bluuebunny/crossref_metadata_2025 huggingface dataset:
Methodologies for the preservation of proliferation associated antigens PCNA, p120, and p105 in tumor cell lines for use in flow cytometry
Authors: Mikulka Warren R. , Bolton Wade E.
Published: June 2005

The retention of antigen expression of PCNA, p120, and p105 in two tumor cell lines (MOLT‐4 and MDA‐MD‐175‐VII) under various conditions of fixation was investigated using flow cytometric analysis. Four currently utilized procedures for fixation/permeabilization of intracellular antigens were compared for their ability to stain the nuclear antigens. A procedure using a brief incubation in a solution of lysolecithin in paraformaldehyde followed by fixation in ice‐cold methanol prior to antibody staining was selected to evaluate reagent protocols aimed at preserving antigen expression. Holding samples overnight at 4°C in 2.5% fetal bovine serum after the lysolecithin/paraformaldehyde and methanol fixation steps prior to staining with monoclonal antibodies resulted in no decline in the percentage of cells positively stained for all three markers with little decrease in intensity of fluorescence and no increase in DNA coefficient of variation (c.v.). Fixed/permeabilized MOLT‐4 cells held longer than 24 h before staining were lower in PCNA fluorescence than freshly stained cells; holding samples of either cell line longer than 48 h resulted in decreased PCNA and p120 staining. Prefixing and holding cells in 50% methanol or 50% ethanol overnight before processing and staining severely depressed PCNA and p120 fluorescence. Prefixing either cell line in a range of concentrations (0.25–1.0%) of paraformaldehyde also resulted in reduced intensity of PCNA and p120 fluorescence along with increased DNA c.v. P105 staining appeared to be relatively unaffected by all prefixation/storage conditions tested, except for a decline of fluorescence when MDA cells were prefixed in 50% ethanol. Cells cryopreserved in liquid nitrogen for 1 week before processing showed <5% loss of PCNA and p120 fluorescence compared to freshly processed cells, but p105 fluorescence dropped 29% in cryopreserved MDA cells. These results underscore the fact that specific protocols for the fixation and storage of biological samples prior to staining and analysis must be determined for the specific nuclear antigen marker under investigation. © 1994 Wiley‐Liss, Inc.
Bladder irrigation specimens assayed by fluorescence in situ hybridization to interphase nuclei
Authors: Wheeless L. L. , Reeder J. E. , Han R. , O'Connell M. J. , Frank I. N. , Cockett A. T. K. , Hopman A. H. N.
Published: June 2005

Bladder irrigation specimens provide a sampling of the entire bladder urothelium and are the most practical sample for longitudinally monitoring patients. This study presents cross‐sectional fluorescence in situ hybridization (FISH) analyses with correlated DNA cytometry data on 76 patients monitored for recurrent bladder tumors. FISH probes complementary to centromeric satellite sequences for chromosomes 1, 7, 9, 11, 15, and 17 were used. Aberrations in copy number were observed for chromosomes 1, 7, 11, and 17 principally in patients with aneuploid tumors. Monosomy of chromosome 9 was observed in 39% of the diploid and 31% of the specimens with high hyperdiploid fraction. Significantly, 24% of patients with a history of bladder cancer but with no clinical evidence of disease exhibited monosomy of chromosome 9. This suggests a persistent and significantly large population of abnormal cells in the absence of clinical evidence of disease. Loss of chromosome 9 relative to DNA ploidy was observed in 24% of patients with no evidence of disease, in 59% of patients with tumor, and in 79% of patients with histologically confirmed transitional cell carcinoma, grades 1–3. Loss of chromosome 15 was also observed in a large percentage of patients. Loss of chromosome 15 was observed in 41% of specimens from patients in whom no tumor was seen, in 38% of specimens from patients with tumor, and in 67% of specimens from patients with histologically confirmed transitional cell carcinoma. Results of this study document the use of bladder irrigaton specimens as a specimen source for FISH analyses. The ability to perform FISH analyses on bladder irrigation specimens from patients followed for recurrent bladder cancer is important in that it offers the potential of studying early genetic events as well as correlating specific aberrations with progression and recurrence. © 1994 Wiley‐Liss, Inc.
A new method for the analysis of plasma cell DNA content in multiple myeloma samples using a CD38/propidium iodide double staining technique
Authors: Orfão Alberto , García‐Sanz Ramón , López‐Berges M. Consuelo , Vidriales M. Belén , González Marcos , Caballero M. Dolores , San Miguel Jesüs F.
Published: June 2005

In the present paper a CD38/propidium iodide double staining technique is described which separately assesses the cell cycle distribution of myelomatous plasma cells from that of the residual normal hemopoietic cells. For this purpose, bone marrow (BM) cells from a group of 42 untreated multiple myeloma patients were analyzed. Of these, 23 cases were aneuploid (55%) and 19 diploid (45%). The use of the CD38/propidium iodide double staining method allowed a clear separation between CD38 strong positive cells from the remaining bone marrow populations, cells sorting experiments confirming that plasma cells were almost exclusively contained in the former fraction where they represented 97 ± 2% of the total cells sorted. In all cases, the S‐phase in plasma cells and in the remaining normal hemopoietic bone marrow cells was assessed, being higher in normal hemopoietic cells (8.0 ± 6.3%) than in plasma cells (3.3 ± 2.6%, P < 0.002). In addition, there was no correlation between the S‐phase of the neoplastic and normal bone marrow cells (r = 0.22; P > 0.10); this work therefore shows that the assessment of the total proliferative rate of bone marrow samples does not reflect either the proliferation of normal cells or that of neoplastic plasma cells but will depend on the proliferative rate and the percentage of each population within the sample, which can be assessed by the technique described here. © 1994 Wiley‐Liss, Inc.
Constitutive expression of P‐glycoprotein as a determinant of loading with fluorescent calcium probes
Authors: Brezden Christine B. , Hedley David W. , Rauth A. Michael
Published: June 2005

Determination of intracellular calcium levels in Chinese hamster ovary (CHO) cells using the fluorescent calcium probe indo‐1AM was hindered by the low level of accumulation of indo‐1 in these cells. CHO cells are known to express basal levels of the multidrug resistance efflux pump P‐glycoprotein (P‐gp). Rhodamine‐123, which is a known substrate of P‐gp, was used to confirm the presence of P‐gp in CHO cells. Verapamil and cyclosporin (CsA), both inhibitors of P‐gp, enhanced accumulation of indo‐1 in these cells and therefore allowed for improved intracellular calcium measurements. P‐gp overexpressing colchicine‐resistant CHO cells (CHRC5) also displayed enhanced indo‐1AM loading with P‐gp inhibitors. Nondetectable levels of P‐gp activity were found in wild‐type CEM‐CCRF cells (human T lymphoblasts), and these cells did not show any difference in indo‐1AM loading in the presence or absence of P‐gp inhibitors. Loading of a second calcium fluorescent probe fluo‐3AM was improved in CHO cells by P‐gp inhibition, whereas the structurally related pH probe BCECF‐AM was minimally affected. Because low levels of P‐gp may be expressed by a range of cell lines and normal tissues, it is suggested that this be considered if difficulties are encountered in loading fluorescent calcium probes. © 1994 Wiley‐Liss, Inc.
Cytogenetic analysis by chromosome painting
Authors: Carter Nigel P.
Published: June 2005

Chromosome painting is a term used to describe the direct visualisation using in situ hybridisation of specific chromosomes in metaphase spreads and in interphase nuclei. Chromosome painting, coupled with fluorescence in situ hybridisation (FISH), is now used routinely to enhance the identification of chromosomal rearrangements, the assignment of breakpoints, and the determination of the origin of extra chromosomal material. Amplification of small numbers of flow‐sorted chromosomes by the polymerase chain reaction allows labelled chromosome paints to be generated in a matter of days. These technologies have enabled the development of reverse chromosome painting, in which the paint is produced from sorted aberrant chromosomes and hybidised back onto normal metaphase spreads to identify directly the composition of the aberrant chromosome. Reverse chromosome painting is able to identify not only the chromosomal origin of marker chromosomes but also the regions and breakpoints involved. In some cases, such as interstitial translocations and complex marker chromosomes, the combination of conventional (forward) chromosome painting and reverse chromosome painting combine to provide a definitive analysis of the rearrangement. With the availability of chromosome paints and painting kits from a variety of commercial sources, multicolour chromosome painting has now become a routine method of analysis in the clinical cytogenetic laboratory. © 1994 Wiley‐Liss, Inc.
Immunophenotyping specimens from HIV‐infected persons: Laboratory guidelines from the Centers for Disease Control and Prevention
Authors: Nicholson Janet K. A.
Published: June 2005

The Centers for Disease Control and Prevention has published laboratory guidelines for enumerating CD4+ T‐cells from human immunodeficiency virus (HIV)‐infected persons to help improve the quality of lymphocyte immunophenotyping. New data that clarify some of the variables in the process of immunophenotyping have been obtained. These include information relevant to both hematology and immunophenotyping procedures as well as quality control parameters that are important in producing reliable results. © 1994 Wiley‐Liss, Inc.
Applications of cytometry to study acute leukemia: In vitro determination of drug sensitivity and detection of minimal residual disease
Authors: Campana Dario
Published: June 2005

Modern clinical applications of cytometry include the determination of the most powerful antileukemic drugs in each patient at the time of diagnosis and the monitoring of residual disease during and off treatment. The precision of in vitro assays to test the susceptibility of cancer cells to cytotoxic drugs depends on the ability to maintain the cells' viability in culture. We found that bone marrow‐derived allogeneic stromal cells are critical to prevent death by apoptosis of acute lymphoblastic leukemia (ALL) cells. Thus, we devised an in vitro drug sensitivity assay in which ALL cells are seeded onto stromal cells and viable leukemic cells are counted at the end of cultures by flow cytometry. Our preliminary results indicate that this assay is suitable for evaluating the drug sensitivity of leukemic lymphoblasts and testing the antileukemic activity of potentially effective compounds which have not yet been administered to patients with ALL. The identification of immunophenotypes expressed on leukemic cells but absent or extremely rare among normal hematopoietic progenitors allows close monitoring of the effects of drug treatment in vivo. Phenotypes that afford a detection level of 1 leukemic cell among 10,000 normal bone marrow cells have been identified in 90% of cases of T‐ALL, 25% of B‐lineage ALL, and 40% of acute myeloid leukemia (AML). In several studies, residual disease emerging during continuation therapy or off treatment almost invariably anticipated overt relapse by 1–7 months. These data indicate the reliability of immunologic techniques to detect occult leukemia. The combined application of drug sensitivity testing and residual disease monitoring may improve the accuracy of clinical management practices, and, ultimately, lead to improved cure rates. © 1994 Wiley‐Liss, Inc.
Cell subset (CS) parameter to record the identities of individual cells in flow cytometric data
Authors: Redelman Doug , Coder David M.
Published: June 2005

The Flow Cytometry Standard (FCS) for cytometric data (Dean et al.: Cytometry 11:323–332, 1990) provides for appending an ANALYSIS section to a data file, but it does not explicitly provide for recording the identities of individual cells. We propose an extension to the FCS definition in order to record the identity of each individual cell in list mode data. In order to do so, one first defines the subpopulations of cells to be identified and one then assigns a number to each defined population. For example, in an analysis in which peripheral blood mononuclear cells are labeled with antibodies to CD3, CD4, and CD8, the “negative” lymphocytes that are labeled with none of the antibodies could be identified as population 1, the CD3−CD4−CD8+ lymphocytes as population 2, etc. As the measured values from each cell are analyzed, the number that identifies the population to which that cell belongs is assigned as the value of an additional parameter. Since this procedure merely adds a new parameter, the only necessary extension to the FCS specification is that a particular name is recognized for this parameter. We propose that CS (abbreviation for cell subset) be recognized as the name and that CS be used as the value for the $PnN (parameter name) keyword in the FCS file TEXT. The CS values that are created can be used to aid in data analysis and can be permanently recorded in a conventional FCS file. A data file that is saved with CS values includes an explicit and integral record of the complete analysis, regardless of the complexity of the analysis. We describe how to create and record the CS parameter, provide examples of how the CS parameter can be used to assist in data analysis, and propose ways in which the CS parameter can be used to monitor quality control © 1994 Wiley‐Liss, Inc.
Detection of intracellular lymphoid differentiation antigens by flow cytometry in acute lymphoblastic leukemia
Authors: Sartor Mary , Bradstock Kenneth
Published: June 2005

The value of flow cytometric detection of the intracellular lymphoid differentiation antigens CD3 and CD22 in the differential diagnosis of acute leukemia was assessed in cases of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and leukemic cell lines. Cells were fixed in 0.25% paraformaldehyde at 4°C for 60 min, permeabilized with 0.2% Tween 20 at 37°C for 15 min, then stained with CD3 or CD22 monoclonal antibodies by indirect immunofluorescence. Cytoplasmic CD22 was detected on greater than 20% (mean 55%; range 20–87%) of blasts from all 20 cases of precursor‐B ALL analyzed. The percentage of cells with cytoplasmic CD22 was greater than that with membrane CD22 in all except 2 cases of precursor‐B ALL. Cytoplasmic CD22 was not detected in 8 cases of precursor‐T ALL, 4 T‐leukemia cell lines, or in 7 cases of AML. In contrast, cytoplasmic CD3 was detectable by flow cytometry in all 8 cases of precursor‐T ALL, but not in precursor‐B ALL, pre‐B leukemia cell lines, or in AML. These results confirm that cytoplasmic CD3 and CD22 are excellent markers of the early T and B lineages in ALL and can be reliably detected by flow cytometry. This technique should be a valuable addition to routine immunophenotyping for classification of acute leukemia. © 1994 Wiley‐Liss, Inc.
Hardware and software requirements for quantitative analysis of comparative genomic hybridization
Authors: du Manoir Stanislas , Kallioniemi Olli‐P. , Lichter Peter , Piper Jim , Benedetti P. A. , Carothers A. D. , Fantes J. A. , García‐Sagredo J. M. , Gerdes T. , Giollant M. , Hemery B. , Isola J. , Maahr J. , Morrison H. , Perry P. , Stark M. , Sudar D. , van Vliet L. J. , Verwoerd N. , Vrolijk J.
Published: June 2005

Recommendations are made for hardware and software capabilities that will permit a level of performance of comparative genomic hybridization (CGH) analysis on metaphase chromosomes that is comparable to the best current practice. Guidelines for interpreting the results of CGH analysis in terms of chromosomal gains or losses are also presented. © 1995 Wiley‐Liss, Inc.
Image analysis in comparative genomic hybridization
Authors: Lundsteen Claes , Maahr Jan , Christensen Britta , Bryndorf Thue , Bentz Martin , Lichter Peter , Gerdes Tommy
Published: June 2005

Comparative genomic hybridization (CGH) is a new technique by which genomic imbalances can be detected by combining in situ suppression hybridization of whole genomic DNA and image analysis. We have developed software for rapid, quantitative CGH image analysis by a modification and extension of the standard software used for routine karyotyping of G‐banded metaphase spreads in the Magiscan chromosome analysis system. The DAPI‐counterstained metaphase spread is karyotyped interactively. Corrections for image shifts between the DAPI, FITC, and TRITC images are done manually by moving the three images relative to each other. The fluorescence background is subtracted. A mean filter is applied to smooth the FITC and TRITC images before the fluorescence ratio between the individual FITC‐ and TRITC‐stained chromosomes is computed pixel by pixel inside the area of the chromosomes determined by the DAPI boundaries. Fluorescence intensity ratio profiles are generated, and peaks and valleys indicating possible gains and losses of test DNA are marked if they exceed ratios below 0.75 and above 1.25. By combining the analysis of several metaphase spreads, consistent findings of gains and losses in all or almost all spreads indicate chromosomal imbalance. Chromosomal imbalances are detected either by visual inspection of fluorescence ratio (FR) profiles or by a statistical approach that compares FR measurements of the individual case with measurements of normal chromosomes. The complete analysis of one metaphase can be carried out in approximately 10 minutes. © 1995 Wiley‐Liss, Inc.
Semiautomated DNA probe mapping using digital imaging microscopy: II. System performance
Authors: Sakamoto Masaru , Pinkel Daniel , Mascio Laura , Sudar Damir , Peters Donald , Kuo Wen‐Lin , Yamakawa Kazuhiro , Nakamura Yusuke , Drabkin Harry , Jericevic Zeljiko , Smith Louis , Gray Joe W.
Published: June 2005

This paper describes an evaluation of a semiautomated, multicolor image‐analysis system to map cloned probes along metaphase chromosomes. Mapping with this system consists of fluorescence in situ hybridization (FISH) for probe localization, automatic acquisition of multicolor images showing total chromosomal DNA and probe location(s), and automatic determination of the fractional locations of the probes along the chromosomes relative to the short arm telomere (FLpter). The system was evaluated by mapping ten phage and ten cosmid probes previously mapped to chromosome 3 with other procedures. The standard deviations of FLpter measurements averaged 3.4 Mb and 2.6 Mb for phage and cosmid probes, respectively. With this variation, the order of two probes mapped in separate hybridizations could be determined with 95% confidence when their separation was greater than 2.5 Mb. In all cases, the probe locations and order were consistent with previous mapping data. FLpter values were converted to band locations using measurements of the band locations made using digital imaging microscopy. This proved superior to conversions made using ISCN ideograms. © 1995 Wiley‐Liss, Inc.
Flow injection fluorescence microscopy applied to a rapid cell surface immunoassay
Authors: Pollema Cy H. , Lernmark Åke , Ruzicka Jaromir
Published: June 2005

A perfusion system for fluorescence microscopy that utilized a flow injection system was developed and used to study cell surface antibody binding on viable cells grown in monolayer cultures on coverslips. A polyclonal cell‐specific antiserum used to probe the cell surface was monitored by indirect immunofluorescence. The flow injection system was completely automatic and allowed controlled perfusion of cell surfaces with the desired sequence of antibodies. The individual steps of the indirect assay were studied to determine the binding behavior of the primary cell surface antibody, the labeled second antibody, and control of nonspecific binding. Under stopped flow conditions, the second antibody was maximally bound within 10 min, while constant mixing of the second antibody solution over the cells resulted in maximal binding within as little as 6 min. A primary antibody contact time of 2 min followed by a wash and then exposure to the second antibody for 2 min showed that this fast automated procedure could distinguish specific from nonspecific binding of cell surface antibodies to the same set of cells for several repeated exposures. The flow injection fluorescence microscopy system can be automated to allow screening for cell surface antibodies and to study their interaction with specific cell surface antigens. © 1995 Wiley‐Liss, Inc.
Flow cytometric assessment of peripheral blood contamination and proliferative activity of human bone marrow cell populations
Authors: Abrahamsen Jenny Foss , Lund‐Johansen Fridtjof , Laerum Ole Didrik , Schem Baard Christian , Sletvold Olav , Smaaland Rune
Published: June 2005

Bone marrow aspiration is superior to bone marrow biopsies due to less discomfort to the volunteer or patient, but it is inferior concerning the reproducibility of cytokinetic information. Therefore, a method that could select aspirates of quality and reproducibility equal to those of biopsies was sought. Low‐density (mononucleated) bone marrow cells were labelled with T200 common leukocyte antigen, CD45, which differentiate cells into erythroid, myeloid, and lymphocyte + monocyte subpopulations based on their immunofluorescence intensity. A hypotonic propidium iodide solution was added, and DNA cell cycle characteristics of the total cells and the subpopulations were obtained. Twenty‐two aspirations were performed on three healthy men. There was a strong negative correlation between the amount of CD45‐gated lymphocytes + monocytes, indicative of peripheral blood cell contamination in the aspirate, and the percentage of total cells and subpopulations in DNA S phase. A marked reduction in the percentage of cells in S phase was observed when the lymphocyte + monocyte counts were higher than 30%; this level was used to exclude aspirates with an unacceptable degree of peripheral blood cell admixture. Twelve of the aspirates were found to be of acceptable quality due to their low lymphocyte + monocyte count. These aspirates were compared with 11 bone marrow biopsy expellates from hematologically normal patients undergoing open cardiac surgery. The 12 aspirates were found to have almost identical mean percent S‐phase cells as the biopsy expellates, both for the total cell population (14% ± 3.45% vs. 15% ± 1.5%) and for the erythroid (24% ± 6% vs. 24.4% ± 3.3%) and myeloid (10% ± 2.4% vs. 10.7% ± 2.5%) subpopulations. The aspirates with an unacceptable degree of peripheral blood cell admixture had a significantly lower mean percent cells in S phase for both the total cells and the subpopulations. It is concluded that bone marrow aspirates of small volumes may give cytokinetic information as reproducible as bone marrow biopsies if the above‐described method is applied. © 1995 Wiley‐Liss, Inc.
Classmode: A new data format for real‐time multiparameter data analysis and data compression
Authors: Keij Jan F. , Groenewegen Ad C. , Jonker Richard R. , Smith Chris R. , Visser Jan W. M.
Published: June 2005

A new data acquisition and analysis format (class‐mode) was developed that allows real‐time data classification in a flow cytometer. In our cytometer, detected events were classified in real time by their presence or absence in a set of look‐up tables (LUT). A modification of the cytometer hardware allows the exclusive transfer of the LUT data to the acquisition/storage computer. Using a combination of 8 LUTs, the analyzed events can be classified into 256 subpopulations. Real‐time data classification results in an increased data transfer rate and a significant compression of the data. © 1995 Wiley‐Liss, Inc.
Sex determination in dioecious plants Melandrium album and M. rubrum using high‐resolution flow cytometry
Authors: Doležel Jaroslav , Göhde Wolfgang
Published: June 2005

High‐resolution flow cytometry was used to analyze nuclear DNA content in dioecious plants Melandrium album and M. rubrum. Very low coefficients of variation (0.53–0.70%) of G0/1 peaks were obtained after the analysis of nuclei isolated by chopping leaf tissues. In both species, females have lower DNA content than males due to sex chromosome heteromorphism. This made it possible to discriminate peaks belonging to female and male nuclei on the same histogram and permitted unambiguous sex identification in both species. The results indicated the possibility of diagnosing sex also in other heterogametic dioecious plants. The assay described here might be interesting in any area where rapid sex identification is required at an early stage of plant development. © 1995 Wiley‐Liss, Inc.
Expression of HSP70 induced in CHO cells by 45.0°C hyperthermia is cell cycle associated and DNA synthesis dependent
Authors: Hang Haiying , Fox Michael H.
Published: June 2005

In response to a heat shock, the synthesis of heat shock proteins with a molecular weight of 72 kD (hsp72) is induced. To determine whether hsp72 expression in Chinese hamster ovary (CHO) cells is cell cycle associated, flow cytometry was used to measure DNA content and heat shock protein levels simultaneously. After 10 min at 45.0°C and incubation at 37°C for several hours, hsp72 was induced to a high level in early S phase. Inhibitors of DNA synthesis, aphidicolin, and 1‐β‐D‐arabinofuranosylcytosine (araC), suppressed the induction of hsp72 but did not inhibit it completely. Hsp73 (constitutive hsp70) was also induced by heat shock, but the induction was uniform through the cell cycle and it was not suppressed by araC. © 1995 Wiley‐Liss, Inc.
Cell cycle analysis of amount and distribution of nuclear DNA topoisomerase I as determined by fluorescence digital imaging microscopy
Authors: Baker Sharyn D. , Wadkins Randy M. , Stewart Clinton F. , Beck William T. , Danks Mary K.
Published: June 2005

Fluorescence digital imaging microscopy (FDIM) has been used to perform a cell cycle analysis of both the amount and the distribution of nuclear DNA topoisomerase I in individual CEM human leukemia cells. Cells were stained by indirect immunofluorescence methods using a polyclonal antiserum generated with a 21‐amino‐acid peptide representing amino acids 219–239 of human topoisomerase I. Immunohistochemical staining was followed by staining with Hoechst dye 33342, allowing DNA content to be determined in each cell. Cell cycle analysis showed that nuclear topoisomerase I content doubled (2.2‐fold increase) as the cells progressed from G1 to G2/M phases of the cell cycle. However, when normalized for nuclear size, topoisomerase I content per nuclear area remained almost constant (1.3‐fold increase). For comparison, we measured the amount of proliferating cell nuclear antigen (PCNA), a protein whose expression fluctuates during the cell cycle. Nuclear PCNA content increased 2.7‐fold from G1 to S phase, then declined in G2/M‐ phases, whereas PCNA content per nuclear area increased 1.7‐fold from G1 to S phase. We also measured topoisomerase I content in leucine‐deprived cells to determine if altered growth conditions affect topoisomerase I protein expression. Compared to CEM cells in logarithmic growth, leucine‐deprived CEM cells had 1.8‐fold less topoisomerase I content per nuclear area. Subnuclear distribution studies of proliferating CEM cells showed topoisomerase I to be localized predominantly in the nucleoli throughout the cell cycle. In contrast, leucine‐deprived cells exhibited a perinuclear distribution of topoisomerase I. Our results show that FDIM is a useful technique in determining the cell cycle position and both the content and the distribution of topoisomerase I as well as other nuclear proteins in individual cells. © 1995 Wiley‐Liss, Inc.
Analysis of antifading reagents for fluorescence microscopy
Authors: Florijn R. J. , Slats J. , Tanke H. J. , Raap A. K.
Published: June 2005

The utility of p‐phenylenediamine, 1,4‐di‐azobicyclo‐(2.2.2.)‐octane, and the commercial products Citifluor, Slowfade, and Vectashield, antifading agents frequently used as mounting media for fluorescence in situ hybridization, was investigated. Fading curves for bound fluorochromes were recorded with digital microscopy, and relative fluorescence intensities of fluorochromes in solution were measured with an aperture defined measurement system. The three commonly used fluorochromes, fluorescein, tetramethyl rhodamine, and coumarin, were studied. Vectashield offered the best antifading properties for all three fluorochromes, although their relative fluorescence intensity was slightly less in Vectashield in comparison with other antifading agents. In Vectashield, fluorescein, tetramethyl rhodamine, and coumarin showed half‐life times of 96, 330, and 106 s, respectively, whereas in 90% glycerol in PBS (pH 8.5), these half‐life time values were 9, 7, and 25 s, respectively. Vectashield is particularly recommended as a mounting medium for quantitative digital imaging microscopy and for multicolor applications, where it is easy to have errors due to differences in fading rates of the fluorochromes. © 1995 Wiley‐Liss, Inc.
Usefulness of the scraping method for DNA flow cytometry in breast tumors
Authors: Cornacchiari A. , Grigolato P. G. , Facchetti F. , Morassi M. L. , Cadei M. , Alpi F. , Battocchio S. , Chirico E.
Published: June 2005

Flow cytometric DNA analysis is an important prognostic tool in breast cancer. We evaluated the possibility of performing DNA analysis on cell suspensions obtained by scraping the cut surface of breast tumors; 31 breast tumor nodules, including six benign and 25 malignant lesions, were studied. From each case, cell suspensions acquired by mechanical mincing of a fresh frozen tissue fragment and by two different scrapings (central and peripheral) from the cut surface of the tumor were analyzed via flow cytometry. In all cases, comparison of the DNA histograms for three samples showed no significant differences in the appearance of debris or in the value of coefficient of variation of the G0–G1 peak. All benign nodules showed a normal DNA stemline in all specimens. In 23 of 25 cases of breast carcinoma, the ploidy of the three preparations was similar, with a concordance in 12/14 (85, 71%) cases in DNA nondiploid tumors. Linear regression analysis showed a good correlation in DNA index between either scraping sample and the tissue fragment (r = .955 and r = .905). The results indicate that the scraping technique provides excellent cell suspensions and DNA histograms comparable to those obtained from mechanical mincing of tissue fragments. The technique minimizes preparation time and avoids consuming much tissue and, thus, is the method of choice when very small cancers have to be analyzed. © 1995 Wiley‐Liss, Inc.
Comparative sperm chromatin structure assay measurements on epiillumination and orthogonal axes flow cytometers
Authors: Evenson Donald , Jost Lorna , Gandour Donna , Rhodes Lori , Stanton Barbara , Clausen Ole Petter , de Angelis Paula , Coico Richard , Daley Andrew , Becker Kevin , Yopp Tim
Published: June 2005

The sperm chromatin structure assay (SCSA) measures the susceptibility of sperm nuclear DNA to acid‐induced denaturation in situ, and was developed on two Ortho flow cytometers, an FC200 [Becton Dickinson Immunocytometry Systems (BDIS), Westwood, MA] and a Cytofluorograf 30 (BDIS), both having orthogonal axes of fluorochrome excitation, emission, and sample flow. Sperm cells are first treated with a pH 1.4 buffer to denature DNA in situ and then stained with the metachromatic dye acridine orange (AO). The metachromatic fluorescence measured reflects relative amounts of denatured (red fluorescence) and native (green fluorescence) DNA present per cell. The extent of DNA denaturation is quantified by the calculated parameter alpha t [αt = red/(red + green) fluorescence]. Alpha t variables important for correlations with fertility and toxicant‐induced chromatin damage include mean (Xαt), standard deviation (SDαt), and cells outside the main population (COMPαt). Mean green fluorescence intensity is an important measure for DNA content and/or degree of sperm chromatin condensation. This study showed that the SCSA can be successfully run on two epiillumination‐type instruments, an Ortho ICP22A (BDIS, San Jose, CA) and Skatron ArgusTM (Tranby, Norway), and two additional orthogonal axes instruments, a Becton Dickinson FACScanTM (BDIS) and a Coulter EliteTM (Coulter Corporation, Hialeah, FL). Epiillumination instruments produced a different fluorescence distribution than orthogonal instruments, but the resulting αt values showed strong conformity and interpretation of results was the same. SCSA values obtained on the Coulter EliteTM were most similar to the Cytofluorograf 30; the FACScanTM green fluorescence distribution was narrower and allowed resolution of cell doublets. Neither orthogonal instrument has the ability to directly calculate αt values Listmode data from these instruments were transferred to an off‐line personal computer (PC) for calculation of αt values using LIST‐VIEWTM software (Phoenix Flow Systems, Inc., San Diego, CA). © 1995 Wiley‐Liss, Inc.
Induction of cell cycle perturbations by the tear gas 2‐chlorobenzylidene malonitrile in synchronously and asynchronously proliferating mammalian cells
Authors: Weller Eva Maria , Kubbies Manfred , Nüsse Michael
Published: June 2005

The effects of the tear gas 2‐chlorobenzylidene malonitrile (CS) on mammalian cell proliferation were studied in detail using bromodeoxyuridine/ Hoechst flow cytometry. In synchronized (G0/G1‐phase) Chinese hamster embryo (CHE) cells, exposure to CS (60 μM) caused a permanent arrest in the G0/G1 phase in 50% of the cells and a delayed G0/G1 phase exit. In asynchronously growing CHE cells, the CS‐induced cell kinetic perturbations varied with the cell cycle stage during treatment. While G1‐phase cells showed a delayed progression through S and G2/M phases, S‐phase cells were mainly inhibited in the G2/M compartment of the first cell cycle. In contrast, CS‐treated, asynchronous, amniotic fluid‐derived, fibroblast‐like (AFFL) cells exhibited a prolonged transit through the G2/M phase of the first cell cycle regardless of the cell cycle stage during treatment. This indicates that the induced cytotoxicity of CS is a function of both the cell cycle phase and the particular type of cells. © 1995 Wiley‐Liss, Inc.
Phenotype transition of CD4+ T cells from CD45RA to CD45RO is accompanied by cell activation and proliferation
Authors: Johannisson Anders , Festin Roger
Published: June 2005

An investigation of proliferation and activation events in subsets of human CD4+ cells, defined by their expression of CD45RA and CD45R0, is reported. A single‐laser based assay for the study of multiple surface antigens and two‐parameter cell cycle analysis was used for sorting of and subsequent analysis of proliferation in CD4+ CD45RA+ CD45R0−, CD4+CD45RA−CD45R0+ subsets and phenotypically intermediate stages. After labelling with BrdUrd, cells were sorted with flow cytometry on the basis of light‐scattering properties and staining with anti‐CD45RA, anti‐CD45R0, and anti‐CD4 markers. Sorted cells were double stained with anti‐BrdUrd‐antibodies and PI, and the frequencies of proliferating cells were determined. After 48 h, the highest rate of proliferation was found among cells with a phenotype intermediate between CD4+ CD45RA+CD45R0− and CD4+CD45RA−CD45R0+. After 72 h of culture, the situation was changed insofar as the point of highest proliferation had shifted towards the CD4+CD45RA−CD45R0+ population. These findings were further corroborated by four‐colour staining with anti‐CD4, anti‐CD45RA, anti‐CD45R0, and Hoechst 33342. This indicates that the phenotype transition is accompanied by cell proliferation. The correlated temporal expression of antigens related to activation (HLA‐DR, CD25, CD69, CD71) and cell adhesion (CD11a, CD54, L‐selectin) in each of the different subsets was also investigated. All the activation markers CD25, CD69, and CD71 show a more heterogeneous pattern of expression among the CD4+ CD45RA−CD45R0+ cells than the CD4+ CD45RA+CD45R0− cells, indicating a subpopulation of CD4+CD45RA−CD45R0+ cells responding more slowly to the mitogenic stimulation. © 1995 Wiley‐Liss, Inc.
Flow cytometric evaluation of erythrocyte response to oxidant stress
Authors: Hammouda Abd‐El‐Rahman M. A. , Fakeir Aisha
Published: June 2005

Erythrocyte response to oxidant challenge has been used as an indicator of cell membrane tolerance to oxidant stress. This study describes a simple technique for evaluation of erythrocyte response to hydrogen peroxide oxidant stress using flow cytometry as an assay method. Challenged cells showed detectable increase in fluorescence over the basalevel. This increase in fluorescence was both concentration and time dependent. The test was standadized using 0.1 mM hydrogen peroxide and 2 h incubation. The standardized test showed a higher fluorescence of challenged umbilical cord erythrocytes compared to challenged adult red blood cells. We recommend further use of this test for examining other populations of red blood cells. © 1995 Wiley‐Liss, Inc.
Specific staining of iododeoxyuridine and bromodeoxyuridine in tumors double labelled in vivo: A cell kinetic analysis
Authors: Pollack Alan , Terry Nicholas H. A. , Wu Catherine S. , Wise Birute M. , White R. Allen , Meistrich Marvin L.
Published: June 2005

The simultaneous and specific staining of iododeoxyuridine (IdUrd) and chlorodeoxyuridine (CldUrd) allows for more accurate estimates of potential doubling time (Tpot). Because CldUrd is not approved for human use, the procedure was adapted for the staining of IdUrd and bromodeoxyuridine (BrdUrd). The fluorescein isothiocyanateconjugated B44 antibody (B44‐FITC) stained both IdUrd and BrdUrd in tumor nuclei labelled singly with one or the other pyrimidine analogue. However, when MCaK tumors in exponential growth in vivo were pulse labelled with both IdUrd and BrdUrd, the staining of BrdUrd was not seen, and the labelling pattern reflected specificity to IdUrd. These observations were confirmed using tumors pulse labelled with IdUrd and/or BrdUrd at 6 h and/or 0.3 h prior to tumor removal in all possible combinations. Simultaneous specific staining of BrdUrd by Br3 and of IdUrd by B44‐FITC was documented by quantification of labelling indices (LIs) from double‐labelled tumors. The specificity of B44‐FITC for IdUrd in double‐labelled tumors was due to a greater affinity of this antibody for IdUrd than for BrdUrd. This technique allowed for two independent estimates of LI and Tpot when tumors were double labelled for 3.0 and 5.5 h. Both IdUrd and BrdUrd are approved for clinical use, and this double‐labelling technique should prove to be valuable for measuring the cell kinetics of solid tumors in vivo. © 1995 Wiley‐Liss, Inc.
Use of the biotinylated antibody DAKO‐ER 1D5 to measure oestrogen receptor on cytokeratin positive cells obtained from primary breast cancer cells
Authors: Brotherick I. , Lennard T. W. J. , Cook S. , Johnstone R. , Angus B. , Winthereik M. P. , Shenton B. K.
Published: June 2005

A method for the use of a biotinylated antibody (DAKO‐ER 1D5) to quantify oestrogen receptors (ER) on tumour cells by flow cytometry is described. ER quantification was determined after treatment with saponin rendering cells permeable to ER antibody. Use of dual parameter labelling was performed utilizing a FITC‐conjugated antibody (NCL‐5D3) directed against cytokeratin 8/18. This allowed selection of breast cancer cells of epithelial origin by gating to exclude contaminating inflammatory and stromal cells. Use of such a gating technique was seen to identify cells with a higher level of ER expression. Using QC quantum bead standards, the number of ER binding sites per cell was assessed. Results were compared with conventional ER quantification using a radio‐ligand binding assay. A high degree of correlation was found between the two methods. The flow cytometric method for ER quantification described is simple, rapid, and reproducible. The assay may be of particular value in measuring ER on urgent clinical samples. Advantages of this assay over the radio‐ligand binding assay include reduction in use of radio‐labelled iodine compounds, a decrease in analysis time, and reduced cost and quantity of material needed for assay. © 1995 Wiley‐Liss, Inc.
Quantitative cytofluorimetric determination of cell membrane‐associated large tumor antigen on SV40‐transformed cells
Authors: Hess Ralf D. , Kuther Markus , Haessler Christel , Paetzold Susanne , Braun Dietmar G. , Brandner Gerhard
Published: June 2005

The aim of this study was to quantitate the number of cell membrane‐located SV40 large tumor antigen (large T) molecules of SV40‐transformed cell lines by cytofluorimetric analysis. Five different SV40‐transformed cell lines were labelled by either a biotin‐ or a fluorescein‐conjugated monoclonal antibody, PAb1605, which is specific for the large T carboxyterminus. The conjugated‐antibody fluorescence signals of the stained large T molecules of transformed cells were measured via cytotluorimetry. Comparison of the fluorescence signals of calibrated beads bearing a known number of fluorescein molecules to the signals of conjugated PAb1605 antibodies bound on microbeads to a defined number of IgG binding sites made it possible to determine the number of antibody‐accessible large T molecules per SV40‐transformed cell. The numbers (×10−4) found per cell were 1.0 (ELONA, hamster), 3.0 (VLM, mouse), 3.5 (mKSA, mouse), 11 (C57SV, mouse), and 5.5 (SV80, human), respectively. Thus, the technique described allows a precise quantitation of surfaceexposed, antibody‐accessible viral antigen expression. © 1995 Wiley‐Liss, Inc.
Scanning microfluorometric analysis of proliferating cell nuclear antigen in formalin‐fixed sections of hyperplastic and neoplastic rat liver
Authors: Kong Jin , Ringer David P.
Published: June 2005

Scanning laser cytometric analysis of fluorochrome‐labeled cells was used to survey and quantitate the distribution of proliferating cells in formalin‐fixed, paraffin‐embedded tissue sections from hyperplastic and neoplastic rat livers. The technique used fluorescent immunochemical staining of proliferating cell nuclear antigen (PCNA) as a marker for proliferating cells and propidium iodide as a fluorescent nuclear counterstain. Use of an antigen retrieval treatment improved detection of PCNA and treatment of tissue sections with crystal violet improved the sensitivity of the method by quenching background autofluorescence. PCNA evaluation of cell proliferation in regenerating rat liver 0–48 h post‐partial hepatectomy showed that 3–43% of cells stained positively for PCNA, a pattern closely correlating with previously reported rates of maximum DNA synthesis. The PCNA staining patterns observed among cells in neoplastic nodules were more focal in distribution and indicated that from 5 to 25% of the cell nuclei per nodular region stained positively for PCNA. This use of image analysis for the rapid identification of proliferating cell areas in fixed, paraffin‐embedded tissue active in neoplastic growth will expedite in situ cytochemical and molecular studies attempting to identify key differences between hyperplastic and neoplastic growth. © 1995 Wiley‐Liss, Inc.
Relationship between DNA ploidy level and tumor sociology behavior in 12 nervous cell lines
Authors: Kiss Robert , Camby Isabelle , Salmon Isabelle , Van Ham Philippe , Brotchi Jacques , Pasteels Jean‐Lambert
Published: June 2005

Cell population sociology was studied in two medulloblastomas and 10 astrocytic human tumor cell lines by means of the characterization of the structure of neoplastic cell colonies growing on histological slides. This was carried out via digital cell image analysis of Feulgen‐stained nuclei, to which the Delaunay triangulation and Voronoi paving mathematical techniques were applied. Such assessments were compared to the DNA ploidy level (assessed by means of DNA histogram typing). The results show that the cell colony architecture characteristics differed markedly according to whether the cell lines were euploid (diploid or tetraploid) or aneuploid (hyperdiploid, triploid, hypertriploid, or polymorphic). In fact, the cell colonies from the euploid cell nuclei populations were larger and more dense than those from the aneuploid ones. Furthermore, for an identical period of culture, the cell lines from high‐grade malignant astrocytic tumors (glioblastomas) exhibited cell colonies that were larger and more dense than those in cell lines from low‐grade astrocytic tumors (astrocytomas). In each of these two groups, the diploid cell nuclei populations exhibited cell colonies larger and more dense than the nondiploid colonies. The present methodology is now being applied in vivo to histological sections of surgically removed human brain tumors in order to distinguish between high‐risk clinical subgroups and medium‐risk subgroups in clearly circumscribed histopathological groups. © 1995 Wiley‐Liss, Inc.
Flow cytometric detection of glutathione S‐transferase isoenzymes by quantitative immunofluorescence under nonsaturating conditions
Authors: Pifferi Antonio , Filippini Cristian , Capolongo Laura , Codegoni Annamaria , Balconi Giovanna , Ubezio Paolo , Tagliabue Giovanna
Published: June 2005

The glutathione (GSH)‐glutathione S‐transferase (GST) detoxification system is an important element in cellular defence against injurious agents and anticancer drugs. GST isoenzymes may represent biochemical markers of neoplastic transformation, and, possibly, drug resistance is associated with altered GST‐isoenzyme levels. The ability to measure GST‐isoenzymes in cell populations would be useful for several biological and clinical applications. We have developed an immunofluorescence flow cytometric method for the simultaneous detection of different GST‐isoenzymes and of DNA in fixed cells. Due to the impossibility of working under saturating conditions for the anti‐GST antibody, a normalizing procedure was developed to permit quantitative analysis of single cells labelled with the anti‐GST antibody at high dilution. A theoretical model and experimental data supported the use of this procedure. The method proposed is general and could be applied to other antibodies in order to obtain quantitative data outside saturating conditions. The method was challenged in different applications in order to compare it with other classical techniques. First, we characterized sublines resistant to different anticancer drugs with respect to variations of GST isotypes. In a second application, we studied the intercellular heterogeneity of GST content in mouse renal cells. In addition, GST was determined in aneuploid cells from solid tumor biopsies by separation from diploid cells on the basis of DNA content. Finally, GST distribution during cell‐cycle progression was studied in two different cell lines by the biparametric analysis of GST/DNA. © 1995 Wiley‐Liss, Inc.