A Suzuki–Miyaura method for labelling proliferating cells containing incorporated BrdU

The Analyst ◽  
2018 ◽  
Vol 143 (5) ◽  
pp. 1224-1233 ◽  
Author(s):  
Ning Yan ◽  
Yujun He ◽  
Hui Wen ◽  
Fangfang Lai ◽  
Dali Yin ◽  
...  
Keyword(s):  
Boronic Acid ◽  
Proliferating Cells ◽  
Reaction Method ◽  
Incorporated Brdu

A Suzuki–Miyaura reaction method was developed to label cellular BrdU with fluorescent boronic acid probes.

scholarly journals Effects of tumour necrosis factor-α on BrdU incorporation in cultured human enterocytes

1995 ◽  
Vol 4 (1) ◽  
pp. 31-37 ◽  
Author(s):  
J. McDevitt ◽  
C. Feighery ◽  
C. O'Farrelly ◽  
G. Martin ◽  
D. G. Weir ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Cell Line ◽  
Cell Number ◽  
Brdu Incorporation ◽  
Similar Response ◽  
Proliferating Cells ◽  
Incorporated Brdu ◽  
Factor Α ◽  
Proliferation Assays ◽  
Necrosis Factor

Bromodeoxyuridine incorporation is a useful method for studying the pattern of DNA synthesis in proliferating cells. The distribution pattern of incorporated BrdU in villus enterocytes of duodenal explants was analysed after exposure to TNFα in organ culture. TNFα caused a consistent, low level uptake of BrdU in the portion of the nucleus close to the nuclear membrane, this pattern was absent from the control cultures. As these epithelial cells are terminally arrested in G0, the BrdU incorporation was thought not to be due to S phase DNA synthesis, but rather a response to the cytotoxic influence of TNFα. Microtitre plate proliferation assays of cell density and DNA synthesis were devised to study the effects of TNFα on confluent monolayers of the human foetal jejunal cell line I407 and the mouse fibrosarcoma cell line L929. Both cell lines showed a similar response to TNFα. Exposure to TNFα alone did not reduce cell numbers but did cause a significant increase in DNA synthesis (p < 0.05). When cycloheximtde was added in tandem with TNFα there was a significant reduction in cell number (p < 0.001) and level of DNA synthesis (p < 0.01) indicative of cell death. The DNA of cells exposed to TNFα and cycloheximide was fragmented when viewed on an electrophoresis gel. The results show that BrdU incorporation might be a good indicator of damage to the DNA of cells after cytotoxic insult. TNFα may be responsible for villus enterocyte damage in enteropathies such as coeliac disease and GVHR of the small bowel.

scholarly journals Ultrastructural study of proliferating cells with an improved immunocytochemical detection of DNA-incorporated bromodeoxyuridine.

1995 ◽  
Vol 43 (1) ◽  
pp. 21-29 ◽  
Author(s):  
R Tamatani ◽  
Y Taniguchi ◽  
Y Kawarai
Keyword(s):  
Ultrastructural Study ◽  
Staining Method ◽  
Improved Method ◽  
Proliferating Cells ◽  
Gold Particles ◽  
Immunocytochemical Detection ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Silver Staining Method ◽  
Incorporated Brdu

We designed an improved method to observe proliferating cells with well-preserved ultrastructure. After IP injection of bromodeoxyuridine (BrdU) into rats, the pituitaries were fixed in 4% paraformaldehyde with 0.05% or 0.2% glutaraldehyde and post-fixed with ferrocyanide-reduced osmium. They were embedded in LR White and polymerized by heat. BrdU incorporated into DNA was detected with a commercial anti-BrdU monoclonal antibody (MAb) by the immunogold or the immunogold-silver staining method. Using these methods, proliferating cells labeled by BrdU were observed with well-preserved ultrastructure. By light microscopy, the number of labeled cells was almost the same regardless of the fixative used. By electron microscopy, localization of gold particles that indicate incorporated BrdU varied according to the cells and was mainly observed in two patterns, one in which gold particles were localized in condensed chromatin scattered in the nucleus and the other in which gold particles were dispersed evenly all over the nucleus. These results showed that with our improved method fine ultrastructure and good immunoreactivity of BrdU can be obtained in proliferating cells. We consider that this method is very useful for ultrastructural study of cell proliferation and differentiation.

scholarly journals Long-term Oral Application of 5-Bromo-2-Deoxyuridine Does Not Reliably Label Proliferating Immune Cells in the LEW Rat

1997 ◽  
Vol 45 (3) ◽  
pp. 393-401 ◽  
Author(s):  
Peter Jecker ◽  
Andrea Beuleke ◽  
Ingeborg Dressendörfer ◽  
Reinhard Pabst ◽  
Jürgen Westermann
Keyword(s):  
Bone Marrow ◽  
Immune Cell ◽  
Proliferating Cells ◽  
Incorporated Brdu ◽  
Spleen And Lymph Nodes ◽  
Kinetic Pattern ◽  
Changes Over Time ◽  
Strain Dependent ◽  
Bone Marrow Blood

To study the lifespan of immune cell populations in the LEW rat, 5-bromo-2-deoxyuridine (BrdU) was administered in the drinking water. After 12 weeks, the epithelium of gut and skin was completely BrdU+. In contrast, thymus, bone marrow, and germinal centers of Peyer's patches contained only a few BrdU+ cells, although most should have been labeled during this time. The lack of labeling was due neither to obvious toxic effects of BrdU on these organs nor to insufficient detection of incorporated BrdU. Analysis of the kinetic pattern of the appearance of BrdU+ cells in bone marrow, blood, spleen, and lymph nodes over 12 weeks revealed that the dosage of BrdU initially was high enough to label the proliferating cells in the bone marrow, but then became too low, although the BrdU uptake of the rats was similar over the entire time. This indicates that in the LEW rat the metabolism of orally applied BrdU changes over time, leading to a reduction in the amount of BrdU available for incorporation into the DNA below a level necessary for labeling all proliferating cells. This effect appears to be species- and strain-dependent, and should be considered when the BrdU technique is used. (J Histochem Cytochem 45:393–401, 1997)

Differential Expression of Type I MAGE in New and Relapsed Multiple Myeloma: Evidence for Association with Proliferation and Progression of Disease.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3397-3397
Author(s):  
Hearn J. Cho ◽  
Scott Ely ◽  
Wayne R. Austin ◽  
Ruben Niesvizky ◽  
Roger Pearse ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Myeloma Cell ◽  
Proliferation Index ◽  
Type I ◽  
Newly Diagnosed ◽  
Tumor Vaccines ◽  
Proliferating Cells ◽  
Myeloma Cells ◽  
Incorporated Brdu

Abstract The type I Melanoma Antigen GEne (MAGE) proteins belong to the Cancer-Testis family of tumor-associated antigens and are found in a broad range of solid and hematologic malignancies. We previously showed that the type I MAGE proteins CT7 (MAGE-C1) and MAGE-A3 were commonly detected in primary myeloma by both RT-PCR and immunohistochemistry (IHC). Higher levels of MAGE protein expression had a positive correlation with abnormally elevated proliferation as measured by the Plasma Cell Proliferation Index (PCPI, percentage of Ki-67+ cells in the CD138+ myeloma cell compartment). These findings suggest that MAGE may play a role in abnormal cell cycle regulation in myeloma. We explored this hypothesis by examining type I MAGE gene expression and proliferation by IHC in 46 newly-diagnosed, untreated and 35 relapsed myeloma patients, based on the clinical observation that relapsed patients exhibit lower response rates to therapy and shorter time to progression, indicative of more aggressive disease. PCPI was significantly higher in relapsed patients (19.0 ± 3.5%) compared to newly diagnosed (6.9 ± 1.3%, p<0.0002). Expression of CT7 and CT10 (MAGE-C2), a type I MAGE not previously associated with myeloma, was stable between newly-diagnosed and relapsed patients (76.0% of new samples vs. 77.1% of relapsed for CT7, 48.5% vs. 50.0% for CT10). In contrast, MAGE-A3 was detected in a significantly greater percentage of relapsed patients (77.1%) compared to newly diagnosed (35.6%, p=0.0003). The link between MAGE expression and unrestricted proliferation was further supported by in vitro studies with human myeloma cell lines. Proliferating myeloma cells were metabolically labeled with the nucleotide analog bromodeoxyuridine (BrdU) followed by intracellular staining and flow cytometry. This assay demonstrated that proliferating myeloma cells that incorporated BrdU into their genomic DNA expressed higher levels of type I MAGE protein compared to non-proliferating cells. Arresting cells at the G1-S interface by double thymidine blockade lead to the accumulation of cells expressing high levels of MAGE, which rapidly entered S phase and incorporated BrdU upon release from block. These results strongly suggest that expression of CT7 and CT10 are relatively early and stable events in the pathogenesis of myeloma, whereas activation of MAGE-A3 expression is associated with disease progression. Furthermore, MAGE expression is correlated with abnormal proliferation in vitro and in vivo, suggesting a potential functional role in the dysregulation of the cell cycle that is a hallmark of this disease. These results support the further exploration of the role of type I MAGE expression in myeloma and the development of therapeutic agents targeting them, especially tumor vaccines.

Requirement of neurotrophin-3 for the survival of proliferating trigeminal ganglion progenitor cells

Development ◽  
1996 ◽  
Vol 122 (8) ◽  
pp. 2405-2414 ◽  
Author(s):  
W.M. elshamy ◽  
P. Ernfors
Keyword(s):  
Cell Death ◽  
Progenitor Cells ◽  
Trigeminal Ganglion ◽  
Physiological Role ◽  
Proliferating Cells ◽  
Neurotrophin 3 ◽  
Dividing Cells ◽  
Incorporated Brdu ◽  
Ganglion Neurons

The aim of this study was to identify the physiological role of neurotrophin-3 (NT-3) in the development of trigeminal ganglion sensory neurons. For this purpose we have analysed mice carrying a deletion in the NT-3 gene (NT-3−/− mice). In these mice, by embryonic day (E) 11.25% of the trigeminal ganglion neurons were absent and one day later, approximately 50% were absent, after which no further significant changes were observed. Mice carrying one functional NT-3 gene (NT-3+/− mice) displayed a less severe deficit than that of NT-3−/− mice. Whereas programmed cell death occurred between E12 and E14 in the control mice, pronounced excessive cell death was apparent prior to this in the NT-3−/− mice. The excessive cell death led to a progressive decline in the number of proliferating cells without a significant change in the fraction of dividing cells and total number of neurons, indicating that the neuronal deficit of NT-3−/− mice was caused by cell death of trigeminal ganglion progenitors. Furthermore, the degenerating cells had incorporated BrdU, a nucleotide analogue which labels proliferating cells, and expressed nestin, a marker for progenitor cells. Only rarely were degenerating cells seen to express peripherin, present in postmitotic neurons. These data provide evidence that NT-3 is a survival factor for trigeminal ganglion progenitor cells, and suggests that limiting amounts of NT-3 could influence progenitor cell numbers during gangliogenesis.

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.

Fast and Reproducible Vascular Neointima Formation in the Hamster Carotid Artery: Effects of Trapidil and Captopril

1995 ◽  
Vol 74 (06) ◽  
pp. 1591-1596 ◽  
Author(s):  
H Matsuno ◽  
J M Stassen ◽  
M F Hoylaerts ◽  
J Vermylen ◽  
H Deckmyn
Keyword(s):  
Carotid Artery ◽  
Enzyme Inhibitor ◽  
Neointima Formation ◽  
Proliferating Cells ◽  
Dental Cement ◽  
Proliferation Indices ◽  
Experimental Drugs ◽  
The Media ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryNeointima formation was induced in the hamster carotid artery by mechanical intraluminal injury with a catheter covered with roughened dental cement. Neointimal thickening occurred as early as 7 days after denudation and further increased during the next 1 to 2 weeks. Proliferation indices of smooth muscle cells (SMCs) showed the highest proportion of proliferating cells in the media and neointima respectively 1 and 5 days after the vascular injury. Transmission and scanning electron microscopy of damaged carotid artery sections as well as immuno-histochemical stainings of von Willebrand factor (vWF) confirmed that reendothelialization was progressive and already complete on day 14, at which time the neointima formation was almost complete.In order to pharmacologically characterize this model further, the effects on neointima formation of trapidil (triazolopyrimidine), a platelet-derived growth factor (PDGF) antagonist, and captopril, an angiotensin converting enzyme inhibitor, were investigated. Trapidil administered orally twice daily at total doses of 25, 50 and 100 mg/kg/day, started 3 days prior to infliction of injury and up to 7 or 14 days after the catheterization, significantly reduced neointima formation. Captopril administered orally three times daily at a total dose of 100 mg/kg/day, equally reduced neointima formation, with 100 mg/kg/day trapidil being more effective than 100 mg/kg/day captopril 7 days after injury. When the treatment by either one of these drugs was arrested on day 7, neointima formation resumed quickly.The hamster appears to be a small, reproducible and fast model for the study of SMC proliferation, requiring only relatively small amounts of experimental drugs. The model furthermore is sensitive to substances known to reduce neointima formation in other animal models.

Leukocytes and Thrombosis

1965 ◽  
Vol 13 (01) ◽  
pp. 035-046 ◽  
Author(s):  
R. L Henry
Keyword(s):  
Tissue Culture ◽  
White Blood Cells ◽  
Polymorphonuclear Neutrophils ◽  
Mononuclear Leukocytes ◽  
Amoeboid Movement ◽  
Proliferating Cells ◽  
Cell Tissue ◽  
Blood Clots ◽  
Platelet Aggregates ◽  
To Come

SummaryWhite blood cells can no longer be considered simple trapped inclusions within thrombi. Their numbers in thrombi relative to blood counts increase with time. They appear to come from the blood flowing past the thrombus. They appear to migrate by amoeboid movement into the thrombic mass. Polymorphonuclear neutrophils have been shown to be lytic to fibrin and other proteins and thus can contribute to thrombus dissolution. There is increasing evidence that neutrophils may impart important cytotrophic function to proliferating cells during thrombus organization. Eosinophils are known to carr profibrinolysin and will release this precursor at sites of fibrin deposition. Mononuclear leukocytes can transform into fibroblasts in tissue culture and I consider a thrombus an ideal tissue culture medium. All of these cells can contribute to thrombus dissolution simply by mechanical weakening of the mass by migration into it, releasing enzymes, and allowing blood flow to carry away pieces of the thrombus as emboli. I extend my perspective on thrombosis by considering these intravascular solids as cell tissue cultures rather than simple blood clots or platelet aggregates.

A Boronic Acid-Based Fluorescence Hydrogel for Monosaccharide Detection

2018 ◽  
Author(s):  
Suying Xu ◽  
Adam Sedgwick ◽  
Souad Elfecky ◽  
Wenbo Chen ◽  
Ashley Jones ◽  
...  
Keyword(s):  
Fluorescent Probe ◽  
Boronic Acid ◽  
Binding Constants ◽  
Strong Fluorescence ◽  
Fluorescence Response ◽  
Turn On ◽  
Fluorescence Turn On ◽  
Hydrogel System

<p>A boronic acid-based anthracene fluorescent probe was functionalised with an acrylamide unit to incorporate into a hydrogel system for monosaccharide detection<i>. </i>In solution, the fluorescent probe<b> </b>displayed a strong fluorescence turn-on response upon exposure to fructose, and an expected trend in apparent binding constants, as judged by a fluorescence response where D-fructose > D-galactose > D-mannose > D-glucose. The hydrogel incorporating the boronic acid monomer demonstrated the ability to detect monosaccharides by fluorescence with the same overall trend as the monomer in solution with the addition of fructose resulting in a 10-fold enhancement (≤ 0.25 M). <b><u></u></b></p>

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