scholarly journals Whole-mount BrdU staining of proliferating cells by DNase treatment: application to postnatal mammalian retina

BioTechniques ◽  
2006 ◽  
Vol 40 (1) ◽  
pp. 29-32 ◽  
Author(s):  
Andrei V. Tkatchenko
Keyword(s):  
Proliferating Cells ◽  
Dnase Treatment ◽  
Whole Mount ◽  
Mammalian Retina ◽  
Treatment Application ◽  
Brdu Staining

scholarly journals Ano1 as a regulator of proliferation

2011 ◽  
Vol 301 (6) ◽  
pp. G1044-G1051 ◽  
Author(s):  
Jennifer E. Stanich ◽  
Simon J. Gibbons ◽  
Seth T. Eisenman ◽  
Michael R. Bardsley ◽  
Jason R. Rock ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Slow Wave ◽  
Interstitial Cells Of Cajal ◽  
Primary Cultures ◽  
Channel Blockers ◽  
Proliferating Cells ◽  
Stromal Tumors ◽  
Whole Mount ◽  
Cells Of Cajal ◽  
Regular Medium

Ano1 is a recently discovered Ca2+-activated Cl− channel expressed on interstitial cells of Cajal (ICC) that has been implicated in slow-wave activity in the gut. However, Ano1 is expressed on all classes of ICC, even those that do not contribute to generation of the slow wave, suggesting that Ano1 may have an alternate function in these cells. Ano1 is also highly expressed in gastrointestinal stromal tumors. Mice lacking Ano1 had fewer proliferating ICC in whole mount preparations and in culture, raising the possibility that Ano1 is involved in proliferation. Cl− channel blockers decreased proliferation in cells expressing Ano1, including primary cultures of ICC and in the pancreatic cancer-derived cell line, CFPAC-1. Cl− channel blockers had a reduced effect on Ano1(−/−) cultures, confirming that the blockers are acting on Ano1. Ki67 immunoreactivity, 5-ethynyl-2′-deoxyuridine incorporation, and cell-cycle analysis of cells grown in low-Cl− media showed fewer proliferating cells than in cultures grown in regular medium. We confirmed that mice lacking Ano1 had less phosphorylated retinoblastoma protein compared with controls. These data led us to conclude that Ano1 regulates proliferation at the G1/S transition of the cell cycle and may play a role in tumorigenesis.

scholarly journals Genital Shedding of Human Immunodeficiency Virus Type-1 (HIV) When Antiretroviral Therapy Suppresses HIV Replication in the Plasma

2020 ◽  
Vol 222 (5) ◽  
pp. 777-786
Author(s):  
Marta Bull ◽  
Caroline Mitchell ◽  
Jaime Soria ◽  
Sheila Styrchak ◽  
Corey Williams ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genetic Distances ◽  
Hiv Replication ◽  
Proliferating Cells ◽  
Limit Of Quantification ◽  
Hiv Rna ◽  
Dnase Treatment ◽  
Infected Cells ◽  
Genital Shedding ◽  
And Control

Abstract Background During antiretroviral treatment (ART) with plasma HIV RNA below the limit of quantification, HIV RNA can be detected in genital or rectal secretions, termed discordant shedding (DS). We hypothesized that proliferating cells produce virions without HIV replication. Methods ART-naive Peruvians initiating ART were observed for DS over 2 years. HIV env and pol genomes were amplified from DS. Antiretrovirals and cytokines/chemokines concentrations were compared at DS and control time points. Results Eighty-two participants had ART suppression. DS was detected in 24/82 (29%) participants: 13/253 (5%) cervicovaginal lavages, 20/322 (6%) seminal plasmas, and 6/85 (7%) rectal secretions. HIV RNA in DS specimens was near the limit of quantification and not reproducible. HIV DNA was detected in 6/13 (46%) DS cervicovaginal lavages at low levels. Following DNase treatment, 5/39 DS specimens yielded HIV sequences, all without increased genetic distances. Women with and without DS had similar plasma antiretroviral levels and DS in 1 woman was associated with inflammation. Conclusions HIV RNA and DNA sequences and therapeutic antiretroviral plasma levels did not support HIV replication as the cause of DS from the genital tract. Rather, our findings infer that HIV RNA is shed due to proliferation of infected cells with virion production.

scholarly journals Cell-specific accumulation of Drosophila proteasomes (MCP) during early development.

1990 ◽  
Vol 111 (6) ◽  
pp. 2275-2282 ◽  
Author(s):  
U Klein ◽  
M Gernold ◽  
P M Kloetzel
Keyword(s):  
Relative Molecular Mass ◽  
Enzyme Complex ◽  
Proliferating Cells ◽  
Protein Subunits ◽  
Multicatalytic Proteinase ◽  
Drosophila Embryogenesis ◽  
Antibody Staining ◽  
Whole Mount ◽  
Specific Accumulation ◽  
Multicatalytic Proteinase Complex

The proteasome (MCP) is a high relative molecular mass multicatalytic proteinase complex composed of nonidentical protein subunits. We have investigated the cellular distribution of the enzyme complex during Drosophila embryogenesis using the proteasome specific antibodies N19-35 and N19-28 for immunocytology. Antibody staining of whole-mount embryos shows that during embryogenesis proteasomes are present in proliferating cells and that their accumulation and turnover is differentially regulated. Our data suggest that the proteasome may serve different proteolytic processes and that the enzyme may be involved in cell-specific proteolytic events required for cell proliferation and morphogenesis during early Drosophila development.

scholarly journals Preparing Planarian Cells for High-content Fluorescence Microscopy Using RNA in situ Hybridization and Immunocytochemistry

2021 ◽  
Author(s):  
Markus Grohme ◽  
Olga Frank ◽  
Jochen Rink
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent In Situ Hybridization ◽  
Histone H3 ◽  
Nuclear Genome ◽  
Quantitative Information ◽  
Single Cell Suspension ◽  
Proliferating Cells ◽  
Rna Transcripts ◽  
Whole Mount

High-content fluorescence microscopy combines the efficiency of high-throughput techniques with the ability to extract quantitative information from biological systems. The planarian community has developed sensitive and robust assays for whole animals, yet cell based assays, despite their practical aspects, have not been explored to the same extent. Here we describe a modular collection of detailed protocols adapted for fixed planarian cells that enable multiplexed measurements of biomarkers in microwell plates. Methods include the detection of RNA transcripts by RNA fluorescent in situ hybridization combined with tyramide signal amplification using hapten-labeled riboprobes. In addition, immunocytochemical protocols for quantifying proliferating cells by the detection of phosphorylated histone H3 as well as 5-bromo-2'-deoxyuridine incorporation into the nuclear genome are described. The assays are compatible with planarians of virtually any size, as the tissue is disaggregated into a single cell suspension before fixation and staining. By sharing many reagents with established planarian whole mount staining protocols, preparation of samples for high-content microscopy adoption requires little additional investment. Recommendations for successful experimental workflows and common sources of errors are discussed.

3-D organization of fibrinogen receptors using computer reconstruction and whole-mount microscopy

1988 ◽  
Vol 46 ◽  
pp. 30-31
Author(s):  
E. T. O'Toole ◽  
R. R. Hantgan ◽  
J. C. Lewis
Keyword(s):  
Whole Blood ◽  
Colloidal Gold ◽  
Blood Platelets ◽  
Cell Contact ◽  
Computer Assisted ◽  
Adherent Cells ◽  
Differential Centrifugation ◽  
Computer Reconstruction ◽  
Sds Page ◽  
Whole Mount

Thrombocytes (TC), the avian equivalent of blood platelets, support hemostasis by aggregating at sites of injury. Studies in our lab suggested that fibrinogen (fib) is a requisite cofactor for TC aggregation but operates by an undefined mechanism. To study the interaction of fib with TC and to identify fib receptors on cells, fib was purified from pigeon plasma, conjugated to colloidal gold and used both to facilitate aggregation and as a receptor probe. Described is the application of computer assisted reconstruction and stereo whole mount microscopy to visualize the 3-D organization of fib receptors at sites of cell contact in TC aggregates and on adherent cells.Pigeon TC were obtained from citrated whole blood by differential centrifugation, washed with Ca++ free Hank's balanced salts containing 0.3% EDTA (pH 6.5) and resuspended in Ca++ free Hank's. Pigeon fib was isolated by precipitation with PEG-1000 and the purity assessed by SDS-PAGE. Fib was conjugated to 25nm colloidal gold by vortexing and the conjugates used as the ligand to identify fib receptors.

Applications of Scanning Microscopy in Biomedical Research

1968 ◽  
Vol 26 ◽  
pp. 354-355
Author(s):  
T. L. Hayes
Keyword(s):  
Information Content ◽  
Biomedical Research ◽  
Biomedical Applications ◽  
Three Dimensional ◽  
Dental Enamel ◽  
Resolving Power ◽  
Whole Mount ◽  
Two Parameters ◽  
Content Information ◽  
Sectioned Tissue

Biomedical applications of the scanning electron microscope (SEM) have increased in number quite rapidly over the last several years. Studies have been made of cells, whole mount tissue, sectioned tissue, particles, human chromosomes, microorganisms, dental enamel and skeletal material. Many of the advantages of using this instrument for such investigations come from its ability to produce images that are high in information content. Information about the chemical make-up of the specimen, its electrical properties and its three dimensional architecture all may be represented in such images. Since the biological system is distinctive in its chemistry and often spatially scaled to the resolving power of the SEM, these images are particularly useful in biomedical research.In any form of microscopy there are two parameters that together determine the usefulness of the image. One parameter is the size of the volume being studied or resolving power of the instrument and the other is the amount of information about this volume that is displayed in the image. Both parameters are important in describing the performance of a microscope. The light microscope image, for example, is rich in information content (chemical, spatial, living specimen, etc.) but is very limited in resolving power.

The application of video-enhanced constrast/differential interference constrast microscopy in conjunction with whole mount electron microscopy to the study of organelle transport

1988 ◽  
Vol 46 ◽  
pp. 66-67
Author(s):  
J. H. Hayden
Keyword(s):  
Electron Microscopy ◽  
Rana Pipiens ◽  
Silver Film ◽  
Immunofluorescence Microscopy ◽  
Organelle Transport ◽  
Linear Element ◽  
Whole Mount ◽  
Carbon Coated ◽  
Linear Elements ◽  
Dic Microscopy

In a previous study, Allen video-enhanced constrast/differential interference constrast (AVEC-DIC) microscopy was used in conjunction with immunofluorescence microscopy to demonstrate that organelles and vesicle move in either direction along linear elements composed of microtubules. However, this study was limited in that the number of microtubules making up a linear element could not be determined. To overcome this limitation, we have used AVEC-DIC microscopy in conjunction with whole mount electron microscopy.Keratocytes from Rana pipiens were grown on glass coverslips as described elsewhere. Gold London Finder grids were Formvar- and carbon coated, and sterilized by exposure to ultraviolet light. It is important to select a Formvar film that gives a grey reflection when it is floated on water. A silver film is too thick and will detract from the image in the light microscope.

The in vivo distribution and dynamics of DNA topoisomerase II in Drosophila embryonic nuclei and chromosomes

1993 ◽  
Vol 51 ◽  
pp. 74-75
Author(s):  
Jason R. Swedlow ◽  
Neil Osheroff ◽  
Tim Karr ◽  
John W. Sedat ◽  
David A. Agard
Keyword(s):  
Topoisomerase Ii ◽  
Biochemical Characterization ◽  
Developmental Cycle ◽  
Dna Topoisomerase Ii ◽  
Chromosomal Distribution ◽  
Dna Topoisomerase ◽  
Ideal System ◽  
Dnase Treatment

DNA topoisomerase II is an ATP-dependent double-stranded DNA strand-passing enzyme that is necessary for full condensation of chromosomes and for complete segregation of sister chromatids at mitosis in vivo and in vitro. Biochemical characterization of chromosomes or nuclei after extraction with high-salt or detergents and DNAse treatment showed that topoisomerase II was a major component of this remnant, termed the chromosome scaffold. The scaffold has been hypothesized to be the structural backbone of the chromosome, so the localization of topoisomerase II to die scaffold suggested that the enzyme might play a structural role in the chromosome. However, topoisomerase II has not been studied in nuclei or chromosomes in vivo. We have monitored the chromosomal distribution of topoisomerase II in vivo during mitosis in the Drosophila embryo. This embryo forms a multi-nucleated syncytial blastoderm early in its developmental cycle. During this time, the embryonic nuclei synchronously progress through 13 mitotic cycles, so this is an ideal system to follow nuclear and chromosomal dynamics.

Quantitative Model-Based Image Analysis of NuMa Distribution Links Nuclear Organization with Cell Phenotype

2001 ◽  
Vol 7 (S2) ◽  
pp. 578-579
Author(s):  
David W. Knowles ◽  
Sophie A. Lelièvre ◽  
Carlos Ortiz de Solόrzano ◽  
Stephen J. Lockett ◽  
Mina J. Bissell ◽  
...  
Keyword(s):  
Image Analysis ◽  
Mammary Epithelial Cells ◽  
Nuclear Organization ◽  
Critical Role ◽  
Quantitative Model ◽  
Mammary Epithelial ◽  
Cell Phenotype ◽  
Proliferating Cells ◽  
Mitotic Apparatus ◽  
Model Based

The extracellular matrix (ECM) plays a critical role in directing cell behaviour and morphogenesis by regulating gene expression and nuclear organization. Using non-malignant (S1) human mammary epithelial cells (HMECs), it was previously shown that ECM-induced morphogenesis is accompanied by the redistribution of nuclear mitotic apparatus (NuMA) protein from a diffuse pattern in proliferating cells, to a multi-focal pattern as HMECs growth arrested and completed morphogenesis . A process taking 10 to 14 days.To further investigate the link between NuMA distribution and the growth stage of HMECs, we have investigated the distribution of NuMA in non-malignant S1 cells and their malignant, T4, counter-part using a novel model-based image analysis technique. This technique, based on a multi-scale Gaussian blur analysis (Figure 1), quantifies the size of punctate features in an image. Cells were cultured in the presence and absence of a reconstituted basement membrane (rBM) and imaged in 3D using confocal microscopy, for fluorescently labeled monoclonal antibodies to NuMA (fαNuMA) and fluorescently labeled total DNA.

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