bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flow cytometric study of differentiating cultures of Bacillus subtilis
Authors: Chung John D. , Conner Stewart , Stephanopoulos Gregory 
Published: June 2005

AbstractWe report on (1) the development of aflow cytometry‐based technique for detecting β‐galactosidase in differentiating cultures of Bacillus subtilis and (2) the application of this technique in the study of early developmental gene expression. The problems associated with generating detectable signals (despite the small size of B. subtilis cells) have been overcome using the fluorogenic substrate 5‐octanolyaminofluorescein di‐β‐D‐galactopyranoside (C8‐FDG). Additionally, to control for background fluorescence during the staining process, we included a control population in the C8‐FDG staning mixture that consists of cells devoid of the lacZ gene prestained with another dye, PKH26. The distinct emission spectra of C8‐fluorescein and PKH26 allow nonspecific C8‐FDG staining in this control population to be monitored using two‐color analysis. This technique has been applied in the study of developmental gene expression in sporulating cultures of B. subtilis, and it has been found that such cultures are heterogeneous, comprising two cell populations. One population is induced for expression of early sporulationg genes, which is determined using lacZ fusions, whereas the other remains uninduced. These results have allowed us to understand better the patterns of gene expression exhibited by wild‐type and mutant cultures early during the development process of spore formation. © 1995 Wiley‐Liss, Inc.
Flow cytometric determination of aminopeptidase activities in viable cells using fluorogenic rhodamine 110 substrates
Authors: Ganesh S. , Klingel S. , Kahle H. , Valet G. 
Published: June 2005

AbstractAminopeptidases (AP) are ubiquitously occuring, nonspecific exopeptidases involved in protein degradation. They cleave the N‐terminal amino acid of peptides and occur in practically all mammalian cells and tissues. Physiological and pathological processes such as metastasis of tumors and inflammation have been thought to involve changes in AP activities. Determination of AP (EC 3.4.11.X) activity in viable cells by flow cytometry was the subject of this study because of its general biological and clinical interest. Different bis‐substituted rhodamine 110 (R110) peptide derivatives were synthesised and used as AP‐ and exopeptidase (EC 3.4.13.X–EC 3.4.14.X) substrates for flow cytometric measurements. Intracellular AP activities in viable lympho‐, mono‐, granulo‐, and thrombocytes were detected by fluorescence increase from R110 following intracellular substrate cleavage. Eukaryotic‐AP do not cleave D‐aminoacids and hence NH2(D‐Leu)2R110 substrate served as negative control. Specific substrate cleavage by AP is shown by complete inhibition of fluorescence generation following preincubation of cells with leucinechloromethylketone inhibitor. R110 AP‐ and exopeptidase substrates are suitable indicators for coupled endopeptidase reactions due to their rapid cleavage and largely pH independent generation of intracellular fluorescence. © 1995 Wiley‐Liss, Inc.
Evaluation of red blood cell lysing solutions for the detection of intracellular antigens by flow cytometry
Authors: Tiirikainen Maarit I. 
Published: June 2005

AbstractWhen analyzing leukocyte cell surface antigens by flow cytometry, leukocytes are usually first labeled in whole blood and the red blood cells are finally lysed with lysing solutions. The erythrocytes are lysed, but the leukocytes are expected to remain intact. Six commercial red blood cell lysing methods were investigated for possible leukocyte permeabilization effect. The effectiveness of permeabilization was studied by propidium iodide staining, and the detectability of intracellular antigens was studied by using monoclonal antibodies toward two model antigens. Most of the lysing methods caused permeabilization of at least part of the leukocytes, but only one method, already found in our previous studies, was applicable for complete permeabilization of leukocytes and for detection of intracellular antigens alone or simultaneously with the cell surface antigens. © 1995 Wiley‐Liss, Inc.
Evaluation of T‐lymphocyte subsets present in semen and peripheral blood of healthy donors: A report from the heterosexual transmission study
Authors: Denny Thomas N. , Scolpino Anthony , Garcia Ambrosia , Polyak Andrew , Weiss Stanley N. , Skurnick Joan H. , Passannante Marian R. , Colon Jose 
Published: June 2005

AbstractThe purpose of this study was to accurately determine the T‐lymphocyte subsets found in semen from health volunteers, to evaluate the impact of repeated ejaculation on the frequency or type of immune cells present in semen, and to compare subset analysis in semen to that in the peripheral blood. To accomplish this, a flow cytometric method was developed to identify and count immunophenotypically distinct cells present in semen. Fresh semen samples and peripheral blood were collected over three consecutive days from nine healthy donors. Donors had normal ejaculate volume, sperm count, sperm motility, morphology, and leukocyte count. No significant intra‐donor differences were seen in these parameters over time. No significant differences were observed in the percentage of CD3+ cells, CD4+ cells, CD8+ cells, and the CD4:CD8 ratio in semen on consecutive days. However, within the CD4+ subset, when naive and memory CD4+ cells were measured, some day to day variability was suggested. No significant differences in CD3+, CD4+, CD8+, CD4/CD8 ratio, or naive and memory subsets were seen in the peripheral blood between sampling days. When semen was compared to peripheral blood some differences in immune subset values were observed, with an increase in the percentage of memory CD4+ cells in semen being the most striking. This finding may be relevant to HIV transmission, since others have shown that this cell may be preferentially infected with HIV and is the primary reservoir for virus in infected individuals. © 1995 Wiley‐Liss, Inc.
Autofluorescence correction for fluorescence in situ hybridization
Authors: Szöllösi János , Lockett Stephen J. , Balázs Margit , Waldman Frederic M. 
Published: June 2005

AbstractOptimal sensitivity of fluorescence in situ hybridization (FISH) requires bright signals and low background fluorescence. Use of locus‐specific probes is especially dependent on high sensitivity. Some tissue preparations show high autofluorescence, masking small or dim signals. We have developed a new method for subtracting autofluorescence from digital images on a pixel‐by‐pixel basis. It is based on the observation that fluorescent labels for FISH have narrower excitation and emission spectra than the chemical components responsible for autofluorescence. Our new approach uses calculation of the ratio of autofluorescence between multiple color images for correction of autofluorescence in each individual image. By subtracting autofluorescence components, we were able to enhance centromeric signals and make previously indistinguishable cosmid signals clearly visible. This image‐processing approach to autofluorescence correction may widen the applicability of gene‐specific probes in FISH analysis of tumor material. © 1995 Wiley‐Liss, Inc.
Genetic and phenotypic heterogeneity of human malignancies: Finding order in chaos
Authors: Shackney Stanley E. , Shankey T. Vincent 
Published: June 2005

AbstractThe presence of cellular heterogeneity within human tumors has been recognized for many years. Current concepts regarding the clonal origin of human neoplasms, and recent advances in the study of successive genetic changes that occur during tumor evolution may now make it possible to understand in greater depth the biological and clinical implications of intra‐tumor heterogeneity at both the phenotypic and genotypic levels. In order to explore these concepts further, and to better identify the potential contributions that flow and image cytometry can make to our understanding of tumor heterogeneity, a session of the 1994 ISAC Congress was dedicated to plenary presentations on human can cell heterogeneity. Here, we provide a brief overview of the genetic evolutionary progression of human cancers, some considerations of clinically important phenotypic and genotypic markers, and an outline that might serve as a basis for framing relevant issues that are ammenable to further study.

All Nature is but Art, unknown to thee;
All Chance, Direction, which thou canst not see;
All Discord, Harmony not understood:
All partial Evil, universal Good.
(Alexander Pope, Essay on Man, end of Epistle 1) © 1995 Wiley‐Liss, Inc.
Geographic distribution of co‐dominant DNA stemlines in breast carcinoma
Authors: Visscher D. , Dolan P. , Ottosen S. , Crissman J. 
Published: June 2005

AbstractBreast carcinomas often contain multiple DNA stemlines in flow cytometric DNA histograms. However, due to mixing during tissue disaggregation the microanatomical relationship between the cells which comprise distinct stemlines is unclear. We performed image cytophotometric DNA analysis (IA) on two separate areas of intact tissue sections of 19 breast carcinomas which were selected on the basis of flow cytometric (FCM) DNA content heterogeneity (i.e., multiple stemlines). For comparison, similar analyses were performed on seven tumors with unimodal FCM DNA histograms. Six of the 7 tumors (86%) with unimodal FCM histograms were also unimodal in both IA DNA histograms. Among tumor with heterogeneous FCM DNA histograms, the presence of multiple stemlines was confirmed in IA DNA histograms in 16/19. In nine of these 16 cases, multiple DNA stemlines having similar DNA indices were present in both areas of neoplasm examined with IA. The remaining seven cases displayed unimodal IA histograms in both areas, however DNA indices differed between the two histograms. These findings imply that cell populations corresponding to flow cytometrically detected DNA stemlines are often intimately admixed, even within geographically separated portions of breast tumors. This pattern suggests that productive interactions between genetically distinct tumor populations may lead to stable co‐dominance of ancestral clones during progression of some breast carcinomas.
Assessing sequential oncogene amplification in human breast cancer
Authors: Janocko Laura E. , Lucke Joseph F. , Groft David W. , Brown Kathryn A. , Smith Charles A. , Pollice Agnese A. , Singh Sarita G. , Yakulis Robert , Hartsock Robert J. , Shackney Stanley E. 
Published: June 2005

AbstractStudies of amplification and/or overexpression of c‐myc, HER‐2/neu, and H‐ras in breast cancer have shown that each is associated with a poor prognosis. The purpose of this study was to explore the possibility that there is a preferred sequence of amplification of these oncogenes in breast cancer. The frequencies of amplification and patterns of co‐amplification of c‐myc, HER‐2/neu, and H‐ras were studied in a group of 84 breast cancers. The data suggested a preferred sequence of amplification that consisted of c‐myc amplification‐HER‐2/neu amplification‐H‐ras amplification. This model was supported by loglinear analysis. In addition, the levels of amplification of JC‐A, a DNA fragment newly isolated from a patient with advanced breast cancer, were studied in 61 of these cases. The data suggested that JC‐A amplification occurred early. Loglinear analysis supported a model in which JC‐A amplification occurred either before or after c‐myc amplification but was unrelated to Her‐2/neu or ras amplification. © 1995 Wiley‐Liss, Inc.
DNA Ploidy and proliferation heterogeneity in human prostate cancers
Authors: Shankey T. Vincent , Jin Jia‐Kuan , Dougherty Scott , Flanigan Robert C. , Graham Sharon , Pyle Joseph M. , Graham Sharon 
Published: June 2005

AbstractDNA ploidy determinations have been shown to have clinical application in predicting disease progression, survival, or response to anti‐androgen therapies in prostate carcinomas. Since intra‐tumor heterogeneity may have a profound effect on DNA measurements, we determined the frequency of DNA ploidy and proliferation (here S‐phase fraction) heterogeneity in early prostatic carcinomas, and estimated the potential impact of heterogeneity on predicting disease course, survival, or response to therapy.Using image and flow cytometric analysis of archival, paraffin‐embedded prostate tumors, we measured DNA ploidy in individual foci of prostatic carcinoma in stage T1a, T1b and T1c disease. Image analysis studies included the use of Feulgen stained tissue sections, and a comparison of these results with flow cytometric DNA ploidy determinations on nuclei isolated from the same tumor foci. Flow cytometry was also used to measure DNA Index and tumor S‐phase fraction, in some cases using multiparameter analysis of isolated nuclei to determine DNA content and the level of the proliferation‐associated antigen, p105.Our results indicate that DNA aneuploid foci of prostate carcinoma are infrequently seen in stage T1a disease (13% of the individuals studied), and that the presence of both DNA diplold and aneuploid foci in the same sample is seen in less than 10% of these individuals. Stage T1b and T1c tumors containing only DNA diploid nuclei are seen, though these are likely most common in low volume, low Gleason grade tumors. By using flow cytometry to compare these results with those using image analysis of the same tumor foci, we demonstrated that the majority (>75%) of these aneuploid tumors are DNA tetraploid. Our data on prostate tumor S‐phase fractions indicate that DNA diploid tumors generally have a lower S‐phase than DNA aneupoloid foci (including comparisons of DNA diploid and aneuploid foci in the same prostate tumor). These results support the model that early prostate tumors are DNA diploid and have a low S‐phase, and that these tumors likely evolve to DNA tetraploid tumors with a similar low S‐phase fraction. © 1995 Wiley‐Liss, Inc.
Heterogeneity of anthracycline retention and response to efflux blockers in human tumors
Authors: Krishan Awtar 
Published: June 2005

AbstractRapid cellular efflux of certain natural products used in cancer chemotherapy leads to reduced cytotoxicity and resistance. Multiple Drug Resistance (MDR) gene related glycoproteins are believed to act as the drug efflux pump. Several non‐cancer therapy drugs (e.g., verapamil, phenothiazines) compete for the p‐glycoprotein pump and thus can block efflux of a chemotherapeutic agent and overcome cellular resistance. Anthracyclines, such as adriamycin, are intrinsically fluorescent and thus their cellular retention can be determined by laser flow cytometry. This method has been used to study heterogeneity in cellular retention of anthracyclines and response to efflux blockers in human tumor cells. These studies have led to generation of clinical protocols where laser flow cytometry is used to monitor and overcome heterogeneity in drug retention of tumor cells from patients treated with a combination of adriamycin and efflux blockers. © 1995 Wiley‐Liss, Inc.
Detection of chromosome aneuploidy in breast lesions with fluorescence in situ hybridization: Comparison of whole nuclei to thin tissue sections and correlation with flow cytometric DNA analysis
Authors: Visscher Daniel W. , Wallis Tracie , Ritchie Carole A. 
Published: June 2005

AbstractWe compared flow‐cytometric DNA histogram pattern to counts of 4 fluorescent‐labelled centromeric probes (chromosomes 1, 7, 8, and 17) in whole nuclei (WN) and in nuclei from the corresponding formalin‐fixed deparaffinized thin tissue section (TS) in 25 breast lesions (9 invasive cardqomas, I duct carcinoma‐in‐situ, 5 fibroadenomas, 10 fibrocystic change). In benign lesions, signal gains (i.e., trisomic nuclei) were never observed in greater than 10% of nuclei from either WN or TS preparations. Loss of signal in benign breast lesions, however, varied considerably (0–43%) between individual case and between chromosome probes. The mean incidence of signal loss in WN of benign lesions ranged from 8.9% (chromosome 7) to 14.4 % (chromosome 1) of nuclei. These signal loss frequencies exceeded those of benign lymphoid control cells. In three benign lesions, signal loss in WN (with one probe) was observed in at least 25% of nuclei. Signal losses in benign TS, on average, were 50–150% greater than in matched WN preparations (chromosome 1–21.7%, chromosome 7–21.5%). Malignant lesions generally, but not always, displayed fewer monosomic nuclei and more trisomic nuclei in WN compared to TS, compatible with a slicing (i.e., nuclear truncation) artifact. Signal counts in carcinomas correlated well with flow cytometrnic DNA index; however, they were also characterized by evidence of genetic instability, manifest as signal gains in a subset of nuclei (10–25%) with individual probes in diploid range cases, as well as intratumoral heterogeneity, reflected as discrepancies in probe counts between WN and TS samples.We conclude that signal losses with centromeric probes are largely, but not entirely, explained by nuclear slicing. The minimum signal loss threshold for establishment of monosomy using interphase cytogenetics is thus unclear, even in WN. Signal gains indicative of trisomy, in contrast, are reliably associated with malignancy and may reflect gross DNA aneuploidy as well as genetic instability.
Near‐IR dyes in three‐color volumetric capillary cytometry: Cell analysis with 633‐ and 785‐nm laser excitation
Authors: Lee Linda G. , Woo Sam L. , Head David F. , Dubrow Robert S. , Baer Thomas M. 
Published: June 2005

AbstractSeveral fluorescent dyes that absorb in the nearinfrared are described. The photostability and aggregation properties of the dyes were examined. Two of the dyes, BHMP and BHDMAP, emit at 805 nm and were useful dyes for protein labeling. A dual‐laser, three‐color scanning instrument was constructed. CD3 + CD4 + and CD3 + CD8 + populations were enumerated in undiluted, whole blood based on the fluorescence of Cys5, Cy5.5 and BHMP. © 1995 Wiley‐Liss, Inc.
Spatial dependence of the optical collection efficiency in flow cytometry
Authors: Goodwin Peter M. , Ambrose W. Patrick , Martin John C. , Keller Richard A. 
Published: June 2005

AbstractThe sensitive flow cytometric detection of fluorescent species in liquid sample streams requires efficient collection of light from small [∼1] picoliter (p1) sample volumes. This is often accomplished with high numerical aperture (NA) imaging collection optics used in combination with a spatial filter. A method to measure the spatial variation of the optical collection efficiency within the sample volume, using a submicrometer light source, is described. Measurements of the relative optical collection efficiency are presented for two optical collection systems used in our laboratory for single molecule detection. The measurements are in qualitative agreement with relative optical collection efficiency calculations using a simple geometrical optics model. Absolute measurements of the peak collection efficiencies for the two collection systems are also presented. These absolute collection efficiency measurements are in good quantitative agreement with ideal collection efficiencies calculated using measured transmissions and rated NAs of the collection optics. The utility of this information for the characterization and optimization of sensitive fluorescence detection apparatus is discussed. © 1995 Wiley‐Liss, Inc.
Cell membrane labeling with fluorescent dyes for the demonstration of cytokine‐induced fusion between monocytes and tumor cells
Authors: Spötl Ludwig , Sarti Alessandra , Dierich Manfred P. , Möst Johannes 
Published: June 2005

AbstractFusion between monocytes and tumor cells has been suggested as a cause for tumor metastasis. The aim of the present study was to establish an in vitro fusion model representing the in vivo situation as close as possible. For this purpose fusion between cells was induced by cytokine containing conditioned medium. In order to prove that hybrid formation between tumor cells and monocytes occurs, a two‐color‐fusion‐assay based on membrane labeling with the fluorochromes PKH 2 (green) and PKH 26 (red) was established. These fusion experiments were analyzed by microscopy and, in addition, by flow cytometry. The attempt to induce fusion between monocytes and several tumor cell lines of hematopoietic origin revealed quite diverse results. The most extensive hybrid formations were seen with TALL, a T‐lymphocytic tumor line. The monocytic tumor line HL60 and the B‐lymphocytic tumor line BL41 also clearly yielded hybrids with monocytes but in smaller numbers. With some other hematopoietic tumor lines no evidence for hybrid formation was detected. These studies indicate that fusion of normal monocytes with certain tumor cells may be induced under conditions that may occur in comparable manner in vivo. © 1995 Wiley‐Liss, Inc.
Heterogeneous calcium flux in peripheral T cell subsets revealed by five‐color flow cytometry using log‐ratio circuitry
Authors: Roederer M. , Bigos M. , Nozaki T. , Stovel R. T. , Parks D. R. , Herzenberg L. A. 
Published: June 2005

AbstractCalcium flux measurements of different subpopulations of cells by flow cytometry are important in understanding complex interactions in the immune system. This paper discusses the use of the difference of Log signals as a preferred method for obtaining this information simultaneously with other immunofluorescence parameters. We describe simple modifications to a commercial instrument that enables the measurement of calcium flux in addition to three immunofluorescence paramein. Finally, we show an application of this technique to measuring calcium flux of T cell subsets in human blood. We show that different subsets of peripheral CD4 T cells have significantly different capabilities to flux calcium after CD3 stimulation. These differences are related to the functional capacities of the cells within these subsets. © 1995 Wiley‐Liss, Inc.
Flow cytometric immunofluorescence and DNA analysis: Using a 1.5 mW helium‐neon laser (544 nm)
Authors: Hudson James C. , Porcelli Ronald T. , Russell Thomas R. 
Published: June 2005

AbstractWe evaluate a 1.5 mW HeNe laser (544 nm) for use on an EPICS Elite with a 76 μm Sortsense flow cell. The two applications chosen were immunofluorescence and DNA analysis. We measured the fluorescence threshhold of phycoerytherin calibration beads to be approximately 336 MESF. Cell analysis with a HeNe laser and Argon laser correlated well for the CD4PE, CD56PE, CD19PE conjugates, with correlation coefficients of 0.98, 0.99, 0.94, respectively. The % positive and mean channel fluorescence were comparable to the results obtained with a 15 mW Argon laser. In addition, a three‐color configuration yielded excellent results. Cell analysis of CD4PE, CD3ECD and CD19Cy‐Chrome with the HeNe laser and Argon laser correlated well with correlation coefficients of 0.96, 0.95, and 0.92, respectively. The histograms showed good separation between the negative cells, the dimly staining cells and the brightly staining cells. Propidium Iodide was chosen for DNA analysis. Full CV values for whole blood DNA fluorescence using the green laser were good at 2.6%. These data indicate the low power 544 nm laser is sufficient to do immunophenotyping and DNA analysis. Results may be explained by higher quantum efficiency and lower background fluorescence. The wavelength of the 544 nm laser is much closer to the excitation peaks of PI, PE, and the tandem dyes ECD and Cy‐Chrome. Also, the Raman scattering of water for the 544 nm laser has a longer wavelength maximum than the emission peaks of PI, PE, and ECD. The major advantages of this laser for the research laboratory are small size, no cooling fan, low power requiremements and low cost. © 1995 Wiley‐Liss, Inc.