Immunocytochemical detection of 5-bromo-2-deoxyuridine incorporation in individual cells

Furth, R. van; Zwet, T L van

doi:10.1016/0022-1759(88)90401-2

Cited by 49 publications

(23 citation statements)

References 11 publications

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“…A 20-60 minute labeling interval is sufficiently brief that cells in the process of recruitment should contribute little to the analysis, yet has been shown to efficiently label dividing cells within the lungs (20) . In vivo labeling of dividing cells with BrdU yields results which are comparable to other methods (35,36) . Thus, it is likely that the BrdU treatment protocol used gave an accurate glimpse of instantaneous lymphocyte proliferation within the lungs.…”

Section: Discussionmentioning

confidence: 69%

Lung Lymphocytes Proliferate Minimally in the Murine Pulmonary Immune Response to Intratracheal Sheep Erythrocytes

Seitzman

Sonstein²,

Kim³

et al. 1998

Am J Respir Cell Mol Biol

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The importance of in situ lymphocyte proliferation for net accumulation of lung lymphocytes during pulmonary immune responses and in immunologic lung diseases remains uncertain. Accordingly, we studied the experimental pulmonary immune response of antigen-primed C57BL/6 mice to intratracheal challenge with the particulate antigen sheep red blood cells (SRBC). Uptake of nucleotide analogs (bromodeoxyuridine in vivo and tritiated thymidine in vitro), expression of the cell activation antigens CD25 and CD69 by flow cytometry, and response to the anti-mitotic agent hydroxyurea (in vivo) were measured. Although many lung lymphocytes and CD4+ T cells were CD25+ and CD69+, indicating recent activation, all techniques demonstrated that lung lymphocytes proliferated minimally in vivo. Blockade of cell division by hydroxyurea administration for 24 hours did not significantly decrease lung lymphocyte accumulation on day 3 after challenge. Lung lymphocytes also proliferated minimally in vitro (even on macrophage removal and despite addition of exogenous IL-2 or IL-4). However, lung lymphocytes responded vigorously to mitogens (immobilized anti-CD3, phytohemagglutinin or concanavalin A), excluding global unresponsiveness to restimulation. Thus, in this model of pulmonary immunity, accumulation of lung lymphocytes does not require local T cell proliferation and presumably depends instead on recruitment.

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Section: Discussionmentioning

confidence: 69%

Lung Lymphocytes Proliferate Minimally in the Murine Pulmonary Immune Response to Intratracheal Sheep Erythrocytes

Seitzman

Sonstein²,

Kim³

et al. 1998

Am J Respir Cell Mol Biol

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“…Bromodeoxyuridine (BrdU), a halogenated nucleoside that can serve as a thymidine (TdR) analog, has been widely used as an alternative to tritiated TdR ([ 3 H]TdR) incorporation to label proliferating cells in cell biology (2,59). BrdU incorporated into de novo DNA can be detected using anti-BrdU monoclonal antibody.…”

mentioning

confidence: 99%

Actively Growing Bacteria in the Inland Sea of Japan, Identified by Combined Bromodeoxyuridine Immunocapture and Denaturing Gradient Gel Electrophoresis

Hamasaki

Taniguchi

Tada

et al. 2007

Appl Environ Microbiol

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A fundamental question in microbial oceanography concerns the relationship between prokaryote diversity and biogeochemical function in an ecosystem context. We combined bromodeoxyuridine (BrdU) magnetic bead immunocapture and PCR-denaturing gradient gel electrophoresis (BUMP-DGGE) to examine phylotype-specific growth in natural marine assemblages. We also examined a broad range of marine bacterial isolates to determine their abilities to incorporate BrdU in order to test the validity of the method for application to diverse marine assemblages. We found that 27 of 29 isolates belonging to different taxa could incorporate BrdU. BUMP-DGGE analysis revealed phylogenetic affiliations of DNA-synthesizing, presumably actively growing bacteria across a eutrophic to mesotrophic transect in the Inland Sea of Japan. We found that the BrdU-incorporating (growing) communities were substantially different from the total communities. The majority (34/56) of phylotypes incorporated BrdU and were presumably growing, and these phylotypes comprised 10 alphaproteobacteria, 1 betaproteobacterium, 11 gammaproteobacteria, 11 Cytophaga-Flavobacterium-Bacteroides group bacteria, and 1 unclassified bacterium. All BrdU-responsive alphaproteobacteria were members of the Rhodobacterales, suggesting that such bacteria were dominant in the growing alphaproteobacterial populations in our samples. The BrdU-responsive gammaproteobacteria belonged to the Oceanospirillales, the SAR86 cluster, the Pseudomonadales, the Alteromonadales, and the Vibrionales. Thus, contemporaneous cooccurrence of diverse actively growing bacterial taxa was a consistent pattern in our biogeochemically varied study area.Bacteria in seawater play important roles in the ocean's food webs. Their activities and responses to organic substrates significantly influence the flux of organic matter and oceanic biogeochemical cycles (3, 4). Because only a small fraction of bacteria in any seawater sample can be isolated and cultivated, culture-independent methods employing molecular approaches have been used to great advantage during the last two decades to study the natural assemblages of marine bacteria (11,19,39). These and other studies have revealed unexpected diversity and dynamics of bacterial community structure in seawater (8,16,47). However, the great challenge remains to relate diversity to ecological function and biogeochemical activities of bacteria. In recent studies workers have combined fluorescence in situ hybridization (FISH) with microautoradiography and found differences in the uptake of specific organic substrates by different phylogenetic groups of bacteria (7,8,40). Stable-isotope probing has enabled PCR-based DNA analysis of bacteria that incorporate specific substrates (45).Bromodeoxyuridine (BrdU), a halogenated nucleoside that can serve as a thymidine (TdR) analog, has been widely used as an alternative to tritiated TdR ([ 3 H]TdR) incorporation to label proliferating cells in cell biology (2, 59). BrdU incorporated into de novo DNA can be detected using ...

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“…This protocol gave much better morphological preservation than those recommended by others, such as sodium hydroxide or hydrochloric acid treatments (cf. VanFurth and VanZwet, 1988;Schutte et al, 1987).…”

Section: Discussionmentioning

confidence: 99%

Accurate determination of the time of cell birth using a sequential labeling technique with [3H]-thymidine and bromodeoxyuridine ("window labeling").

Repka

Adler²

1992

J Histochem Cytochem.

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During tissue embryogenesis, precursor cells divide actively and eventually withdraw from the mitotic cycle before differentiation. Accurate information about the time of terminal mitosis ("birthdate") of precursors is of vital importance for studying relationships between cell proliferation and differentiation. Methods presently available for birthdate determination, based on "pulse" or "cumulative" labeling with either tritiated thymidine ('HT) or bromodeoxyuridine (BrDU) incorporated into DNA during the mitotic cycle, allow only the approximate timing of terminal mitosis. To overcome this limitation, we have developed a "window labeling" technique based on the sequential administration of 3HT and BrDU. Chick retinal precurxu cell cultures were fmt exposed to 3HT and, after a specified time interval, also to BrDU. After 6 days the cultures were fiied and processed for BrDU immunocytochemistry and 3HT autoradiography.

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Immunocytochemical detection of 5-bromo-2-deoxyuridine incorporation in individual cells

Cited by 49 publications

References 11 publications

Lung Lymphocytes Proliferate Minimally in the Murine Pulmonary Immune Response to Intratracheal Sheep Erythrocytes

Lung Lymphocytes Proliferate Minimally in the Murine Pulmonary Immune Response to Intratracheal Sheep Erythrocytes

Actively Growing Bacteria in the Inland Sea of Japan, Identified by Combined Bromodeoxyuridine Immunocapture and Denaturing Gradient Gel Electrophoresis

Accurate determination of the time of cell birth using a sequential labeling technique with [3H]-thymidine and bromodeoxyuridine ("window labeling").

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