Utility and sensitivity of anti BrdU antibodies in assessing S‐phase cells compared to autoradiography

Raza, Azra; Ucar, Kalust; Bhayana, Ranjan; Kempski, Maureen; Preisler, Harvey D.

doi:10.1002/cbf.290030212

Cited by 22 publications

(7 citation statements)

References 4 publications

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“…gain or loss of chromosomes) from manual handling. It is unlikely that any cells had progressed into early S-phase, as even short BrdU/EdU incorporation periods (1 min) are clearly detectable [53], For ELISA, it was necessary to perform high-throughput mitotic shake-off, slightly increasing contamination with S-phase cells compared to manual selection. S-phase cells contain fewer fully methylated histones [54,55], reducing histone methylation in nuclear G 1 cell extracts.…”

Section: Discussionmentioning

confidence: 99%

Quiescence Loosens Epigenetic Constraints in Bovine Somatic Cells and Improves Their Reprogramming into Totipotency

Kallingappa

Turner

Eichenlaub

et al. 2016

Biology of Reproduction

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Reprogramming by nuclear transfer (NT) cloning forces cells to lose their lineage-specific epigenetic marks and reacquire totipotency. This process often produces molecular anomalies that compromise clone development. We hypothesized that quiescence alters the epigenetic status of somatic NT donor cells and elevates their reprogrammability. To test this idea, we compared chromatin composition and cloning efficiency of serum-starved quiescent (G0) fibroblasts versus nonstarved mitotically selected (G1) controls. We show that G0 chromatin contains reduced levels of Polycomb group proteins EED, SUZ12, PHC1, and RING2, as well as histone variant H2A.Z. Using quantitative confocal immunofluorescence microscopy and fluorometric enzyme-linked immunosorbent assay, we further show that G0 induced DNA and histone hypomethylation, specifically at H3K4me3, H3K9me2/3 and H3K27me3, but not H3K9me1. Collectively, these changes resulted in a more relaxed G0 chromatin state. Following NT, G0 donors developed into blastocysts that retained H3K9me3 hypomethylation, both in the inner cell mass and trophectoderm. G0 blastocysts from different cell types and cell lines developed significantly better into adult offspring. In conclusion, serum starvation induced epigenetic changes, specifically hypotrimethylation, that provide a mechanistic correlate for increased somatic cell reprogrammability.

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Section: Discussionmentioning

confidence: 99%

Quiescence Loosens Epigenetic Constraints in Bovine Somatic Cells and Improves Their Reprogramming into Totipotency

Kallingappa

Turner

Eichenlaub

et al. 2016

Biology of Reproduction

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“…Like [3H]-thymidine, BrdU can be used in vivo [16][17][18][19] and in vitro [20]. The sensitivity [14,[21][22][23] and specificity [22] of BrdU immunodetection for labeling S-phase cells are comparable to [3H]-thymidine autoradiography.…”

Section: Introductionmentioning

confidence: 88%

Kinetics of Bromodeoxyuridine Uptake by Smooth Muscle Cells after Arterial Injury

London

Mayberg²

1994

J Vasc Res

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The time course of 5-bromo-2’-deoxyuridine (BrdU) uptake by medial and neointimal smooth muscle cells was examined in rat carotid arteries at periods of 1-20 days after balloon catheter injury. DNA-incorporated BrdU was determined by the indirect immunoperoxidase technique in both plastic- and paraffin-embedded specimens. The BrdU labeling index (LI) peaked at 48 h in the media (19.9% paraffin, 9.4% plastic) and at 5 days in the neointima (55.4% paraffin, 60.2% plastic). Immunohistochemistry for the endogenous marker proliferating cell nuclear antigen was variable and generally less reliable than BrdU. Using total viable medial smooth muscle cell number as an index of medial injury, the LI for BrdU was directly proportional to the extent of injury at 48 h, and correlated with loss of actin immunoreactivity. BrdU immunohistochemistry is a reliable alternative to [³H]-thymidine autoradiography for short-term labeling in this model. Proliferation of smooth muscle cells in this model is likely related to the degree of injury to the media.

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“…Raza et al reported that the BrdU LI and the tritiated thymidine LI were almost the same 7 . Using enzymatic treatment and acid hydrolysis, BrdU-incorporated cells were detected even in formalin-fixed samples 8 .…”

Section: Discussionmentioning

confidence: 93%

Immunohistochemical Demonstration of S-phase Cells in Canine and Feline Spontaneous Tumors by Anti-bromodeoxyuridine Monoclonal Antibody

et al. 2005

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Bromodeoxyuridine (BrdU) labeling of S-phase cells, which are DNA-synthesizing cells, was demonstrated in 30 canine and 6 feline neoplasms. Simultaneously, the optimization of the BrdU-labeling method, including tissue fixation, administration dose of BrdU, and the immunohistochemical procedure, was examined. BrdU-incorporated cells in a neoplasm of a dog administered 3 mg/kg of BrdU could be detected. Although BrdU-positive cells could be visualized in 70% ethanol fixed samples, we could satisfactorily demonstrate BrdU-immunoreactivities in samples fixed in 10% buffered formalin and subjected to enzymatic treatment and acid hydrolysis. The BrdU-labeling index (LI) of malignant neoplasms tended to be higher than that of benign neoplasms. The mean BrdU LI of malignant neoplasms was significantly higher than that of benign neoplasms (P<0.05). The present study demonstrated that the BrdU-labeling method may be useful for detecting S-phase cells in canine and feline tissues. (J Toxicol Pathol 2005; 18: 135-140)

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Utility and sensitivity of anti BrdU antibodies in assessing S‐phase cells compared to autoradiography

Cited by 22 publications

References 4 publications

Quiescence Loosens Epigenetic Constraints in Bovine Somatic Cells and Improves Their Reprogramming into Totipotency

Quiescence Loosens Epigenetic Constraints in Bovine Somatic Cells and Improves Their Reprogramming into Totipotency

Kinetics of Bromodeoxyuridine Uptake by Smooth Muscle Cells after Arterial Injury

Immunohistochemical Demonstration of S-phase Cells in Canine and Feline Spontaneous Tumors by Anti-bromodeoxyuridine Monoclonal Antibody

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