of cells with reduced drug retention was evaluated in solid tumours. After a 1 h incubation with daunorubicin (DNR), the right angle scatter (RAS), forward angle scatter (FAS), and specific fluorescence (Fluo) were measured in sensitive and resistant cells; only Fluo was related qualitatively, but not quantitatively, to resistance. Various incubation conditions were examined. When the pH of the incubation medium increased, the DNR retention increased in sensitive and resistant cells. In contrast, when the cell concentration increased, the DNR retention decreased. Using sensitive and resistant cell lines, a proportion of resistant cells lower than 10% can be detected in a mixture. To analyse cells from solid tumours, the cells were dissociated by repeated fine needle aspirations. Tumours from 22 patients have been processed with this technique; 8 samples were classified as S (sensitive); 2 as R (resistant); and 12 as I (intermediate).

Further experiments were run to study and improve the method. Another method of detection of dead cells was tested. The intra‐assay variability of the technique was found to be less than 10%. When the study was performed with different fragments of the same tumour, the variation, corresponding to the tumour heterogeneity, rose to 21 to 36%. The inter‐assay reproducibility was too high, so a variant of this technique has been adapted, using verapamil or cyclosporin A, which is able to block DNR efflux; this new method allows tumour cells to be used as their own controls. © 1992 Wiley‐Liss, Inc.
Cell cycle analysis of asynchronous cell populations by flow cytometry using bromodeoxyuridine label and Hoechst‐propidium iodide stain
Authors: Ormerod Michael G. , Kubbies Manfred
Published: August 2005

Continuous labelling of cells with deoxybromouridine (BrdUrd) followed by staining with a bis‐benzimidazole (Hoechst 33258) and a phenanthridinium (propidium iodide or ethidium bromide) allows the cells to be separated by flow cytometry according to the extent of their DNA replication. This BrdUrd‐Hoechst/PI method has been used mainly to observe perturbations of the cell cycle in synchronously growing cells. In this paper we demonstrate that, when the method is applied to asynchronously dividing cells, more extensive information can be derived about the effects of cytotoxic and other treatments on the kinetics of the cell cycle. The interpretation of the data is explained, the effects of different types of cytotoxic agent are described, and the method is compared briefly to other methods for following cell cycle kinetics. © 1992 Wiley‐Liss, Inc.
Flow cytometric measurement of lipid peroxidation in vital cells using parinaric acid
Authors: Hedley David , Chow Sue
Published: August 2005

A method for measuring lipid peroxidation using time resolved flow cytometry is described. Because of its chemical nature, the naturally fluorescent fatty acid cis‐parinaric acid is readily consumed in lipid peroxidation reactions. It could be loaded into Chinese hamster ovary cells in a time and concentration dependent manner at 37°C, with 5 μM for 60′ giving consistent, bright fluorescence without evidence of cytotoxicity. Examination of cells by fluorescence microscopy showed diffuse staining of surface and internal membranes. Cells were maintained at 37°C while being examined in an Epics Elite flow cytometer equipped with a 325nm HeCd laser, and parinaric acid fluorescence at 405nm was measured over time. Addition of the oxidant tert‐butyl hydroperoxide resulted in a burst of intracellular oxidation, shown by simultaneously loading the cells mrith dichlorofluorescein, and loss of parinaric fluorescence over time. This was followed by cell death, indicated by loss of forward light scatter and uptake of propidium iodide.

Pretreatment of the cells with the antioxidant α‐tocopherol, 200 μM, reduced the rate of loss of parinaric acid fluorescence and delayed the onset of cell death. Simultaneous biochemical determination of the lipid peroxidation breakdown product malondialdehyde confirmed a close temporal relationship with loss of parinaric acid fluorescence, both with and without α‐tocopherol pretreatment and suggested that the flow cytometric assay for lipid peroxidation is of comparable sensitivity.

The mitochondrial stain dodecyl acridine orange and the cyanine dye DiOC(6)3 were combined with cis‐parinaric acid staining and could be excited by the latter using resonance energy transfer. Because these two probes show a degree of organelle specificity, they can be used to measure the loss of parinaric acid due to lipid peroxidation at defined subcellular sites. Although our own interest in the method is to examine the actions of redox cycling anticancer drugs and the integrity of host antioxidant defences as a possible mechanism of drug resistance, it appears to be a versatile technique for investigating an important process of cell injury that is difficult to study using standard biochemical assays. © 1992 Wiley‐Liss, Inc.
Streptavidin‐based quantitative staining of intracellular antigens for flow cytometric analysis
Authors: Srivastava Praveer , Sladek Todd L. , Goodman M. Nadine , Jacobberger James W.
Published: August 2005

A streptavidin–biotin‐based three‐step immunolabeling protocol for quantitative staining of intracellular antigens for flow cytometric analysis was evaluated using simian virus 40 (SV40) large T antigen. The concentration as well as the quantity of antibody used required optimization. The optimum labeling conditions varied moderately with cell lines that express T antigen levels over a 40–50‐fold range. The procedure resulted in specific fluorescence 2.4 times higher than that using a comparable two‐step indirect immunofluorescence technique. The gain in resolution was shown to be greater when staining cells with lower antigen levels. In the analysis of background fluorescence, the principal components were, as for the two‐step technique, autofluorescence and propidium spectral overlap. While streptavidin does add to the background, the increase is relatively small. Decreasing the propidium concentration from 50 μg/ml to 5 μg/ ml was found to reduce significantly the level of background from this source. Theoretical aspects of quantitative staiaing and of resolution versus quantification are discussed. © 1992 Wiley‐Liss, Inc.
Quantitation of resistance of leukemic cells to cytosine arabinoside from BrdUrd/DNA bivariate histograms
Authors: Lacombe F. , Belloc F. , Dumain P. , Puntous M. , Lopez F. , Bernard P. , Boisseau M. R. , Reiffers J.
Published: August 2005

The cytotoxicity of ara‐C derives from an inhibition of DNA synthesis after incorporation of ara‐CTP into DNA. The rate of DNA synthesis can be determined from the amount of bromodeoxy‐uridine (BrdUrd) incorporated into cells after a short exposure to BrdUrd. We developed a computer program to quantify the inhibition of the rate of DNA synthesis by analysis of the distribution of BrdUrd/ DNA. Inhibition was evaluated in ara‐C‐sensitive and resistant cells after incubation with different doses of ara‐C. An index of resistance to ara‐C (RI) was expressed as the ratio of the amount of BrdUrd incorporated into S phase cells incubated with ara‐C to that incorporated in the absence of ara‐C. In the ara‐C‐sensitive and resistant HL60 cells, a linear relationship between RI and log ara‐C concentration was observed. Small numbers of slightly resistant cells in mixtures of ara‐C‐sensitive and resistant cells could be determined using this method, making it suitable for clinical use to test the resistance of leukemic cells to ara‐C. © 1992 Wiley‐Liss, Inc.