bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurement of tumor necrosis factor activity by flow cytometry
Authors: Lévesque Ann , Paquet Alain , Pagé Michel
Published: June 2005

Tumor necrosis factor‐α (TNF‐α) is a monokine of 17 kDa produced by activated macrophages and various cells involved in the immune system. We propose a new method for the measurement of TNF activity using flow cytometry. After an incubation with TNF, L929 cells were harvested and treated with a calcein‐AM and ethidium homodimer‐1 solution. Nonfluorescent calcein‐AM is hydrolyzed by intracellular esterases to yield fluorescent calcein. The ethidium homodimer‐1 is a high‐affinity red fluorescent DNA dye that is internalized only through altered cell membranes. A very good correlation was observed between the calcein fluorescence intensity and the number of viable cells as well as the ethidium fluorescence and the number of cells with altered membranes. The assay is sensitive, inexpensive, and correlates with the already reported crystal violet assay while measuring membrane alteration by TNF. It allows the simultaneous measurement of total living and dead cells. There is no interference with culture medium components. This method is rapid and may be used for routine measurement of TNF activity. © 1995 Wiley‐Liss, Inc.
Use of a spherical multiparameter transducer for flow cytometry
Authors: Leif R. C. , Cayer M. L. , Dailey W. , Stribling T. , Gordon K.
Published: June 2005

Fused silicon dioxide, multiparameter flow transducers with 50 μm internal square cross section and ∼60 μm length can simultaneously measure DC and RF impedance as well as fluorescence and multiple‐angle light scattering. A spherical version of such a transducer was mounted in an EPICS CVA flow‐cell housing and was installed on a research prototype equipped with an argon‐ion laser. The signal that was produced by the spherical transducer with EPICS DNA‐Check beads was 1.73 times greater than that produced with the standard cylindrical flow cell. Similarly, with EPICS Immuno‐Brite beads, the average ratio was 1.96.

The Coulter impedance and light‐scattering measurements were similar to those produced with the conventional cylindrical outside flow cell, although the internal cross section of the sphere was square and that of the cylinder was circular. The theoretical arguments of Leif and Wells have been demonstrated to be correct. At present, monolithic, spherical fused‐silica transducers are the optimal design for combined electrooptical, multiparameter flow cytometry analyzers. © 1995 Wiley‐Liss, Inc.
Methods for cell proliferation analysis by fluorescent image cytometry
Authors: Souchier Catherine , Ffrench Martine , Benchaib Mehdi , Catallo Regine , Bryon Paul Andre
Published: June 2005

Methods were developed for multimodal microscopic image analysis in order to identify and analyze one cell type under various microscopic conditions. Our purpose was to quantify both total DNA content using propidium iodide (PI) stain and S‐phase fraction using the bromodeoxyuridine (BrdUrd) incorporation technique in cell population subsets. The model chosen was plasma cells in bone marrow triply labelled with fluorescein isothiocyanate (FITC) for intracytoplasmic immunoglobulins, with amino‐methylcoumarin‐acetate (AMCA) for BrdUrd, and with PI for DNA. Image analysis included three phases. First, plasma cells were recognized on FITC images, and the centroid positions were stored. Second, plasma cell nuclei were geodesically reconstructed from these stored positions using PI images in which DNA content was measured, and the nuclear mask outlines were stored. Third, BrdUrd incorporation level of plasma cells was measured on AMCA images inside PI nuclei masks and stored. Image DNA vs. BrdUrd scatterplots were obtained for cells selected according to the expression of intracytoplasmic immunoglobulin. Thus, both ploidy and proliferation could be independently evaluated on a subset of the cellular population. © 1995 Wiley‐Liss, Inc.
Further characterisation of the in situ terminal deoxynucleotidyl transferase (TdT) assay for the flow cytometric analysis of apoptosis in drug resistant and drug sensitive leukaemic cells
Authors: Chapman Rachel S. , Chresta Christine M. , Herberg Ali Alejo , Beere Helen M. , Heer Sukhbindar , Whetton Anthony D. , Hickman John A. , Dive Caroline
Published: June 2005

Apoptosis, originally defined by specific morphological changes, is characterised biochemically by non‐random cleavage of DNA. Depending on cell type, this DNA cleavage proceeds from 300 and 50kbp fragments prior to, concomitantly with, or in the absence of 180bp integer fragmentation. Incorporation into fragmented DNA of biotin‐labelled nucleotides by terminal deoxynucleotidyl transferase (TdT) has recently become a standard flow cytometric assay for the identification and quantitation of apoptosis. Nucleotide incorportion is visualized using avidin‐tagged fluorescein isothiocyanate (FITC) (Gorczyca et al.: Cancer Res 53:1945–1951, 1993; Jonker et al.: Cytometry (Suppl 13):Abstr 99A, 1993). Here, we characterise this assay further in three different haemopoietic cell lines. Drug‐induced DNA damage is not identified by the TdT assay unless it is coupled to the apoptotic response. This was demonstrated using cells in which activation of the oncogenic Abelson‐encoded protein tyrosine kinase suppressed drug‐induced apoptosis, but did not inhibit drug‐induced DNA damage (by melphalan, hydroxyurea, or etoposide). Furthermore, the TdT assay identifies DNA fragments formed during apoptosis induced by etoposide and N‐methylformamide in HL60 and MOLT‐4 cells, including those high molecular weight DNA fragments formed in MOLT‐4 cells which were not further cleaved to 180–200bp integer fragments. Our results support the use of flow cytometry and the TdT assay to reliably measure apoptotic cells in heterogeneous cell samples. © 1995 Wiley‐Liss, Inc.
Estimation of fetal hemoglobin levels in individual red cells via fluorescence image cytometry
Authors: Horiuchi Kazumi , Osterhout Melissa L. , Kamma Hiroshi , Bekoe Nicoletta A. , Hirokawa Kevin J.
Published: June 2005

A method for estimating fetal hemoglobin (Hb F) levels in individual red blood cells was developed. Cell smears were prepared using a slide maker to ensure uniform thickness and were then stained with immunofluorescence. An antifading gel was applied to preserve a stable fluorescence. The total fluorescence intensities from the same number of red cells in different slide specimens correlated with their hemolysate Hb F levels, which were determined via column chromatography (R = 0.95). Hb F level in individual cells was estimated from fluorescence intensity and cell area, which were determined via image analysis techniques and the hemolysate Hb F level. Blood from a normal subject, a subject with hereditary persistence of fetal hemoglobin, and from sickle cell patients with varying Hb F levels was analyzed. Our analyses showed a wide distribution of Hb F among cells for the normal subject and a gaussian distribution with a peak at the hemolysate Hb F level for the subject with hereditary persistence of fetal hemoglobin. The Hb F distributions were unique to the patients with sickle cell disease. Because Hb F level in individual sickle cells is crucial to the inhibition of cell sickling, the unique Hb F distribution may be important in determining the clinical course of this disease. © 1995 Wiley‐Liss, Inc.
Organic material concentration in auditory outer hair cells measured by laser interferometry
Authors: Laffon E. , Dilhaire S. , Aurousseau C. , Dulon D. , Claeys W.
Published: June 2005

Outer hair cells (OHC) of the mammalian cochlea are quasicylindrical cells of different length, which play a major role in hearing at threshold. Their particular shape allows the use of a noninvasive laser interferometric technique of isolated cells in vitro in order to measure the organic material concentration (OMC), hence the density of each cell body. In most (95%) of the OHCs isolated from the same guinea pig, when the cell diameter is normalized, the results show that the cell body OMC does not vary with cell length. In different animals, the respective normalized OMC mean values can vary between 70 kg/m3 and 103 kg/m3. A few OHCs with morphological particularities often possess cell body OMCs > 103 kg/m3. The results of the interferometric measurements in isolated OHCs confirm that density variations in the cell bodies are not involved in a sound frequency coding. The in vitro OMC variotions of the OHCs could be related to the isolation procedure; however, they could also correlate with actual in vivo OMC variations. © 1995 Wiley‐Liss, Inc.
Changes in intracellular calcium concentration and pH of target cells during the cytotoxic process: A quantitative study at the single cell level
Authors: Radoškević Katarina , de Grooth Bart G. , Greve Jan
Published: June 2005

This study reports on the changes in intracellular calcium concentration ([Ca2+]in) and intracellular pH ([pH]in) that occur in K562 target cells during interaction with human Natural Killer (NK) cells. The data were obtained using a quantitative fluorescence microscope and fluorescent ratio probes specific for [Ca2+]in (Fura‐2‐AM) and [pH]in (BCECFAM). Results demonstrate that two types of target cell response to the attack by an NK cell can be distinguished. The target cell either dies immediately, due to the complete breakdown of the membrane impermeability, or the initial membrane damage (i.e., increased membrane permeability) is repaired and the cell “escapes” immediate death. During both responses an increase of [Ca2+]in takes place in the target cells. In the cells that die immediately, however, [Ca2+]in reaches higher levels (approximately 1,400 nM) than in the cells that restore the initial damage (approximately 700 nM). Changes in target cell [pH]in are also detected during both responses. The direction of the change (acidification or alkalinization) as well as the level of the change depend on extracellular pH ([pH]ex). Also, [pH]in remains changed during the time the cells were followed (10 min). The programming time (i.e., the time from the initiation of the cytotoxic process to the time that a change in the physiological parameter was detected) of the killing process that leads to an immediate target cell death appears to be shortest at [pH]ex 7.3–7.6 (approximately 3 min). © 1995 Wiley‐Liss, Inc.
Standardization of a flow cytometric method for measurement of low‐density lipoprotein receptor activity on blood mononuclear cells
Authors: Løhne Kari , Urdal Petter , Leren Trond P. , Tonstad Serena , Ose Leiv
Published: June 2005

Flow cytometric methods for measurement of low‐density lipoprotein (LDL) receptor activity on peripheral blood mononuclear cells (PBMC) may be used to identify patients with familial hypercho‐lesterolemia (FH). However, cellular LDL receptor activities measured in FH heterozygotes may over‐lap with those of healthy subjects. Analytical variation is probably responsible for some of this over‐lap. We have examined several technical details that may affect analytical variation. In each analysis, we included one standard and two control cell preparations. These were cells isolated from three donors and stored in aliquots at –135°C. Use of standard cells reduced between‐series analytical variation of the controls by approximately 50%. Preincubation‐conditions used to induce the maximum number of receptors, the concentration of fluorochrome 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine‐perchlorate (DiI)‐LDL, labelling time, and conditions during storage of labelled cells before flow cytometry were also examined in order to reduce analytical variation. Having standardized the assay, we found among 20 healthy subjects a median receptor activity of 100% vs. 51% among 26 patients who fulfilled clinical criteria for FH. However, four of the patients showed distinctly normal receptor activities, which may suggest either the presence of some other biochemical defect or that in vivo dysfunctional receptors may be measured as normal in some patients with our assay. © 1995 Wiley‐Liss, Inc.
Preferred genetic evolutionary sequences in human breast cancer: A case study
Authors: Shackney Stanley E. , Smith Charles A. , Pollice Agnese A. , Janocko Laura E. , Singh Sarita G. , Groft David W. , Brown Kathryn A. , Hartsock Robert J.
Published: June 2005

Multiparameter flow cytometry studies were performed on the cells of an aggressive human breast cancer at the time of diagnosis and at relapse. The aneuploid cells that overexpressed large amounts of both HER‐2/neu and ras survived intensive chemotherapy and were responsible tumor relapse. At relapse, these cells were shown to overexpress simultaneously at least five oncogenes: HER‐2/neu, ras, EGF receptor, p53 and c‐myc. A partial reconstruction of the genetic evolutionary sequence in this tumor indicated that HER‐2/neu overexpression was an early step in the sequence. Subsequent HER‐2/neu overexpression, EGF receptor overexpression and p53 protein overexpression were each associated with ras overexpression. The data suggest that ploidy and oncogene overexpression cannot be used as independent clinical prognostic factors. The ability to characterize tumors according to the degree of advancement in the genetic evolutionary might serve as a basis for genetic staging for adjuvant therapy. © 1995 Wiley‐Liss, Inc.
Analytical approaches relating genetic evolutionary pathways to prognostic factors
Authors: Rohloff Albert C. , Sakach Joseph M. , Shackney Stanley E.
Published: June 2005

Human solid tumors accumulate multiple genetic abnormalities as they progress to advanced stages. Multiparameter flow cytometry measurements of individual cells within each tumor may be useful in describing the genetic pathways taken by individual tumors during the course of their genetic evolution. In this paper, we analyzed correlated cell‐bycell measurements of cell DNA content, HER‐2/neu protein content, and ras protein content obtained by multiparameter flow cytometry studies of primary breast cancers from 92 patients. These laboratory findings were correlated with established clinical prognostic factors for each patient at the time of diagnosis, using a stepwise multiple analysis ofvariance (MANOVA). The stepwise MANOVA successively splits a group of patients into two mutually exclusive dissimilar groups, selecting the clinical prognostic factor that is most effective in doing so. Using this criterion, formation of the first three groups that were judged most dissimilar on the cytometry parameters was based on the number of positive nodes at the time of diagnosis. We show that ploidy, HER‐2/neu protein content, and ras protein content, as measured by multiple parameter flow cytometry, are correlated with nodal status and other known clinical prognostic factors. The cellby‐cell multiparameter data suggest that for some individual tumors there are multiple genetic evolutionary pathways. Multiple genetic evolutionary pathways are also suggested by the MANOVA analysis. Focusing on the identification and analysis of genetic evolutionary pathways within individual tumors and across patients appears to offer a promising approach for defining the prognosis of early cancers. © 1995 Wiley‐Liss, Inc.
Heterogeneity of chromosome 17 and erbB‐2 gene copy number in primary and metastatic bladder cancer
Authors: Sauter Guido , Moch Holger , Gasser Thomas C. , Mihatsch Michael J. , Waldman Frederic M.
Published: June 2005

To study the relationship of tumor genomic heterogeneity with bladder cancer phenotype and p53 gene alterations, 138 primary bladder tumors were examined by dual labeling fluorescence in situ hybridization (FISH) using probes for chromosome 17 centromere (p17H8) and p53 (17p13.1). The number of different aneusomic populations >5% (and monosomic populations >20%) of cells served as a marker for heterogeneity. Nuclear p53 overexpression and Ki67 labeling index (Ki67 LI) were determined by immunohistochemistry. The number of aneusomic populations was 0 in 53 tumors, 1 in 18, 2 in 47, 3 in 9, and >3 in 11 tumors. Presence of aneusomy was associated with tumor grade and stage (P < 0.0001 each). Ki67 LI was low in disomic tumors (11.0 ± 7.7), higher in tumors with 1–3 aneusomic populations (17.4 ± 11.3), and highest in tumors with >3 aneusomic populations (25.8 ± 10.9; P = 0.02 for >3 vs. 1–3 populations). Aneusomy and heterogeneity were associated with p53 alterations. Aneusomy was seen in 35% of tumors with neither p53 expression nor p53 deletion but in 97% of tumors with both p53 deletion and expression. Nine of 11 tumors with > 3 aneusomic populations exhibited both p53 deletion and overexpression.

To study genomic heterogeneity in tumor progression, two recurrences and three metastases of a tumor with known erbB‐2 amplification were examined for centromere 17 and erbB‐2 copy number. A considerable heterogeneity in centromere 17 and erbB‐2 gene copy number was found in both recurrences and metastases, indicating a marked genomic instability in these metastatic cells. These results show that genomic heterogeneity is common in bladder cancer. Highly heterogeneous tumors might represent a particularly aggressive subtype of bladder carcinoma. © 1995 Wiley‐Liss, Inc.
Heterogeneous cell kinetics in tumors analyzed with a simulation model for bromodeoxyuridine single and multiple labeling
Authors: Baisch H. , Otto U. , Hatje U. , Fack H.
Published: June 2005

Bromodeoxyuridine (BrdUrd) labeling of DNA and flow cytometry measurement of bivariate BrdUrd‐DNA content distributions yield proportions of cells in the cycle phases. After application of BrdUrd, with time, these proportions change according to the cell kinetic parameters of the investigated cell line or tumor. In a previous study of S‐phase transit time using the relative movement method, we obtained better fits with S‐duration distributions rather than constant values (Baisch and Otto: Cell Prolif 26:439–448, 1993). Now, we have developed a simulation model using asymmetric phase duration distributions in all phases of the cell cycle to fit the experimental data after single or multiple BrdUrd labeling. The model includes transit of cells from proliferating to quiescent compartments in all phases. The results yield the phase duration distributions, mean and median percentages of quiescent cells in all phases, growth fraction, and potential doubling time. The model was used to fit data of five renal cell carcinomas xenotransplanted into nude mice that were obtained after single and multiple labeling up to 93 hours. The estimated phase duration distributions varied from narrow to extremely asymmetric. In particular, TG2M duration and asymmetry were nearly as large as those of G1 phase in some tumors. The contribution of inter‐and intratumoral heterogeneity cannot be separated by the simulation model, but evidence of intratumoral heterogeneity is provided by DNA content distributions at extended time spans after BrdUrd labeling. © 1995 Wiley‐Liss, Inc.
Heterogeneity of bromodeoxyuridine sensitivity of cultured cells from melanoma metastases
Authors: Poot Martin , Hiller Karl‐Heinz , Heimpel Simone , Schuster Armin , Köhler Julia , Hoehn Holger
Published: June 2005

Continuously growing cell cultures, testing positive for tyrosinase activity, were derived from two brain and three lymph‐node metastases of five patients with malignant melanoma. These cell cultures were analyzed regarding their proliferation rate with continuous bromodeoxyuridine (BrdUrd) labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry. Melanoma cell cultures are more sensitive toward BrdUrd in comparison to human diploid fibroblast cultures: 50% growth inhibition at 360 ± 130 μM BrdUrd (range: 130–520; n = 11) vs. 650 ± 50 μM BrdUrd (n = 3) for fibroblasts. Moreover, BrdUrd sensitivity in melanoma cells is oxygen dependent: 50% growth inhibition at 200 ± 55 μM (range: 65–400 μM) for 20% oxygen vs. 360 ± 130 μM BrdUrd for 5% oxygen. The cell cycle kinetic mechanism of BrdUrd‐induced growth inhibition is accumulation of cells in the G2 phase. Cultures from a single metastasis showed up to a 3‐fold variation in BrdUrd sensitivity. In one of the brain metastases two populations of different ploidy level (pseudotriploid vs. pseudotetraploid) and BrdUrd sensitivity could be resolved. Thus, continuous BrdUrd labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry is a powerful tool to detect heterogeneity in proliferative capacity and drug sensitivity of cell populations within one tumor biopsy. © 1995 Wiley‐Liss, Inc.
Flow cytometric determination of glutathione in clinical samples
Authors: Chow Sue , Hedley David
Published: June 2005

Glutathione‐based processes are believed to be important determinants of resistance to cancer chemotherapy, and measurement of glutathione (GSH) in tumor tissue is therefore of clinical relevance. Flow cytometric methods have been developed for measuring cellular GSH content that appear to correlate well with biochemical determinations. These flow cytometric techniques are rapid, allow tumor cells to be distinguished from stromal elements, and show considerable intratumoral heterogeneity in human tumor cell GSH content. Although large prospective studies are required to determine the correlations between GSH content and treatment outcome in cancer patients, this approach is a powerful alternative to standard biochemical assays for GSH. © 1995 Wiley‐Liss, Inc.
Use of a multiparametric panel to target subpopulations in a heterogeneous solid tumor model for improved analytical accuracy
Authors: O'Rrien Melissa C. , Gupta Ravinder K. , Lee Song Y. , Bolton Wade E.
Published: June 2005

The exclusion of non‐tumor and dead cells from the analysis of live tumor cells can significantly improve the accuracy of prognostic indicators such as proliferative and DNA indexes. To target live breast tumor cells in a heterogeneous breast tumor model, we have designed a panel consisting of the DNA‐specific dye DAPI and epithelial tissue‐specific (cytokeratin), tumor‐associated (MC5), proliferation‐associated (proliferating cell nuclear antigen), and viability‐associated (tubulin) markers. The breast tumor model consisted of a mixture of equal numbers of live and dead MDA‐MB‐175‐VII (breast tumor) cells, live CEM (leukemic) cells, and live peripheral blood mononuclear cells.

Targeting the live MDA cells in the mixture by gating on tubulin, cytokeratin, and MC5 resulted in a sevenfold increase in PCNA positivity (from 3% ungated to 22.3%), a 60% decrease in the %S‐phase fraction (from 37.2% ungated to 15%), and elimination of extraneous hypodiploid and diploid components, enriching the tetraploid MDAs. These results are consistent with those obtained for unmixed live MDA cells. The combined utilization of this panel and “cumulative” electronic gating of the targeted population increases the number of relevant parameters that can be analyzed per sample and the accuracy of the resultant data. © 1995 Wiley‐Liss; Inc.
Cytogenetic heterogeneity and histologic tumor growth patterns in prostatic cancer
Authors: Alers J. C. , Krijtenburg P. J. , Vissers C. J. , Bosman F. T. , Van Der Kwast Th. H. , Van Dekken H.
Published: June 2005

Twenty‐five prostatic adenocarcinomas were studied for the presence of intratumoral cytogenetic heterogeneity by interphase in situ hybridization (ISH) to routinely processed tissue sections. ISH with a chromosome Y‐specific repetitive DNA probe provided a model to investigate patterns of chromosomal heterogeneity within and between different pathological grades. The Gleason grading system was used, since it is based on a detailed classification of growth patterns. Heterogeneity with respect to ploidy of the tumor was examined by ISH with a repetitive DNA probe specific for chromosome 1. The ploidy status of these cancers was confirmed by DNA flow cytometry (P < 0.001). Cytogenetic heterogeneity at the (Y) chromosomal level was observed between Gleason areas, within one area, and even within single tumor glands. The different patterns of chromosomal heterogeneity were seen in all tumor grades and stages. Differences in ploidy status were also found following the aforementioned histological patterns, again, in all grades and stages. Intraglandular heterogeneity was most frequently seen. No correlation was found between cytogenetic heterogeneity and proliferative activity (Kl‐67 immunostaining). In contrast to current views on clonality, suggesting regional separation of subclones with different DNA content, this study demonstrates that these subclones can be interspersed. © 1995 Wiley‐Liss, Inc.
Flow cytometer and imaging device used in combination
Authors: Kubota Fumio , Kusuzawa Hideo , Kosaka Tokihiro , Nakamoto Hiroyuki
Published: June 2005

This report presents a flow cytometer devised by the authors and equipped with a CCD camera to obtain images of selected particles. Each image is linked to a point in the scattergram from which it can be retrieved from memory. The system uses an argon ion laser for flow‐cytometric analysis and a diode‐pumped pulse laser (532nm) to generate CCD images. Aggregated platelets in a blood sample and casts in a urine sample can be detected by this system. A fast flow rate is suitable for efficient flowcytometric detection, and image resolution is sufficiently adequate for recognizing aggregated platelets and casts. Flow‐cytometric and morphological data are used together to distinguish different types of cells in the same region on the scattergram and identify cells present in extremely small numbers among the majority. © 1995 Wiley‐Liss, Inc.
Novel flow‐cytometric method for separating cell types in differentiated F9 embryoid bodies
Authors: Burdsal Carol A. , Pedersen Roger A. , Hyun William C. , Latimer Jean J.
Published: June 2005

The differentiation of F9 teratocarcinoma cells mimics the formation of a mouse embryonic tissue, the primitive endoderm. In vitro, small aggregates of F9 cells, termed embryoid bodies, differentiate in response to retinoic acid and develop a surface epithelium that is characterized by the production of α‐fetoprotein. In the present study, cellular autofluorescence profiles obtained by fluorescence‐activated cell sorting demonstrated that undifferentiated embryoid bodies were composed of a single type of cell. In contrast, retinoic acid‐induced embryoid bodies were composed of two cell types: a major population displaying autofluorescence levels similar to those of cells from undifferentiated embryoid bodies and a second population displaying higher autofluorescence. RNA analyses demonstrated that the transcription of α‐fetoprotein was associated only with the more highly autofluorescent population, indicating that flow cytometry provides a novel mechanism for the separation of undifferentiated cells from differentiated endoderm cells in F9 embryoid bodies. © 1995 Wiley‐Liss, Inc.
Factors affecting flow cytometric detection of apoptotic nuclei by DNA analysis
Authors: Elstein Kenneth H. , Thomas David J. , Zucker Robert M.
Published: June 2005

Apoptotic thymocyte nuclei normally appear on a flow cytometric DNA histogram as a subdiploid peak. We observed that addition of a specific RNase A preparation to the detergent‐based lysing buffer increased the fluorescence of toxicant‐induced apoptotic nuclei to the level of untreated diploid unclei. The chelating agent EDTA partially inhibited the RNase effect, suggesting contaminating divalent cations may have been involved. Moreover, spectrofluorometric analysis revealed that addition of RNase or divalent cations decreased the amount of DNA present in the lysate. This suggested that the upscale fluorescence shift was due to a decrease in the ability of the lysing buffer to extract DNA, possibly as a result of cation‐induced chromatin condensation, rather than increased accessibility of fluorochrome binding sites due to apoptotic degeneration. Moreover, during a 16‐h culture, we observed a similar, but time‐dependent, upscale shift in the fluorescence of thymocytes undergoing apoptosis either spontaneously or as a result of exposure to 1 μM tributyltin methoxide (TBT), 2% ethanol, 2% methanol, or 1 μM dexamethasone phosphate (DEX). This commonality of effect suggests that a similar magnitude of chromatin reorganization occurs in apoptotic cells in prolonged culture regardless of the method of apoptotic induction. These findings should alert investigators to potential inaccuracies in the flow cytometric quantitation of apoptosis in in vitro systems employing prolonged toxicant exposures or complex lysing cocktails that may contain active contaminants. © 1995 Wiley‐Liss, Inc.*
Flow cytometric detection of human immunodeficiency virus type 1 proviral DNA by the polymerase chain reaction incorporating digoxigenin‐ or fluorescein‐labeled dUTP
Authors: Yang Gang , Olson John C. , Pu Ruilian , Vyas Girish N.
Published: June 2005

Serological assays are routinely used in the laboratory diagnosis of human immunodeficiency virus type‐1 (HIV‐1) infection, but the polymerase chain reaction (PCR) is ultimately the most sensitive and direct method for establishing definitive diagnosis. As an alternative to the conventional radioactive PCR procedure we have developed and evaluated a pair of rapid nonradioisotopic flow cytometric detection methods. Using heminested PCR we directly incorporated fluorescein‐12‐dUTP (fluo‐dUTP) or digoxigenin‐11‐dUTP (dig‐dUTP) into the PCR‐amplicons. The labeled amplicons were hybridized with biotinylated antisense and sense probes, followed by capture of the hybrid DNA using streptavidin‐coated beads which were finally analyzed in a flow cytometer by (1) direct detection of the fluorescence intensity of the amplicons incorporating fluo‐dUTP and (2) immunodetection of the amplicons incorporating dig‐dUTP by anti‐digoxigenin IgG labeled with fluorescein isothiocyanate (FITC). Although both assays were functionally comparable with radiolabeled probe in reliably detecting as low as five copies of HIV‐1 proviral DNA sequences, the immunodetection of dig‐dUTP consistently yielded higher mean channel fluorescence and gave a stable signal over an extended period of 12–14 weeks. In testing a panel of 20 pedigreed PBMC specimens from blood donors with or without HIV‐1 infection, the results of both flow cytometric assays were identical with those of the conventional radioactive procedure. Therefore, we conclude that the dig‐dUTP incorporation in amplicons, hybridization with a pair of sense‐antisense biotinylated probes and immunodetection of hybrids by flow cytometric analyses is the nonisotopic method of choice for PCR‐diagnosis of HIV‐1 infection. © 1995 Wiley‐Liss, Inc.
Application of flow cytometry in toxinology: Pathophysiology of human polymorphonuclear leukocytes damaged by a pore‐forming toxin from Staphylococcus aureus
Authors: Meunier O. , Falkenrodt A. , Monteil H. , Colin D. A.
Published: June 2005

The pore‐forming activity of leukocidin (PVL) secreted by Staphylococcus aureus has been investigated on human white cells by flow cytometry techniques. This two‐component toxin induced morphological modifications of neutrophils and monocytes as detected by forward light scattering measurements, but was inactive on lymphocytes. These modifications were the consequence of pore formation through the cell membrane leading to its permeabilization. In the absence of calcium, PVL formed pores large enough to allow ethidium ions to penetrate the cells and become fluorescent by intercalating nucleic acids. In the presence of calcium, the pores were too small for ethidium entry but allowed an influx of calcium as shown by the increase in fluorescence of Fluo‐3 loaded in the cells. This increase in intracellular calcium concentration induced the activation of neutrophils by PVL as shown by the liberation of their granule content measured by a decrease in side light scattering. Furthermore, ethidium fluorescence was used to discriminate the cells sensitive to PVL, and the analysis of differentiated HL‐60 cells and cells obtained from a case of chronic myeloid leukemia led to the conclusion that myeloid cells become sensitive to PVL during differentiation to the metamyelocyte stage. © 1995 Wiley‐Liss, Inc.
BPV E1 protein alters the kinetics of cell cycle entry of serum starved mouse fibroblasts
Authors: Belyavskyi Michail , Miller Jane , Belyavskaya Elena , Wilson Van
Published: June 2005

A stable bovine papillomavirus E1 expressing cell line (C2E1) was used to investigate the effects of E1 protein on the requirement for growth factors during serum‐induced reentry from quiescence to proliferation. Flow cytometric bivariate DNA/PCNA analysis was utilized to study the expression of proliferating cell nuclear antigen (PCNA) concomitant with this transition. C2E1 cells, unlike the control cells (CNEO), were able to reenter the cell cycle when stimulated with low serum (1%). Stimulation with 10% serum revealed that C2E1 cells entered the first cell cycle faster than CNEO, indicating that E1 protein decreased the time of progression from G0 stage upon serum activation. It was also shown that PCNA expression started earlier in C2E1 cells than in CNEO cells after quiescent cells were stimulated with 10% serum. Addition of 1% serum was able to induce PCNA expression in C2E1 but not in CNEO cells in the first 24 h after stimulation. Using Triton X‐100 treatment, it was found that the distribution between bound and unbound forms of PCNA was altered in E1‐expressing cells compared to CNEO cells. Based on these results, it is suggested that E1 might possess mitogen‐like properties. © 1995 Wiley‐Liss, Inc.
Identification of resting cells by dual‐parameter flow cytometry of statin expression and DNA content
Authors: Pellicciari C. , Mangiarotti R. , Bottone M. G. , Danova M. , Wang E.
Published: June 2005

Statin, a 57‐kDa nuclear protein, has been recognized as a unique marker of quiescent (G0) cells; specific monoclonal antibodies (MoAb) against statin have been produced and used to label resting cells in tissue sections and in cultured cells. We present an improved method for the identification of G0 cells by dual‐parameter flow cytometry of statin expression and DNA content. The appropriate technical conditions were set up by using resting and cycling human fibroblasts as a model cell system. Several fixatives proved to be suitable for the immunocytochemical detection of statin; among them, 70% ethanol was selected because this fixation procedure is suitable for DNA staining with intercalating dyes and is routinely used for the immunolabeling of proliferation markers (such as proliferating cell nuclear antigen (PCNA) and Ki‐67) and of bromodeoxyuridine (BrdUrd) incorporation. Following cell permeabilization with detergent, exposure to the antistatin antibody (S‐44), and indirect fluorescein isothiocyanate immunolabeling, cells were counterstained for DNA with propidium iodide and analyzed by dual‐parameter flow cytometry. In cells from several animal sources (rat thymocytes and C6 glioma cells, mouse 3T3 cells, and human MCF‐7 cells), under different experimental conditions, the expression of statin was found to correlate inversely with that of PCNA and Ki‐67, and with the BrdUrd labeling index. In dual‐parameter flow scattergrams, G0 (statin positive) cells can be discriminated from the potentially cycling (statin negative) G1 cells, i.e., within a cell fraction having the same DNA content. This approach can be envisaged as a powerful tool both for monitoring changes in the resting cell fraction and for Investigating the process of G0–G1 transition in unperturbed and drug‐treated cell populations. © 1995 Wiley‐Liss, Inc.
Simultaneous triple staining for hypoxia, proliferation, and DNA content in murine tumours
Authors: Webster Lynne , Hodgkiss Richard J. , Wilson George D.
Published: June 2005

Hypoxia and proliferation rate are two important biological factors influencing the outcome of radiotherapy regimes for solid tumours. Hypoxic cells are more resistant to radiation than aerobic cells and a rapidly dividing tumour may repopulate faster during treatment. Clinical trials are underway to assess the importance of both these parameters. In this article we describe a method to simultaneously measure hypoxia and proliferation using multiparameter flow cytometry. Hypoxic cells were detected using a bioreductively bound marker with an immuno‐recognisable side‐chain, NITP and proliferation was measured by bromodeoxyuridine (BrdUrd) incorporation. These parameters were related to cell cycle position by measuring total DNA content with 7‐aminoactinomycin D. The data were analysed using single laser excitation on a bench top flow cytometer. Simultaneous measurement of the three parameters shows the presence of cells which have incorporated BrdUrd and are also hypoxic by the criterion of NITP binding. The murine SaF tumour has a relatively constant aneuploid labelling index of 24%. However, the level of aneuploid hypoxia was variable ranging from 0.8 to 40.9% with a mean value of 15.6%. Within the BrdUrd labelled population there is a range of hypoxia from 2.8 to 28.5% (mean 15.1%); this represents 0.7 to 6.6% of the total tumour population. There are approximately twice as many oxygenated cells than hypoxic cells actively in the cell cycle. In vivo tumours contain cells with S phase DNA content which do not incorporate BrdUrd. This cell population has equivalent proportions of hypoxic and oxic cells. However, there are up to 12‐fold more hypoxic cells in the unlabelled S than the BrdUrd labelled population. These data show that proliferation and hypoxia can be measured simultaneously using flow cytometry and the technique may form the basis of a predictive assay for these two important biological determinants of radiotherapy outcome. © 1995 Wiley‐Liss, Inc.
Isolation of full‐size mRNA from ethanol‐fixed cells after cellular immunofluorescence staining and fluorescence‐activated cell sorting (FACS)
Authors: Esser Charlotte , Göttlinger Christoph , Kremer Joachim , Hundeiker Claudia , Radbruch Andreas
Published: June 2005

Preparation of intact, full‐size RNA from tissues or cells requires stringent precautions against ubiquitous and rather stable RNases. Fluorescence‐activated cell sorting (FACS) usually aims at the isolation of cells according to cell surface markers on living cells, from which RNA can be obtained by standard protocols. The separation of cells according to intracellular immunofluorescence markers, such as intranuclear, intracytoplasmic, or secreted molecules, requires permeation of the cell membrane for the staining antibodies, which is usually achieved by fixation. However, commonly used fixatives such as ethanol, methanol, or formaldehyde do not inactivate RNases completely, thereby hampering the analysis of complete RNA molecules from fixed cells. We report isolation of intact, fullsize RNA suitable for Northern blotting from cells that were fixed by 95% ethanol/5% acetic acid containing RNase inhibitors, stained intracellularly, and sorted by FACS. © 1995 Wiley‐Liss, Inc.
Evaluation of sensitivity of flow cytometry in detecting aneuploidy in wheat using disomic and ditelosomic wheat–rye addition lines
Authors: Pfosser Martin , Heberle‐Bors Erwin , Amon Amelia , Lelley Thomas
Published: June 2005

Flow cytometric DNA analysis was used to study changes in nuclear DNA content induced by the addition of complete or telosomic rye chromosomes into the genome of common wheat (Triticum aestivum L.). The DNA content of each addition line was determined by comparison with an internal reference value and was expressed as a difference with respect to the original wheat parental line. A 1.84% difference in the DNA content could be detected. Nuclei were flow sorted and the presence of rye chromatin in the nuclei with the higher DNA content was demonstrated by Southern hybridization. Flow cytometry was proven to be sensitive enough to detect the small DNA content deviations that are expected to occur in aneuploid plants of wheat. © 1995 Wiley‐Liss, Inc.
Alterations of T‐cell receptor variable region expression in human immunodeficiency virus disease
Authors: McCoy J. Philip , Overton W. Roy , Blumstein Linda , Baxter John D. , Gekowski Kathleen M. , Donaldson Milton H.
Published: June 2005

Since only a small percentage of CD4+ lymphocytes is infected at any one time during the course of human immunodeficiency virus (HIV) disease, a question central to the pathogenesis of HIV is whether or not the depletion of CD4+ lymphocytes is a random or selective event. The majority of peripheral blood T lymphocytes use α and β variable chains as components of their T‐cell receptor (TCR) complex. Depletion of CD4+ T lymphocytes from the peripheral blood may be dependent on the Vb̃ chain expressed by the CD4+ cell, based on the hypothesis that HIV may encode a superantigen. Peripheral blood from normal controls and HIV+ patients was studied for alterations in the expression of various Vb̃ chains of the TCR. Three‐color flow cytometry was used to determine the expression of Vb̃2, Vb̃3, Vb̃8, Vb̃ 13, and Vb̃ 19 on all lymphocytes and on both CD4+ and CD8+ lymphocytes independently. Alteration of the Vb̃ chains in HIV+ disease was analyzed as a function of absolute CD4 count and Centers for Disease Control (CDC) stage of the patient. These data suggest that the loss of T helper (CD4) lymphocytes during the course of HIV disease may be a selective event. These data are consistent with the hypothesis that selective depletion of CD4+, Vb̃19+ lymphocytes may be due to the encoding of a superantigen by HIV. Furthermore, using multicolor flow cytometry and stratifying patients by absolute CD4 counts (or stage of disease) may reveal immunologic changes that might otherwise be overlooked. © 1995 Wiley‐Liss, Inc.
Flow cytometry: Its use in pediatric renal transplantation utilizing polyclonal induction
Authors: Bunchman Timothy E.
Published: June 2005

Induction protocols for pediatric renal transplant recipients commonly utilize polyclonal or monoclonal agents in sequence with additional immunosuppression. Polyclonal agents Minnesota antilymphoblast globulin (MALG), anti‐thymocyte globulin (ATGAM) effect both T and B cells while monoclonal agents (OKT3) are T‐cell specific. Flow cytometric analysis of T‐cell subsets has become the marker of adequacy of immunosuppression during OKT3 therapy, yet to date no marker exists to measure the adequacy of immunosuppression with polyclonal agents. At the University of Michigan, flow cytometric analysis in pediatric renal transplant recipients undergoing polyclonal induction reveals that CD3, CD4, and CD8 suppression initially occurs. As opposed to OKT3, a rebound of CD3 cells occurs despite daily use of a polyclonal agent in sequence with additional immunosuppressives. During these analyses, a single kidney was lost which flow cytometry failed to predict. No significant difference in flow cytometric patterns occurred when comparing ATGAM to MALG induction. Flow cytometry utilized during polyclonal induction in pediatric renal transplant recipients revealed a variety of T‐cell suppression patterns but failed to demonstrate persistence of suppression. Despite this lack of suppression as indicated by flow cytometry, a 97% 1 year allograft survival rate exists in the pediatric transplant program at the University of Michigan Medical Center. Therefore, the role of flow cytometry during polyclonal induction has yet to be well defined. © 1995 Wiley‐Liss, Inc.
Immunologic differentiation of absolute lymphocyte count with an integrated flow cytometric system: A new concept for absolute T cell subset determinations
Authors: Mercolino Thomas J. , Connelly Mark C. , Meyer Eric J. , Knight Marilyn D. , Parker John W. , Stelzer Gregory T. , Dechirico Gioacchino
Published: June 2005

We describe a method to obtain results for immune status monitoring that uses a three‐test panel, comprised of isotype control and 2 specific Mab tests (CD4/CD8/CD3 and CD16/CD19/CD3), in conjunction with a flow cytometer that directly measures absolute counts. Automated software is used for lineagespecific gating of three‐color immunofluorescence to determine lymphocyte and lymphocyte subset counts. The autogating function of this software is shown to yield equivalent results to manual analysis by an expert user, and to be effective when as few as 25 target cells are present. The software is also shown to perform automatic quality control checks of the sample preparation, reagent, and automated analysis. We demonstrate that the sum of T (CD3 +), B (CD19 +), and natural killer (NK, CD16 + CD3−) cells, as a determination of all lymphocytes, correlates well with lymphocytes measured using a light scatter differential. Moreover, T + B + NK lymphocyte count is shown to be less error‐prone than lymphocyte count from light scatter differential, and to minimize errors that arise from between‐technician variation in sample preparation. Our data suggest that the new approach that we describe could offer an alternative to the traditional two‐stage methods for measuring absolute counts of lymphocyte subsets for immune status monitoring. As such this method could reduce, through objective automated analysis, testing cost and complexity, without sacrificing the quality of results. © 1995 Wiley‐Liss, Inc.
Expression of the fas antigen in patients infected with human immunodeficiency virus
Authors: McCloskey Thomas W. , Oyaizu Naoki , Kaplan Mark , Pahwa Savita
Published: June 2005

Lymphocytes from patients with HIV infection have been shown to undergo accelerated apoptosis. Fas antigen is a cell surface protein known to initiate an apoptotic signal. Therefore, we undertook a study to examine the expression of the Fas antigen during HIV infection. Using three color flow cytometry, expression of the Fas antigen on lymphocytes of 23 HIV infected individuals (CDC category 2, CD4 200–499 cells/m̈L, n = 10; CDC category 3, CD4 < 200 cells/m̈L, n = 13) and 10 healthy controls was examined. Both CD3+CD4+ and CD3+CD8+ subsets were examined for their expression of this marker. In lymphocytes of healthy controls, 47% of the CD3+CD4+ and 45% of the CD3+CD8+ cells were Fas antigen positive. This percentage was significantly increased in CD4 cells from HIV infected patients belonging to CDC category 3, but was unchanged from normal values in CDC category 2 subjects. The increase in the percentage of CD4+ T cells expressing Fas antigen in patients correlated significantly with the decrease in circulating CD4 T cell count (P < 0.009). In addition, by examining mean fluorescence intensity, we found that the amount of Fas expression per cell was increased threefold in CD3+CD4+ cells and increased twofold in CD3+CD8+ cells in category 3 patients. These results demonstrate that an increase in Fas antigen expression occurs during HIV infection. © 1995 Wiley‐Liss, Inc.
Flow cytometric and clinicopathologic study of 197 hydatidiform moles with special reference to the significance of cytometric aneuploidy and literature review
Authors: Fukunaga Masaharu , Endo Yasuhiko , Ushigome Shinichiro
Published: June 2005

In order to evaluate the significance of cytometric aneuploidy in molar placentas, we analyzed 197 hydatidiform moles by flow cytometry using formalin‐fixed, paraffin‐embedded tissues. Of 150 complete moles (CMs), 110 were diploid, 26 were tetraploid, and 14 were aneuploid (non‐triploid/tetraploid aneuploid). Of 47 partial moles (PMs), 44 were triploid and 3 were diploid. We could not find any histologic differences among the diploid, tetraploid, and aneuploid CMs. We found that flow cytometric DNA analysis was very helpful to differentiate CM from PM. Persistent diseases developed in 12 of 69 CMs (17.4%) (9 of 47 diploid and 3 of 14 tetraploid CMs) and in none of 26 PMs (0%). Four diploid and 2 tetraploid CMs were invasive and one each with diploid and tetraploid CM developed choriocarcinoma and none of 8 aneuploid CMs had sequelae; however, there was no correlation between DNA ploidy and clinical outcome in the CMs. These results suggest that cytometric aneuploidy (non‐diploidy) in CMs is not an independent predictor of persistent disease. © 1995 Wiley‐Liss, Inc.