bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flow cytometric analysis of DNA replication during the differentiation of 3T3‐L1 preadipocytes
Authors: Gratzner Howard G. , Ahmad Patricia M. , Stein Jeanne , Ahmad Fazal 
Published: August 2005

AbstractWe have employed a flow cytometric method of monitoring DNA synthesis in order to study the stimulation of DNA replication during the induction of adipocyte differentiation in the preadipocyte cell line 3T3‐L1. When confluent monolayers of this cell line are treated with a mixture of methyl isobutyl xanthine, dexamethasone, and insulin (MDI), they undergo at least one round of cell division, which is followed by the de novo synthesis of many enzymes that are involved in the formation of lipid. If the MDI‐treated cells are incubated in iododeoxyuridine (IdUrd)‐or bromodeoxyuridine (BrdUrd)‐containing medium and subsequently stained with antibodies specific for these base analogues and with propidium iodide (PI), the kinetics of the induction of replication can be monitered. No differences in the patterns of IdUrd incorporation versus PI staining were observed between exponentially growing 3T3‐L1 cells and those that had been stimulated to replicate DNA with MDI. In addition, we developed a flow cytometric (FCM) method for staining fatty acid synthetase and localizing the antigen in the G1 phase. We have thus demonstrated the feasibility of this methodology for correlating by FCM the production of enzymes such as fatty acid synthetase with IdUrd and BrUrd incorporation. The technique should permit studies of the inhibition of differentiation of adipocytes by halogenated pyrimidines.
DNA Replication and differentiation in rat myoblasts studied with monoclonal antibodies against 5‐bromodeoxyuridine, actin, and α2‐macroglobulin
Authors: Kaufman Stephen J. , Robert‐Nicoud Michel 
Published: August 2005

AbstractDuring the differentiation of skeletal muscle, mononucleate myoblasts proliferate, then stop replicating, spontaneously fuse, and express a large number of genes which encode the muscle phenotype. We have used monoclonal antibodies specific for 5‐bromodeoxy‐uridine, myoactin, and equine α2‐macroglobulin to follow and establish the sequence of events that surround the transition from a replicating to a differentiating population. Triple‐label immunofluorescence microscopy was used to visualize the changes in DNA synthesis, formation of myoactin fibers, and the cessation of endocytosis of α2‐macroglobulin that accompany myogenesis. Our results indicate that myoblasts cease actively endocytosing α2‐macroglobulin after stopping DNA synthesis but prior to fusion. Formation of myoactin fibers rarely occurs in mononucleate myoblasts and only in post‐mitotic cells, but they are common in multinucleate myotubes. We suggest that the regulation of DNA synthesis is critical to normal myogenesis and that detection of incorporated BrdUrd by immunofluorescence, in conjunction with other antibodies and nucleic acid probes, is a convenient method with which to study and sequence the molecular events in single cells as they relate to the transition in DNA synthesis that accompanies differentiation.
Cytokinetic properties of asynchronous and cytosine arabinoside perturbed murine tumors measured by simultaneous bromodeoxyuridine/DNA analyses
Authors: Pallavicini M. G. , Summers L. J. , Dolbeare F. D. , Gray J. W. 
Published: August 2005

AbstractThis paper describes the determination of cytokinetic properties of asynchronous and cytosine arabinoside‐(Ara‐C) treated KHT tumors growing in vivo using the bromodeoxyuridine (BrdUrd)/DNA analysis technique. The cytokinetic properties of asynchronously growing tumors were estimated by computer analysis of sequential BrdUrd/DNA distributions measured at 2‐ to 3‐h intervals after administration of a single i.p. injection of BrdUrd. The cytokinetic properties of the AraC‐treated tumors were estimated by computer analyysis of BrdUrd/DNA distribution sured at 2‐ to 3‐h intervals after Ara‐C treatment. BrdUrd was injected 30 min prior to tumor harvest. The cytokinetic properties of the cells rendered nonclonogenic by Ara‐C were followed in BrdUrd/DNA distributions measured at 2‐ to 3‐h intervals after Ara‐C treatment of tumors that were labeled with BrdUrd 30 min prior to Ara C injection. The Gl‐, S‐, and G2M‐phase durations were estimated to be 7.6, 10.9, and 2.0 h prior to Ara‐C; decreasing to 1.2, 4.1, and 1.4 after Ara‐C. The growth fraction was estimated to be 0.8 prior to Ara‐C. Complete recruitment of the normally noncycling subpopulation was observed after Ara‐C treatment. Ara‐C‐killed cells were removed from the tumor within 24 h following Ara‐C injection. These cytokinetic properties were similar to those reported in other studies.
The labelling index of human and mouse tumours assessed by bromodeoxyuridine staining in vitro and in vivo and flow cytometry
Authors: Wilson G. D. , McNally N. J. , Dunphy E. , Kärcher H. , Pfragner R. 
Published: August 2005

AbstractBromodeoxyuridine (BrdUrd) incorporation and flow cytometry were used to measure human tumour kinetic parameters in vitro and in vivo. The technique was validated by comparison of labelling index estimates of mouse tumours in vivo and in vitro using BrdUrd and flow cytometry Nvith tritiated thymidine (3HdThd) autoradiography. Similar labelling indices were obtained with both in vivo and in vitro incorporation into DNA of the two different precursors. Measurements of human tumour labelling indices were similar following in vitro incubation with either BrdUrd or 3HdThd.The use of BrdUrd allowed the visualization of a population of S‐phase cells that did not appear to incorporate BrdUrd or 3HdThd. The human tumour labelling indices obtained with BrdUrd incorporation were similar to previously reported values using autoradiography studies. Preliminary studies demonstrated that significant human tumour labelling could be achieved with an intravenous injection of 500 mg BrdUrd.
Automatic classification of chromosomes as part of a routine system for clinical analysis
Authors: Lundsteen Claes , Gerdes Tommy , Maahr Jan 
Published: August 2005

AbstractA procedure for automatic classification of G‐banded human chromosomes has been implemented on a semiautomated system for routine clinical analysis. Chromosomes represented by their density profiles are described by so‐called weighted density distribution (WDDs) by application of a number of weighting functions and classified by a parametric discriminant analysis. During 16 mo of routine use of the system, 2,794 metaphases (127,925 chromosomes) from amniotic fluid have been karyotyped by the system with an error rate of 8–9%. This corresponds to 4–5 errors per metaphase. These errors can immediately be corrected by the operator on a displayed karyogram with a light pen.
Effect of section thickness on quality of flow cytometric DNA content determinations in paraffin‐embedded tissues
Authors: Stephenson Robert A. , Gay Helen , Fair William R. , Melamed Myron R. 
Published: August 2005

AbstractDNA content determinations were carried out by flow cytometry on nuclear suspensions prepared from the same paraffin embedded tissue block for each of eight surgically resected human carcinomas at section thicknesses of 5,10,20,30,40,50, and 100 μm. Flow cytometric DNA determinations were also obtained on fresh tissue specimens in four of the eight carcinomas. As section thickness decreased below 50 μm, there was a progressive increase in the histogram baseline noise at low DNA values and a decrease in the relative peak height of aneuploid DNA. The former was attributed to an increase of nuclear fragments in thinner sections, and the latter to the greater probability of transection of the larger aneuploid cells within a specimen. Both artifacts were minimized at section thickness of 50 μm or greater.
Flow cytometric discrimination of mitotic cells: Resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde
Authors: Larsen Jo̊Gen Knud , Munch‐Petersen Birgitte , Christiansen Jette , Jo̊gensen Knud 
Published: August 2005

AbstractCells in mitosis can be flow cytometrically discriminated from G1, S, and G2 cells by analysis of a nuclear suspension prepared with nonionic detergent, fixed with formaldehyde, and stained with mithramycin, propidium iodide, or ethidium bromide. With these DNA‐fluorochromes, the fluorescence is quenched by formaldehyde less in mitotic nuclei than in interphase nuclei. Mitotic nuclei have a 20–40% increased mithramycin fluorescence and 30–60% decreased light scatter in comparison to those of G2 nuclei. There is a high correlation (r = 0.95; P < 0.001) between microscope counts of mitotic figures in smear preparations of the initial cell suspension and the flow cytometrically estimated fraction of nuclei with increased mithramycin flourescence. Flow sorting (FACS) demonstrates that the mitotic nuclei are confined to the peak of increased mithramycin fluorescence and decreased light scatter. The method has been applied to cultures of Yoshida ascites tumor cells, JB‐1 retculosarcoma cells, and PHA‐stimulated human lymphocytes, incubated in the presence or absence of vinblastine for mitotic arrest. In a heteroploid mixture of fixed Yoshida (near‐diploid) and JB‐1 (hypotetraploid) nuclei, the mitotic fractions of the two cell lines could be estimated separately when analyzed with mithramycin flourescence versus light scatter or with mithramycin fluorscecence versus propidium iodide fluorescence.
Flow cytometric and radioisotopic determanations of platelet survival time in normal cats and feline leukemia virus‐infected cats
Authors: Jacobs R. M. , Boyce J. T. , Kociba G. J. 
Published: August 2005

AbstractThis study demonstrates the potential usefulness of a flow cytometric technique to measure platelet survival time in cats utilizing autologous platelets labeled in vitro with fluorescein isothiocyanate (FITC). When compared with a 51Cr method, no significant differences in estimated survival times were found. Both the 51Cr and FITC‐labeling procedures induced similar changes in platelet shape and collagen‐induced aggregation. Platelets labeled with FITC had significantly greater volumes compared with those of glutaraldehyde‐fixed platelets. These changes were primarily related to the platelet centrifugation and washing procedures rather than the labels themselves. This novel technique potentially has wide applicability to cell circulation time studies as flow cytometry equipment becomes more readily available.In a preliminary study of the platelet survival time in feline leukemia virus (FeLV)‐infected cats, two of three cats had significantly reduced survival times using both flow cytometric and radioisotopic methods. These data suggest increased platelet turnover in FeLV‐infected cats.
Cell size, DNA, and cytokeratin analysis of human head and neck tumors by flow cytometry
Authors: Bijman J. Th. , Wagener D. J. Th. , Wessels J. M. C. , van den Broek P. , Ramaekers F. C. S. 
Published: August 2005

AbstractCell subsets have been discriminated in cell suspensions derived from 37 human head and neck tumors by means of light scatter, DNA, and cytokeratin flow cytometry (FCM). Cell dispersion was performed overnight at 4°C in two different enzyme mixtures, i.e., trypsin/dithioerythritol and collagenase/DNase, under slight agitation of sliced tumor tissue. Cells were examined before and after fractionation on a discontinuous low‐density bovine serum albumin (BSA) gradient.Forward and right‐angle light scatter FCM of 23 tumor specimens revealed four main subpopulations with different size and structure. Fractionation of primary cell suspensions on a BSA gradient at unit gravity separated debris, small cells and large cells. DNA FCM of the enriched populations demonstrated a relation between large cells and DNA aneuploidy. Epithelial cells, as recognized by cytokeratin antibodies, were also related with large cells.The results demonstrated the usefulness of light scatter, DNA, and cytokeratin analysis of crude and fractionated tumor cell suspensions for assessment of the efficacy of a particular dispersion technique and to obtain information of the cell subsets dispersed.
A pulse generator simulating slit‐scan chromosome analysis signals
Authors: Weier H.‐U. L. , Eisert W. G. 
Published: August 2005

AbstractA simple circuit is described for generating a variety of electronic pulses to test hardware and software for slit‐scan chromosome analysis in a flow cytometer. The pulse shape can be changed to have different numbers of local minima, thereby simulating fluorescence pulses from acrocentric, monocentric, and dicentric chromosomes. Long pulses simulate aggregates of chromosomes. The pulse repetition rate as well as the pulse amplitude is variable. Although the circuitry is built with only three integrated circuits, the pulse‐to‐pulse variation in shape and height is quite small. After digitization of the analog signals, the constructed histograms of pulse integrals show a relative coefficient of variation below 1%. This signal generator provides a valuable tool for a number of electronic test applications that would otherwise require expensive standard particles analyzed in a well‐tuned flow cytometer.
A protocol for papanicolaou staining of cytologic specimens following flow analysis
Authors: Berkan Todd K. , Reeder Jay E. , Lopez Peter A. , Gorman Kevin M. , Wheeless Leon L. 
Published: August 2005

AbstractA protocol has been developed for restaining cytologic specimens that have been analyzed on a multidimensional slit‐scan flow system. The technique involves Papanicolou staining of cells on a membrane filter that has been previously stained with acridine orange and fixed with glutaraldehyde buffer. The specimen and staining solutions were sequentially added to a 5‐micrometer pore size, 47‐mm diameter Gelman “Metricel” filter while it remained in a glass filtration apparatus.The practice of retaining the filter in the filtration apparatus throughout the staining procedure minimizes cell loss and eliminates specimen cross contamination when compared with conventional filter dip staining. The availability of this postflow specimen Papanicolaou staining protocol permits accurate determination of the performance characteristics of a multidimensional slit‐scan flow system and should be useful whenever staining of a limited number of cells with minimal cell loss is desired.
Griffonia simplicifolia I isolectin as a functionally monovalent probe for use in flow cytometry
Authors: McCoy J. Philip , Shibuya Naoto , Riedy Mary C. , Goldstein Irwin J. 
Published: August 2005

AbstractThe inherent tendency of lectins to agglutinate cells has limited their use as reagents for the detection of carbohydrate groups on cell surfaces by flow cytometry. In the current study, we demonstrate a method for theuse of a fluoresceinated tetrameric isolectin (Griffonia simplicifolia I‐A3B, FITC‐GS I‐A3B) as a functionally monovalent, nonagglutinating probe in flow cytometry. This isolectin contains three A subunits and one B subunit. Both types of subunits bind α‐D‐galactopyranosyl (α‐D‐galp‐) end groups with similar affinities; however, the A subunits have a 1,000‐fold greater affinity for N‐Acetyl‐D‐galactosamine (GalNAc) than does the B subunit. The addition of low (1–2 mM) concentrations of GalNAc to the FITC‐GS I‐A3B isolectin results in blockage of the three A subunits without significantly affecting the B subunit; this yields a functionally monovalent probe for the detection of cell surface α‐D‐Galp end groups. This approach has been used to examine two types of cells: Ehrlich ascites tumor cells and rat alveolar macrophages, both of which are known to express cell surface α‐D‐Galp end groups. Lectin binding, as determined by number of positive cells and fluorescence intensity, was dependent upon concentration of the lectin and haptenic sugar. Specificity of the staining was demonstrated by the ability of methyl α‐D‐galactopyranoside (Met α‐D‐Galp) to abolish the binding of the lectin to the cells. Elimination of both GalNAc and Met α‐D‐Galp from the staining solution resulted in agglutination of the cells, indicating that the A subunits were active in the absence of GalNAc.
Isolation of viable type II alveolar epithelial cells by flow cytometry
Authors: Wilson Julie S. , Steinkamp John A. , Lehnert Bruce E. 
Published: August 2005

AbstractWe developed a new method for isolating viable type II cells from fractionated and unfractionated lung cell suspensions by flow cytometry using acridine orange (AO). Fischer‐344 rat lungs were dispersed into single‐cell suspensions by a technique that yields a high number of cells (4–5 × 108 cells/lung, =85% viable), =11% of which are type II cells. Elutriated fractions from the lung cell preparation and parent, unfractionated cell suspensions were incubated with 1.0–0.02 μg/ml AO and analyzed by flow cytometry. Parameters analyzed included axial light loss (ALL) and red fluorescence (RF). Based on their unique RF, attributable to AO staining of type II cell lamellar bodies, and their ALL characteristics, type II pneumocytes were sorted from elutriated fractions to greater than 95% purity. Using the same approach, type II pneumocytes were sorted from unfractionated lung cell suspensions at ≤85% purity. The viabilities of the type II alveolar epithelial cells isolated by this method range from 85% to 95%, and the ultrastructural features of the sorted cells were unaltered by AO labeling or sorting.
Differentiation of HL‐60 promyelocytic leukemia cells: Simultaneous determination of phagocytic activity and cell cycle distribution by flow cytometry
Authors: Blair Owen C. , Carbone Rocco , Sartorelli Alan C. 
Published: August 2005

AbstractPhagocytosis of fluorescent microspheres by HL‐60 promyelocytic leukemia cells following induction of differentiation with dimethyl sulfoxide (DMSO) was monitored using flow cytometry. Initiation of phagocytic capability following initiation of differentiation with 1.5% DMSO coincided with the attainment of respiratory burst activity as measured by NBT (nitro blue tetrazolium) reduction; the degree of phagocytic activity was dependent upon parameters such as microsphere size, microsphere number, and exposure time. Ingestion of fluorescent microspheres did not interfere with the measurement of DNA content using propidium iodide; thus, simultaneous determination of phagocytic activity and the cell cycle phase was possible. Accumulation of cells in the G1/G0 phase of the cell cycle following DMSO treatment was correlated with the acquisition of the capacity to phagocytize. Analysis of two‐parameter correlated data also indicated that phagocytosis is coupled with residence in the G1/G0 phase of the cell cycle, further suggesting that the ability to phagocytize fluorescent microspheres is associated with end‐stage differentiation.
Sequential monitoring of peripheral blood lymphocyte subsets in rats
Authors: Thompson Sandy C. , Bowen Kerry M. , Burton Robert C. 
Published: August 2005

AbstractThe methodology of enumeration of T cell subsets in humans is well established and has widespread clinical application. Sequential monitoring of lymphocyte subsets in small animals requires a consistent and reliable methodology for staining and enumerating lymphocyte populations. The optimal conditions for enumerating subclasses of rat peripheral blood lymphocytes with the Spectrum III flow cytometer are described. The sources of biological variation and technical error that were considered in optimizing the techniques and the limitations in applying them are discussed.
Multidimensional slit‐scan detection of bladder cancer preliminary clinical results
Authors: Wheeless L. L. , Berkan T. K. , Patten S. F. , Reeder J. E. , Robinson R. D. , Eldidi M. M. , Hulbert W. C. , Frank I. N. 
Published: August 2005

AbstractA multidimensional slit‐scan flow system was developed for the automated recognition of abnormal cells derived from cancer of the uterine cervix and its precursors. It provides great sensitivity in both its ability to recognize cellular abnormality and to deal with the myriad potential causes of false alarms in an automated flow system. While its initial application was the automated recognition of the spectrum of neoplasia in gynecologic cytology samples, a preliminary study was carried out using specimens obtained from the urinary bladder. Cellular material was collected by bladder irrigation and stained with the fluorochrome acridine orange. One hundred fifty‐three bladder irrigation specimens, including 115 abnormal specimens containing cells derived from neoplastic lesions of the bladder epithelium, were analyzed. For the purposes of this study, abnormal specimens from the urinary bladder included specimens containing cells derived from the following lesions of the urothelium: dysplasia (atypical hyperplasia), carcinoma‐in‐situ, and transitional cell carcinoma, grades 1–3. Approximately 50,000 cells were analyzed for most specimens. Of the 38 presumed normal specimens (specimens containing only normal urothelial components), four were instrument classified abnormal. For the 69 specimens containing cells derived from transitional cell carcinoma, grade 1, 1–2, 2, 66 were correctly classified as abnormal while three were classified as normal. There were no false negatives in the 17 specimens containing cells derived from transitional cell carcinoma, grade 2–3, 3, or in the two specimens containing cells derived from carcinoma‐in‐situ. Of the eight specimens containing cells derived from dysplasia (atypical hyperplasia), seven were instrument classified abnormal. This system has demonstrated utility in the study and recognition of the cellular components of urothelial cancer and its precursors present in the urinary bladder.
Flow cytometers using optical waveguides in place of lenses for specimen illumination and light collection
Authors: Shapiro Howard M. , Hercher Michael 
Published: August 2005

AbstractOptical waveguides represent alternatives to lenses for delivery of light to, and collection of light from, small regions of space such as the observation point(s) of a flow cytometer. The flow chamber and directly associated illumination and collection optics of a flow cytometer can be built using fiber optic waveguides in fixed positions or as an integrated optical assembly incorporating deposited or implanted waveguides, providing illumination and collection geometries and efficiencies comparable to conventional designs, with the advantage that the optical waveguide design, once assembled, never again requires alignment of the optics. A prototype apparatus in which fiber optics were used to illuminate and collect light from a point inside a thin‐walled, round quartz capillary was observed to measure fluorescence of polystyrene spheres with precision close to that obtained from a conventional flow cytometer using the same laser source. The design of the fiber optic system is readily adaptable to construction of multiple illumination beam instruments; other optical waveguide flow cytometer designs that dispense with the capillary share this advantage and can also provide orifices for electronic volume sensing and for jetting prior to droplet sorting.