bluuebunny/crossref_metadata_2025 huggingface dataset:
Interlaboratory study of cellular fluorescence intensity measurements with fluorescein‐labeled microbead standards
Authors: Vogt Robert F. , Cross G. David , Phillips Donald L. , Henderson L. Omar , Hannon W. Harry
Published: August 2005

To determine the precision of cellular fluorescence intensity (FI) measurements derived from labeled microbead standards, FI results were compared from 43 different flow cytometers in 34 laboratories. All laboratories analyzed prepared aliquots of fluoresceinated calf thymocyte nuclei (Fluorotrol), human lymphocytes stained with fluoresceinated anti‐CD4 antibody, and fluoresceinated microbeads used as both internal and external standards. Measurements were conducted by most laboratories on the third and fourth days after sample preparation. Results for percent of events within the gates and the histograms returned by participants indicated that the samples had remained stable and that gated populations had been properly identified. All standard curves showed strong linearity, and the pooled results from all standards produced a best‐fit curve that was in close agreement with the assigned values. Nonetheless, results for cellular FI were highly variable, with CVs of 20–34%. Agreement within lab/instrument was much better, with CVs ranging from 3.0 to 9.9%. The overall variability was not obviously attributable to differences in the types of cytometer, nor could it be explained by attributes of the standard curves or any other single variable examined. However, the application of a corrective factor based on FI results for Fluorotrol allowed a two‐fold improvement in the precision of FI measurements on CD4‐stained lymphocytes, with an overall CV of 11%. Uncharacterized differences in the operating conditions of flow cytometers can influence cellular FI measurements, but consistent results can be obtained if a stained cellular calibrator is analyzed in addition to the proper microbead standards.
Two‐ and three‐color immunofluorescence using aminocoumarin, fluorescein, and phycoerythrin‐labelled antibodies and single laser flow cytometry
Authors: Delia Domenico , Martinez Erundina , Fontanella Enrico , Aiello Antonella
Published: August 2005

Antibodies coupled to 7‐aminocumarin (AMCA) emit a bright blue fluorescence under ultraviolet (UV) excitation and are therefore ideal for three‐color immunofluorescence (IF) with fluorescein (FITC) and phycoerythrin (PE) labeled reagents; however, due to the different absorption spectra, the use of these fluorophores for multicolor flow‐cytometric analysis requires a double light excitation source (e.g., two‐laser system).

We report a strategy which uses a single argon‐ion laser to simultaneously excite AMCA, FITC, and PE, thus allowing the flow cytometric analysis of three immunological parameters. When the UV‐visible argon‐ion laser is fitted with an appropriate set of mirrors, the 351.1–363.8 nm (UV) and 488 nm wavelengths (accounting for 80 mW and 520 mW, respectively) are simultaneously generated; these lines can then be exactly focused on the same observation point by an achromatic cylindrical lens.

A number of comparative analysis were performed with this instrumental set up to verify the sensitivity of AMCA IF and its possible application for multicolor immunophenotypic evaluation of blood cell subsets.

When AMCA‐ and FITC‐labeled anti‐mouse Ig antibodies were assessed for their ability to detect limiting amounts of mouse monoclonal antibody bound to cells, the former was less sensitive than the latter. A number of factors, including differences in excitation energy (80 mW for AMCA and 520 mw for FITC) and extinction coefficients (1.9 × 104 for AMCA and 6 × 104 for FITC) could explain this result.

Single‐color IF analysis of blood cells stained with monoclonal antibodies specific for T‐cells (CD3, CD4, and CD8) and B‐cells (CD19) gave, for each marker, similar results, regardless of the fluorochrome used; similarly, two‐color IF using AMCA/FITC, AMCA/PE, and FITC/ PE combinations provided comparable results. AMCA fluorescence was minimally detected in the green (requiring only 5% color compensation) but not in the red fluorescence channel. Triple‐color detection of CD3, CD4 (or CD8), and CD 19 positive blood cell subsets was also achieved.

Our data indicate that multiple‐color IF analysis of blood T‐ and B‐cell subsets can be performed by flow cytometry and single‐laser dual‐wavelength excitation using antibodies labeled with AMCA, FITC, and PE. Improvements of the fluorescent reagents and of the optical assembly of the instrument may be required to further increase the resolution of AMCA IF.
Imaging cytometry by multiparameter fluorescence
Authors: Galbraith William , Wagner Marc C. E. , Chao Jean , Abaza Mohammed , Ernst Lauren A. , Nederlof Michel A. , Hartsock Robert J. , Taylor D. Lansing , Waggoner Alan S.
Published: August 2005

A system is described for performing multicolor fluorescence image cytometry of cell preparations. After the setting up stage, the operation is automatic: the microscope fields are found and focused; then images are acquired for each fluorophore, corrected and analyzed, without any operator interaction. Human peripheral blood lymphocytes on microscope slides were used as a test system. In these experiments, three fluorescent antibodies were used to identify lymphocyte sub‐populations, and a DNA content probe was used to identify all nucleated cells. The cell subset percentages determined by image cytometry were comparable to percentages obtained when cells from the same preparation were analyzed by flow cytometry. Multicolor fluorescence imaging cytometry can potentially be extended to the analysis of cells in smears, fine needle biopsies, imprints, and tissue sections.
Time window analysis and sorting
Authors: Dunne John F.
Published: August 2005

Flow cytometric hardware and procedures were developed to continuously analyse and to sort a particular time window in a kinetic response. The technique uses balanced air pressure to drive a stimulus‐bead mixture from a vial to a t‐junction where it mixes passively with cells. The t‐junction is distanced from the flow cell and air pressure regulated so that the stimulation occurs at a fixed and adjustable time before the cells are interrogated by the laser beam. Practical applications of the device demonstrate utility with cells whose reponses are seen in seconds or minutes. The device is easily implemented on any sorting flow cytometer.
Tumor cytokinetic effects of acute starvation versus polyamine depletion in tumor‐bearing mice
Authors: Westin Thomas , Gustafsson Bertil , Hellander Karin , Reinholdtsen Lola , Tibell Lena , Lundholm Kent , Edström Staffan
Published: August 2005

Previous investigations in our laboratory have demonstrated that both acute host starvation and polyamine depletion by means of the irrevesible ODC‐inhibitor (ODC = ornithine‐decarboxylase) fluoro‐methylornithine (DFMO) lead to pronounced growth retardation of rapidly proliferating tumors. The aim of this investigation was to elucidate how these different interventions affect cell kinetics and cell cycle phases in vivo. Adult nongrowing mice (C57Bl/J) bearing a poorly differentiated rapidly growing methylcholanthrene induced sarcoma were used. Combined measurements of bromodeoxyuridine incorporation into DNA and flow cytometric techniques were used.

Starvation and DFMO treatment resulted in a prolonged cell cycle transit compared to freely fed animals. Tumor cells from DFMO‐treated mice demonstrated an increased time for DNA synthesis and a relatively larger accumulation of cells in the G2M phase, whereas tumor cells from starved animals were accumulated in the G0G1 phase. The fractional cell loss of tumor cell during proliferation was calculated to be around 18% higher in DFMO‐treated animals compared to starved and freely fed tumor‐bearing mice.

This study demonstrates that different mechanisms are involved in tumor growth supression from substrate deficiency (starvation) and from inhibition of polyamine synthesis.
Characterization of effector‐target conjugates for cloned human natural killer and human lymphokine activated killer cells by flow cytometry
Authors: Callewaert Denis M. , Radcliff Gilbert , Waite Ross , LeFevre Janet , Poulik M. Dave
Published: August 2005

The present studies demonstrate that the intracellular fluorochromes calcein and hydroethidine can be used for quantification of effector‐target conjugates involving cloned human natural killer (NK) or interleukin‐2 (IL‐2) activated human lymphokine activated killer (LAK) cells by dual color flow cytometry without potential artifacts that might result from extensive modification of effector and/or target cell membranes. Cloned NK cells and LAK cells form conjugates with cultured cell lines regardless of susceptibility to lysis. The strength of the interactions in these conjugates was investigated using a variable speed vortexer. Even relatively gentle vortexing disrupted most conjugates involving fresh human peripheral blood lymphocytes (PBL) but only about one‐fourth of conjugates between K‐562 cells and human PBL that had been cultured with or without IL‐2 by this treatment.

The rate of conjugate formation for LAK cells was determined to be about 3 times faster than for cloned NK cells, and both rates are considerably faster than the reported rate of formation of cytotoxic T lymphocyte (CTL) target conjugates. The differences in the rate of conjugate formation are apparently not related to target cell specificity, since LAK cells form conjugates with susceptible and resistant cell lines at comparable rates. When effector‐target conjugates are incubated at 37°C in the absence of calcium—thereby precluding lysis—the percentage of conjugated LAK or cloned NK cells decreases logarithmically with time. These results suggest that an initial equilibrium between free and conjugated lymphocytes gradually shifts in favor of unconjugated cells.
Assessment of radiation‐induced DNA strand breaks and their repair in unlabeled cells
Authors: Affentranger Maja Item , Burkart Werner
Published: August 2005

Radiation induced damage, i.e., the induction of DNA strand breaks, was studied on the level of single, unlabeled cells. DNA strand breaks were determined by direct partial alkaline unwinding in intact cell nuclei followed by staining with acridine orange, a development of a proposal: first described by B. Rydberg (Int J Radiat Biol 46:521–527, 1984). The ratio of green fluorescence (doublestranded DNA) to red fluorescence (single‐stranded DNA) in single cells was taken as a measure of DNA strand breaks. CHO‐K1 and M3‐1 cells irradiated with X‐rays show a dose dependent induction of DNA strand breaks. Incubation at 37°C after irradiation leads to repair of breaks. A repair halflife of about 10–11 min can be determined.

Cell cycle specific differences in the induction of DNA strand breaks or repair behavior are not detectable at the resolution achieved so far.

This new method offers two major advantages: the resolution of DNA damage and repair on the level of single cells and no need for labeling, thereby allowing for DNA damage and repair to be assessed in biopsy material from tumor patients.
Improved staining method for the simultaneous flow cytofluorometric analysis of DNA content, S‐phase fraction, and surface phenotype using single laser instrumentation
Authors: Toba Ken , Winton Elliott F. , Bray Robert A.
Published: August 2005

We have developed an improved technique for triple staining that permits the simultaneous flow cytofluorometric analysis of cell surface antigens, bromodeoxyuridine incorporation into DNA, and DNA quantification using 7‐amino‐actinomycin D. PHA‐activated human peripheral blood lymphocytes were incubated with bromodeoxyuridine and stained for cell surface phenotype with phycoerythrin‐labeled monoclonal antibodies. Stained cells were fixed serially with 1% paraformaldehyde and 45% ethanol. Fixed cells were sequentially stained with an anti‐BrdUrd monoclonal antibody followed by a FITC‐conjugated goat anti‐mouse antibody and incubated with 7‐amino‐actinomycin D. Hypotonic buffer was employed for all procedures after fixation. Stained‐fixed cells were analyzed by flow cytofluorometry for simultaneous green (525 nm), orange (570 nm), and red (> 650 nm) fluorescence. Utilizing this staining technique, we were able to analyze simultaneously cell phenotype, DNA synthesis, and total cellular DNA content with single laser excitation.
In vivo measure of average bacterial cell size from a polarized light scattering function
Authors: Bronk Burt V. , Van De Merwe Willem P. , Stanley Marc
Published: August 2005

A particular combination of elements of the Mueller matrix for scattering of polarized light given by
(S34 + S14)/(S11 + S13)  (S34/S11)‡
is measured vs angle at a wavelength of 633 nm for randomly oriented suspensions of several species of bacteria in different stages of growth. (This combination of elements is dominated in the present measurements by the behavior of the normalized S34 matrix element, as is indicated by the notation defined on the right side of the equation.) The resulting graph in each case shows an oscillating function of angle. This function is compressed toward smaller angles when the bacteria are in the exponential phase of growth in comparison with results for a suspension of the same bacteria in the stationary (starving‐smaller cells) phase of growth. Microscopic measurements were made to determine, for each case, the average dimensions of the bacterial population. Graphs were then plotted of the peak positions from the Mueller matrix function plots vs either cell length or cell diameter. The function was shown to be strongly correlated with cell diameter under the conditions of this experiment and poorly correlated with cell length. The measurements were shown to have a sensitivity to changes in average diameter of about 20 nm.
Measuring percent lymphocytes by flow cytometry to calculate absolute lymphocyte subset counts for HIV + specimens
Authors: Gale Howard B. , Henry Keith
Published: August 2005

A comparison was made of lymphocyte percentages from an automated hematology analyzer (ELT 15) vs. a fluorescence flow cytometer (Cytofluorograf). The hematology values were consistently higher than the flow (by greater than 10% for 13 of 50 HIV + specimens). The findings were similar to three other pairings: H*1 vs. Cytofluorograf, Cell Dyn 3000 vs. Cytofluorograf, and Cell Dyn 3000 vs. EPICS Profile. In another comparison manual percent lymphocytes matched much better with flow values. Factors of sample preparation and instrument analysis as they relate to nonrandom cell loss and lymphocyte resolution are examined.
Dead cell discrimination with 7‐amino‐actinomcin D in combination with dual color immunofluorescence in single laser flow cytometry
Authors: Schmid Ingrid , Krall Wanda J. , Uittenbogaart Christel H. , Braun Jonathan , Giorgi Janis V.
Published: August 2005

Identification of nonviable cells in immunofluorescently stained cell populations is essential for obtaining accurate data. Fluorescent non‐vital DNA dyes, particularly propidium iodide (PI), have been used routinely in flow cytometry for discrimination of dead cells from viable cells on the basis of fluorescence. We describe here the use of an alternative DNA dye, 7‐amino‐actinomycin D (7‐AAD), which can replace PI for the exclusion of nonviable cells. As an example, we present in this paper the utilization of 7‐AAD on various leukemic cell lines for dead cell exclusion whenever the viable cell population could not be discriminated reliably from nonviable cells on the light scatter histogram; 7‐AAD is suitable for dead cell discrimination in lengthy experiments because it is efficiently excluded by intact cells and has a high DNA binding constant. In addition, the dye is valuable in combination with phycoerythrin (PE)‐fluorescence dual‐color flow cytometry on a single argon laser instrument, since its emission in the far red can easily be separated from the emission of PE; 7‐AAD was used on fluoresceinisothiocyanate (FITC) and PE surface‐labeled human thymocytes for characterization of the dying subpopulation of cells which is undergoing programmed cell death. In this heterogeneous cell preparation, the spectral properties of the dye permitted the classification of viable and nonviable cell subpopulations by multiparameter analysis.
Evaluation of breast carcinoma chemosensitivity by flow cytometric DNA analysis and computer assisted image analysis
Authors: Briffod Marianne , Spyratos Frédérique , Hacène Kamel , Tubiana‐Hulin Michèle , Pallud Claude , Gilles Frédéric , Rouëssé Jacques
Published: August 2005

Flow cytometric (FCM) DNA and SPhase (S%) analyses were compared to computerized image analysis (SAMBA 2005) in 27 breast carcinomas (T3, N0‐N1, M0) treated by 3 cycles of preoperative Adriamycin, vincristine, cyclophosphamide, methotrexate, 5‐fluorouracil (AVCMF) chemotherapy (CT). Twelve carcinomas had shown objective regression and 15 no, regression. Samples studied were obtained by sequential fineneedle cytopunctures. Comparing DNA profiles obtained by both methods before and after the first cycle, it appears that tumors can be divided into 3 groups. In the first group (10 cases), no changes were observed after the first cycle of CT. These tumors before treatment had either single DNA peak without cells in S% and G2M or a major peak with a small S% and G2M peak. The second group (9 cases) showed some changes in DNA profiles with an increased G2M peak but no additional values; these tumors before treatment had a small S% and a G2M peak. In the third group (8 case), before treatment, all were non‐diploid with high S% and high G2M. After the first cycle, all showed obvious changes in DNA profiles with a decrease of the G0/G1 peak and an increased S% and G2M with dispersed additional values along the scale in (G2M) × 2 and (G2M) × 4 regions. When changes were compared to tumor regression in the 1st and 2nd groups, 1/10 and 3/9 cases, respectively, were evaluated as objective regression. In the third group, all had objective regression (p < 0.001). In most cases, a good correlation was observed with both methods. Nevertheless, peak position definition was better with FCM than with SAMBA, but SAMBA permits a better estimation of changes in DNA patterns induced by CT. Furthermore, SAMBA gives information on changes in other morphometric parameters, such as nuclear area that significantly increased in chemosensitive cells (p = 0.003), and in chromatin textural parameters. Therefore, FCM and SAMBA give additional information on the early effect of CT on primary tumor cells and could help to select responsive tumors.
Analyzing multivariate flow cytometric data in aquatic sciences
Authors: Demers Serge , Kim Junhyong , Legendre Pierre , Legendre Louis
Published: August 2005

Flow cytometry has recently been introduced in aquatic ecology. Its unique feature is to measure several optical characteristics simultaneously on a large number of cells. Until now, these data have generally been analyzed in simple ways, e. g., frequency histograms and bivariate scatter diagrams, so that the multivariate potential of the data has not been fully exploited. This paper presents a way of answering ecologically meaningful questions, using the multivariate characteristics of the data. In order to do so, the multivariate data are reduced to a small number of classes by clustering, which reduces the data to a categorical variable. Multivariate pairwise comparisons can then be performed among samples using these new data vectors. The test case presented in the paper forms a time series of observations from which the new method enables us to study on the temporal evolution of cell types.
Experimental and clinical evaluation by flow cytometry for the mechanism of combination therapy (cisplatin and peplomycin)
Authors: Kawamoto Keiji , Wada Yasuhiro , Kumazawa Hirofumi , Yamashita Toshio , Kumazawa Tadami , Matsumura Hiroshi
Published: August 2005

Pharmacologic effects of cisplatin (CDDP) and peplomycin (PEP) on tumor cell kinetics were studied bath in vitro and in vivo with the aid of flow cytometry (FCM). Double staining with propidium iodide (PI) and fluorescein isothiocyanate (FITC)‐labeled bromodeoxyuridine (BrdU) was used to analyze the cell cycle, and the number of viable cells was determined with fluorescein diacetate (FDA). Effects of combining the 2 agents were also studied to establish the most effective method of combination therapy. Furthermore, these agents were tried clinically on the basis of experimental results. Results showed that CDDP exerted its action at the S and G2M phases in the cell cycle and PEP at the G2M phase. Among the combination regimens in the experiments with CDDP, PEP, and CDDP + PEP as analyzed by FCM, the strongest block on the G4M phase was shown in the one at a 2‐day interval, resulting in the most effective killing of the tumor cells. Clinical trial of the combination therapy showed the same results as the in vitro experiment; the therapy proved useful for improving the patient's clinical condition and the results obtained with CT imaging and pathology.
Chromosome detection by in situ hybridization in cancer cell populations which were flow cytometrically sorted after immunolabeling
Authors: Beek Johan L. M. , Hopman Anton H. N. , Peter Vooijs G. , Ramaekers Frans C. S.
Published: August 2005

We examined the feasibility of performing non‐radioactive in situ hybridization (ISH) in flow cytometrically sorted (tumor) cells with a chromosome #1 specific centromere probe. The study was performed in a model system of HL60 cells mixed with different quantities of HeLa cells. These latter cells were sorted directly onto poly‐l‐lysine coated glass slides on the basis of their keratin content, a cytoskeletal component not present in HL60 cells. Overall morphology of the separated HeLa cells was excellent and, after the ISH procedure, the appropriate number of ISH spots was observed in more than 85% of the sorted cells. This percentage did not differ significantly in cell mixtures with different percentages of HeLa cells (down to 1%). Sorting of HeLa cells in different phases of the cell cycle, and subsequent ISH, revealed the same spot number for chromosome #1 in all cell cycle stages, including mitosis. In the latter phase of the cell cycle we did not find a duplication of the chromosome #1 centromere, not even after sorting of the mitotic cells on the basis of specific labeling with an antibody to mitotin. The early G2 mitotin negative fraction, however, showed a significant percentage of cells with a duplicate spot number, most likely representing a tetraploid cell fraction in this HeLa cell culture. The protocol that evolved from these model studies was applied to cell suspensions of malignant body cavity effusions as well as solid bladder carcinomas. In several of these cases numerical chromosome aberrations could be detected by ISH more evidently after sorting on the basis of keratin labeling.
Flow‐cytometric analysis of the effects of triethylenemelamine on somatic and testicular tissues of the rat
Authors: Bickham John W. , Sawin Virginia L. , Burton David W. , McBee Karen
Published: August 2005

The effects of short‐term (24 h) exposure to triethylenemelamine on cellular DNA in five tissues (bone marrow, spleen, kidney, large intestine, and testis) of the rat were studied using flow cytometry. Mean coefficients of variation of the G1 peaks were increased in both the low and high dosage groups relative to controls. Bone marrow exhibited the highest degree of effect, possibly due to the rapid rate of cell division in that tissue, and spleen was next highest. Thus, hematopoietic tissues are highly responsive to short‐term, acute exposure to this mutagen. The results of the flow‐cytometry assay closely paralleled a simultaneous chromosomal assay conducted on bone marrow of the same rats. These data are interpreted to be consistent with the hypothesis that the observed increase in mean coefficients of variation is due to the clastogenic effects of the mutagen and subsequent unequal distribution of DNA among the daughters of affected cells.
Three probe flow cytometry of a human foam‐cell forming macrophage
Authors: Hassall D. G.
Published: August 2005

A human cell line THP‐1 was differentiated into macrophages expressing the scavenger receptor for uptake of modified lipoproteins. The cells were exposed to native low‐density lipoprotein (n‐LDL), acetylated‐low‐density lipoprotein (AcLDL), oxidised‐LDL, or 25‐OH cholesterol, leading to the accumulation of cholesteryl esters within the cells. Harvested macrophages were studied using three separate probes: (1) 1,1′‐dioctadecyl‐3,3,3′,3′‐ tetramethylindocarbocyanine perchlorate (diI‐labelled) LDL to study lipoprotein uptake; (2) the lipophilic fluorescent dye Nile Red to study cholesteryl ester accumulation within the cells; and (3) the polyene antibiotic Filipin III to study free cholesterol homeostasis. Cells were analysed using fluorescence flow cytometry and the three signals analysed separately. THP‐1 macrophages incubated with diI‐labelled modified lipoproteins produced a concentration dependent increase in the fluorescence emissions, consistent with accumulation of the labelled particles. Macrophages exposed to unlabelled modified LDLs were demonstrated, by staining with Nile Red, to accumulate cholesteryl esters within their cytoplasm and to alter their cholesterol content as judged by staining with Filipin. The foam‐cell forming macrophage and its response to modified lipoproteins is considered a key step in the development of atherosclerosis. The use of these three probes during the formation of foamcells in vitro offers a way of studying their behaviour at the single cell level.
DNA stainability in aneuploid breast tumors: Comparison of four DNA fluorochromes differing in binding properties
Authors: Myc Andrzej , Traganos Frank , Lara Jonathan , Melamed Myron R. , Darzynkiewicz Zbigniew
Published: August 2005

The aim of this study was to evaluate whether or not the differences in chromatin structure between diploid stromal cells or lymphocytes, which are often used as DNA ploidy standard, and aneuploid breast tumor cells can significantly affect the estimates of the DNA index of these tumors. To this end, the DNA content estimates of 34 aneuploid breast tumors, differing in size, degree of differentiation, and presence or absence of estrogen and progesterone receptors and metastases, were compared using four common DNA fluorochromes: DAPI, Hoechst 33342, propidium iodide, and acridine orange. These dyes differ in their mode of interaction with DNA (binding to minor groove or intercalation) and for each of them binding to DNA is restricted to a different degree by nuclear proteins. It was expected, therefore, that if differences in chromatin structure play a role in DNA content estimates, the DNA index of the measured tumors may vary depending on the dye. The cell nuclei were isolated from the tumors using a detergent‐based procedure and stained with each of the dyes and the DNA index was estimated using peripheral blood lymphocytes as a DNA content standard. For each of the tumors, the DNA index estimates with all four dyes correlated very well. When the results obtained with individual dyes were compared in pairs, the correlation coefficients (r) of DNA indices were all above 0.96 (correlation at p < 0.001). The best concordance was seen between specimens stained with Hoechst 33342 and DAPI (r = 0.99), and the least between those stained with Hoechst 33342 and propidium iodide (r = 0.96). The data indicate that DNA content analysis of unfixed nuclei, utilizing the above fluorochromes, is not significantly biased by differences in chromatin structure of the measured cells.
DNA ploidy in cell nuclei from paraffin‐embedded material–comparison of results from two laboratories
Authors: Fosså Sophie D. , Berner Aasmund , Wæhre Håkon , Heiden Thomas , Holm Juul Mina E. , Ouden Dies Van Den , Pettersen Erik O. , Wang Naining , Tribukait Bernhard
Published: August 2005

In 49 pairs of contiguous sections from paraffin‐embedded prostatic cancer tissue, the DNA indices (DIs) were determined by flow cytometry (FCM) at 2 different laboratories. In 3 of 45 pairs of evaluable nuclear suspensions, DIs of 1.1 (DNA aneuploid) were found at Laboratory 1, whereas all 3 tumours were classified as DNA diploid at Laboratory 2. In the remaining 42 specimens, the correlation between the DIs was excellent, though the application of strictly defined DNA ploidy ranges led to different DNA ploidy allocation in 3 cases.

It is concluded that in 85–90% of the cases, reliable DIs can be obtained by FCM done in paraffin‐embedded material at different laboratories. Slight technical variations aiad interpretation differences may lead to different, ploidy allocation in 10–15% of the cases.
BrdUrd/DNA flow cytometry analyses demonstrates cis‐diamminedichloroplatinum (II)‐induced multiple cell‐cycle modifications on human lung carcinoma cells
Authors: Demarcq Catherine , Bastian Gérard , Remvikos Yorghos
Published: August 2005

The treatment of cultured human cells by cis‐diamminedichloroplatinum (II) (cis‐DDP) has been shown to induce complex modifications in the cell cycle. Using dual parameter DNA/BrdUrd flow cytometric analysis, we were able to monitor the cell cycle traverse of a pulse‐labeled cohort of cells in an asynchronous culture of the A549 cell line (human lung adenocarcinoma). Two major modifications of the cell cycle following cis‐DDP treatment were observed: (1) after 24 h of treatment, the labeling index was significantly increased and was linked with a prolonged S‐phase; the S‐phase delay occurred rapidly after cis‐DDP and was dose dependent but not exposure time dependent; (2) an accumulation of cells at the S/G2 transition with an onset approximately 12 h after cis‐DDP contact, which was found to be dependent on both dose and duration of exposure. The cytokinetic results also predict maximal sensitivity to cis‐DDP for G1 cells and minimal for G2 cells. In our model the late S/G2 accumulation was always preceded by a slowing down of the S‐phase. However, only the former should be the correct indicator of cytotoxicity since it was correlated with cell survival as evidenced by d colony formation assay, under all treatment conditions.
Non‐random distribution of abnormal mitoses in heteroploid cell lines
Authors: Dooley William C. , Allison David C.
Published: August 2005

We have performed absorption‐cytometric DNA measurements of the DNA contents of the G0/G1, G2, metaphase, and telophase cells of the heteroploid MCa‐11 and HL‐60 lines, as well as the WCHE‐5 line which has a narrowly restricted number of chromosomes. We found that morphologically unbalanced mitoses occurred much more frequently in telophase‐cell pairs of the heteroploid MCa‐11 and HL‐60 lines than in those of the chromosomally stable WCHE‐5 line. Furthermore, the morphologically unbalanced mitoses represented unequal segregation of DNA into each of the daughter telophase nuclei. Such mitotic segregation errors (MSE) occurred almost exclusively in telophase cells with DNA contents which were above, or below, the DNA content of the modal telophase population. The net effect of these non‐random, unbalanced divisions of heteroploid cells with non‐modal DNA contents is to produce one daughter cell with a DNA content that tends to return to the modal DNA content peak.
Semi‐automated detection of aberrant chromosomes in bivariate flow karyotypes
Authors: Boschman Gert A. , Manders Erik M. M. , Rens Wim , Slater Rosalyn , Aten Jacob A.
Published: August 2005

A method is described that is designed to compare, in a standardized procedure, bivariate flow karyotypes of Hoechst 33258 (HO)/ Chromomycin A3 (CA) stained human chromosomes from cells with aberrations with a reference flow karyotype of normal chromosomes. In addition to uniform normalization of normal and abnormal flow karyotypes, the main purpose is detection of structurally abnormal chromosomes in often complex karyotypes of tumor cells. The method, which has been implemented in a computer program, consists of a comparison of individual chromosome peaks with the positions of peaks in the flow karyotype constituted by normal chromosomes and takes into account the natural variability in base composition of normal chromosomes among healthy individuals. Flowkaryotypes are normalized using an iterative fitting procedure, using corrections for (1) amplification of HO and CA fluorescence, (2) cross‐talk between the fluorescence signals of HO and CA, and (3) offset of the HO and CA origin. Flow karyotypes of two cell lines, one with a simple deletion and the other with more complex karyotypic changes, were analyzed. The results of flow analysis were found to be in general agreement with the cytogenetic analysis of quinacrine banded karyotypes.
Influence of different cell extraction methods on cytometric features
Authors: Thunnissen F. B. J. M. , Perdaen H. , Forrest J.
Published: August 2005

The purpose of this study was to investigate the influence of different cell extraction procedures on relative nuclear DNA content (IOD), nuclear area, and texture features of Feulgen‐stained nuclei. In imprints and smears of fineneedle aspirates and suspensions from one human liver specimen, 50 diploid, 50 tetraploid, and 25 octaploid nuclei were measured from each slide. In addition, for DNA measurements, the progressive mean of IOD and tetraploid/diploid and octaploid/diploid ratios was calculated. The results show that the progressive mean of the IOD is constant after measuring 25–30 nuclei. For the three type of specimens, the IOD of diploid nuclei varied slightly. The average coefficient of variation was about 5% for the fineneedle aspirates, imprints, and suspensions. For all tissue sampling methods, the 99% confidence limits of the tetraploid/diploid ratio and octaploid/diploid ratio were within the range of 1.9–2.1 and 3.7–4.3, respectively. The nuclear area and most of the texture features showed a significant difference (p < 0.01) between the three sampling methods in all nuclear populations. In conclusion, different tissue sampling methods have little or no effect on DNA‐related IOD measurements, whereas the outcome of nuclear area and texture features is very dependent of the cell extraction procedure.
Effect of ionic strength in immunocytochemical detection of the proliferation associated nuclear antigens p120, PCNA, and the protein reacting with Ki‐67 antibody
Authors: Bruno Silvia , Gorczyca Wojciech , Darzynkiewicz Zbigniew
Published: August 2005

This study was aimed at revealing whether or not ionic interactions between the epitope of the antigen detected by Ki‐67 antibody, or the proliferation‐associated proteins PCNA or p120, and neighboring cellular constituents impede detectability of these antigens in HL‐60 cells by indirect immunofluorescence assay. To this end, the ionic strength (NaCl concentration) of the solutions in which cells were suspended during their fixation with 0.5% paraformaldehyde was increased, to up to 1.65 M NaCl, to weaken the intra‐ and/or intermolecular ionic interactions during the process of crosslinking, and the cells were then immunostained. Fluorescence of cells reacting with Ki‐67 antibody was maximally increased after their treatment with 1.15 M NaCl; the average increase was nearly 110% above the level seen with the standard methodology utilizing 0.15 M NaCl. The increase was greater for cells in the G1 phase of the cell cycle compared to cells in S or G2. Fluorescence of cells stained with the PCNA antibody was maximally enhanced after cell treatment with 0.65 M NaCl. The enhancement, however, varied depending on the source of the antibody; it was nearly 200% in the case of the antibody provided by Boehringer and over 100% by DAKO. Detection of the nucleolar antigen p 120 was not significantly affected by 0.65–1.65 M NaCl. The data indicate that ionic interactions between cellular constituents indeed play a role in masking the epitope of PCNA and the antigen detected by Ki‐67. These results also suggest that to enhance the detectability of the intracellular proteins, the current immunocytochemical methods should be modified and customized for each particular antigen in terms of selection of the optimal ionic strength during the fixation procedure.
NADH‐dependent dehydrogenase activity estimation by flow cytometric analysis of 3‐(4,5‐dimethylthiazolyl‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction
Authors: Huet Olivier , Petit Jean Michel , Ratinaud Marie Hélène , Julien Raymond
Published: August 2005

MTT reduction is usually analysed by colorimetric assay to study mitochondrial dehydrogenase activity as a test of cytotoxicity. This enzymatic reaction produces dark‐blue granules of formazan, which increase cell refringency. In this work, we define the conditions for MTT use in quantitative flow cytometric analysis. MTT reduction provides a nonfluorescent dye usable by this technique to study an intracellular NADH‐dependent dehydrogenase activity in vital cells. We observe that formazan production increases asymptotically with cell concentration and that this temperature‐dependent Michaelis enzymatic reduction is produced essentially by mitochondrial dehydrogenases. In isolated mitochondria from rat hepatocytes and in whole L1210 murine leukemiia cells, the Michaelis constants (KM) observed in the presence of respiratory substrates were, respectively, 10 μM And 500 μM. The inhibition of mitochoAldrial protein synthesis by chloramphenicol, which induces a rise of MTT reduction due to the correlative stimulation of glycolysis (Pasteur effect), is a limit of the MTT assay as a cytotoxicity test.
Method to improve the sensitivity of flow cytometric membrane potential measurements in mouse spinal cord cells
Authors: Seamer Larry C. , Mandler Raul N.
Published: August 2005

We have developers a technique to improve the sensitivity of relative membrane potential measurements in mouse spinal cord cells using the fluorescent, anionic, voltage sensitive dye, DiBa‐C4(3) (Oxonol) and flow cytometry. In order to attribute cellular fluorescence primarily to membrane potential, signal variability due to cell size and shape was reduced by dividing the log fluorescence signal from each cell by either its log forward angle light scatter or log side scatter signals. The use of these ratios in place of log oxonol fluorescence reduced the coefficient of variation of the distributions while leaving the changes in mean fluorescence largely unaffected. Kolmogorov‐Smirnov analysis of pre‐ vs. postkainate stimulation (an excitatory amino acid) showed improved sensitivity of the assay with the use of this ratio technique.
Quantification by laser scan microscopy of intracellular doxorubicin distribution
Authors: de Lange J. H. M. , Schipper N. W. , Schuurhuis G. J. , ten Kate T. K. , Van Heijningen Th. H. M. , Pinedo H. M. , Lankelma J. , Baak J. P. A.
Published: August 2005

Changes in intracellular drug localization accompany doxorubicin resistance in multidrug resistant tumor cells. The purpose of this study was to develop a method to quantify these changes and so detect different levels of resistance. Tumor cells were incubated with the fluorescent anthracycline doxorubicin (excitation at 480 nm; emission maximum at 560–590 nm) and were quantified using laser scanning microscopy. The fluorescent mode was used to record the intracellular drug distribution, whereas the absorption mode was used to define the nuclear and cytoplasmic boundaries. The cell compartments were delineated interactively on an image processing system and the ratio nuclear fluorescence/cytoplasmic fluorescence (N/C ratio) was determined. N/C ratios were: 1.8 in the Chinese hamster ovarian cell line AUXB1 and 0.1 in its MDR subline CHRC5; 3.8 in the human squamous lung cancer cell line SW‐1573 and 1.8 and 0.4 in its MDR sublines SW‐1573/2R120 and SW‐1573/2R160, respectively; and 3.6 in the human myeloma cell line 8226/S and 2.1 and 1.0 in its MDR sublines 8226/Dox4 and 8226/Dox40, respectively.

The doxorubicin distribution was independent of the doicorubicin concentration within a range from 1–32 μM. Furthermore, the progressive mean of the nuclear/cytoplasmic: doxorubicin fluorescence ratio showed that a minimal sample size of 30 cells is necessary for reliable results. The results of two independent assessments showed a high reproducibility (r = 0.97). Thus, with the method described in this paper, it is possible to detect relatively low levels of doxorubicin resistance (factor 8). © 1992 Wiley‐Liss, Inc.
Spontaneous evolution of nuclear size and DNA content of human non‐small‐cell‐lung cancers grafted onto nude mice
Authors: Kruczynski Anna , Pauwels Olivier , Wright Michel , Delsol Georges , Kiss Robert
Published: August 2005

We monitored the biological evolution of four human non‐small‐cell lung cancers labeled KLX6, KLX7, KLX9, and KLX14 that had been grafted onto nude mice. This monitoring was carried out by means of digital cell image analysis which assessed morphometric (nuclear area, NA) and densitometric (nuclear DNA content, DI) features on Feulganstained nuclei from imprint smears. In each of the four models, the biological evolution of the NA and DI was characterized through their progression during serial passaging onto nude mice. The results show that of the four lung cancer models analyzed, one (KLX6) remained definitively stable with respect to its DNA content (DI assessments), while the other three, i.e., KLX7, KLX9, and KLX14, varied significantly. As assessed by the morphometric parameter, i.e., the NA, the four xenografted lines also varied significantly over serial passaging. In conclusion, we show that digital cell image analyses of Feulgen‐stained cell nuclei including morphoinetric and densitometric parameters are a powerful tool for monitoring the biological evolution of human lung cancers grafted onto nude mice. We think therefore that the ploidy level of a tumor might be dependent upon its age and/or its related clinical stage. © 1992 Wiley‐Liss, Inc.
Methods for topographical analyses of intra‐nuclear BrdUrd‐tagged fluorescence
Authors: Usson Yves , Humbert Catherine
Published: August 2005

The observation of BrdUrd staining in the nuclei of cells from exponentially growing populations reveals different typical replicating patterns. We propose a methodological approach to order and characterize BrdUrd intranuclear distributions. First, visual ordering of the patterns is assessed using a spectral analysis coupled to a k‐nearest neighbors clustering technique. Subsequently, nine topographical features are introduced to characterize the spatial distribution of BrdUrd‐tagged fluorescence in the nuclei of proliferating cells. These topographical features are based on a structural approach. The localization of fluorescence spots is expressed in terms of the normalized distance from the nuclear border and its standard deviation. These topographical features are simple to calculate and easy to relate to visual experience. © 1992 Wiley‐Liss, Inc.
Differential analysis of animal bone marrow by flow cytometry
Authors: Martin Ronald A. , Brott David A. , Zandee Joyce C. , McKeel Mildred J.
Published: August 2005

A simple procedure was developed for rapid analysis of animal bone marrow by flow cytometry using the lipophilic cationic dye 3,3′‐dihexyloxacarbocyanine iodide (DiOC6(3)). The batch process allows differentiation of bone marrow cells into lymphoid, erythroid, and myeloid populations and enables classification of erythroid and myeloid cells into proliferating and maturing subpopulations. From these data, myeloid:erythroid (M:E) ratios and maturation indices for erythroid and myeloid cells (EMI and MMI, respectively) can be derived. This procedure provides the opportunity to analyze bone marrow quantitatively and offers distinct advantages to current manual methods in terms of simplicity, throughput, and reproducibility. The method has been tested successfully using marrow from Wistar rats, B6C3F1 mice, beagle dogs, and cynomolgus monkeys. This technique facilitates the evaluation of bone marrow samples taken from preclinical safety studies or from animal colonies of large size. © 1992 Wiley‐Liss, Inc.
Molecular extinction coefficients of lead sulfide and polymerized diaminobenzidine as final reaction products of histochemical phosphatase reactions
Authors: van Noorden Cornelis J. F. , Jonges Geertruida N.
Published: August 2005

Molar extinction coefficients of precipitated lead sulfide (PbS) and polymerized diaminobenzidine (polyDAB) have been determined at wavelengths of 450 nm and 480 nm, respectively, for quantitative his tochemical analysis of phosphatase reactions. These values are essential for the conversion of cytophotometric (mean in tegrated) absorbance values to absolute units of substrate converted per unit time and volume of tissue. This conversion allows direct comparison of histochemical and biochemical data. The molar extinction coefficient of PbS at 450 nm was found to be 3,800 and therefore, per mole phosphate liberated, the molar extinction coefficient is 5,700 because 3 moles phosphate are captured by 2 moles lead at neutral or alkaline pH. Parallel experiments with the cerium‐DAB method revealed that the molar extinction coefficient of polyDAB at 480 nm is 5,500 with respect to liberated phosphate. The molar extinction coefficients were applied for comparison of data from biochemical and histochemical assays of glucose‐6‐phosphatase activity in rat livers. A significant correlation was found between both sets of data. The values were in the same order of magnitude with histochemical values approximately 1.4 times higher than biochemical values. © 1992 Wiley‐Liss, Inc.
Flow cytometric analysis of peripheral blood erythrocyte chimerism in α‐thalassemic mice
Authors: van den Bos Cor , van Gils Francis C. J. M. , Bartstra Rolf W. , Wagemaker Gerard
Published: August 2005

A rapid and reliable method for longitudinal studies on the degree of red cell chimerism following bone marrow transplantation of α‐thalassemic recipient mice is presented. Blood obtained by tail clipping from transplanted mice was analyzed by measuring forward light scatter (FLS) distribution of red cells using a flow cytometer. Amplification and threshold of FLS were specifically adjusted. For flow cytometric analysis, the red cells needed to be suspended in hypotonic saline (103 mmol/1 NaCl). Osmotic fragility testing showed that lysis of erythrocytes did not significantly influence the measurements. Flow cytometric measurement allowed for a rapid determination of the degree of red cell Chinerism.
Growth behavior of human pancreatic carcinoma xenograft correlates with nuclear features
Authors: Coen H. , Kint H. , Kuyper C. F. , Jonges G. N. , Dumarey N. , Pauwels M. , Kloppel G. , Roels F.
Published: August 2005

Two human pancreatic ductal adenocarcinomas with different growth rates were serially transplanted into nude mice. Feulgen‐stained 4 μm sections and imprints from the xenografts were studied with a VICOM automated image analysis system. After pooling the results from two passages, with three mice in each passage, it was shown that of 23 nuclear parameters measured the following were correlated with a fast tumor growth rate: in sections, a decrease in heterogeneity of the chromatin and an increase in perimeter and nuclear area; in imprints, an increase in lesser diameter, in mean grey level difference between second neighboring pixels, and in total integrated optical density (DNA content). Several parameters differed significantly between passages, and between animals in the same passage. These findings suggest that the growth speed of pancreatic tumors may be predicted by nuclear parameters. © 1992 Wiley‐Liss, Inc.