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Brdu brdc

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BrdU:BrdC is a laboratory consumable product used for cellular and molecular biology research. It consists of two key components: Bromodeoxyuridine (BrdU) and Bromodeoxyinosine (BrdC). These molecules can be utilized to facilitate the study of DNA synthesis and cell proliferation processes.

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Lab products found in correlation

Alexa fluor 488 labelled ttaggg 3, by Panagene (2 mentions) Cy3 labelled pna ccctaa 3 probes, by Panagene (2 mentions) Axiovert 200m microscope, by Zeiss (2 mentions) Colcemid, by Roche (1 mentions) Exoiii, by Promega (1 mentions) Alexa 488, by Eurogentec (1 mentions) Hoechst 33258, by Merck Group (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Fv1000, by Olympus (1 mentions)

3 protocols using brdu brdc

1

Chromosome-specific CO-FISH Assay

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The CO-FISH assay was adapted from previous studies [35 (link), 36 (link)]. Briefly, exponentially growing cells were cultured overnight in the presence of 10 μM BrdU:BrdC (3:1) (Sigma-Aldrich) at 37 °C to allow for one round of replication. Colcemid (Roche) was then added at a concentration of 0.1 μg/ml for 4 h to arrest cells at prometaphase. After fixation of cellular preparations on slides and Hoechst 33258 (Sigma-Aldrich) staining, the newly synthesized strands were degraded following UV light exposure and treatment with 10 U/μl ExoIII (Promega). Hybridization was performed using fluorescent centromeric PNA probes against CENP-B box motif sequences. The PNA probe labeled with Cy3 (ATTCGTTGGAAACGGGA; PNABio Inc) hybridizes with the leading strand and the reverse PNA probe labeled with Alexa-488 (TCCCGTTTCCAACGAAT; Eurogentec) hybridizes with the lagging strand. DNA was counterstained with DAPI (Sigma-Aldrich). Metaphases were captured on a confocal laser microscope (FV1000 Olympus) and images were analyzed using Image J software (NIH). Quantitation to measure for SCE between α satellite sequences (C-SCE) was done by counting the number of CO-FISH signals showing C-SCE over the total number of CO-FISH signals observed for each metaphase.
Racca C., Britton S., Hédouin S., Francastel C., Calsou P, & Larminat F. (2021). BRCA1 prevents R-loop-associated centromeric instability. Cell Death & Disease, 12(10), 896.
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2

Measuring Telomere Sister Chromatid Exchanges

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Chromosome-Orientation-FISH (CO-FISH) was used to measure T-SCE 24 (link). Briefly, cells were cultured in DMEM medium containing 3:1 ratio of BrdU:BrdC (Sigma) at a final concentration of 10 μM for approximately 20 h, and subsequently with 0.1 μg/ml colcemid for approximately 2 h before harvest. T-SCE frequencies were measured 64 h after transfection with the indicated siRNAs. Metaphase spreads were prepared as described for Q-FISH, stained with Hoechst 33258, exposed to UV light, and digested with exonuclease III to remove newly synthesized DNA strands. Hybridization and wash conditions were identical to those described for telomere-FISH. Alexa Fluor 488-labelled (TTAGGG)3 and Cy3-labelled PNA (CCCTAA)3 probes (0.5 μg/ml, Panagene) were used for the detection of leading and lagging strands, respectively. Images were acquired on a Zeiss Axiovert 200M microscope. T-SCE was scored as a CO-FISH telomere signal split between the two chromatids of a metaphase chromosome.
Kim J., Sun C., Tran A.D., Chin P.J., Ruiz P.D., Wang K., Gibbons R.J., Gamble M.J., Liu Y, & Oberdoerffer P. (2019). The macroH2A1.2 histone variant links ATRX loss to alternative telomere lengthening.. Nature structural & molecular biology, 26(3), 213-219.
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3

Measuring Telomere Sister Chromatid Exchanges

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Chromosome-Orientation-FISH (CO-FISH) was used to measure T-SCE 24 (link). Briefly, cells were cultured in DMEM medium containing 3:1 ratio of BrdU:BrdC (Sigma) at a final concentration of 10 μM for approximately 20 h, and subsequently with 0.1 μg/ml colcemid for approximately 2 h before harvest. T-SCE frequencies were measured 64 h after transfection with the indicated siRNAs. Metaphase spreads were prepared as described for Q-FISH, stained with Hoechst 33258, exposed to UV light, and digested with exonuclease III to remove newly synthesized DNA strands. Hybridization and wash conditions were identical to those described for telomere-FISH. Alexa Fluor 488-labelled (TTAGGG)3 and Cy3-labelled PNA (CCCTAA)3 probes (0.5 μg/ml, Panagene) were used for the detection of leading and lagging strands, respectively. Images were acquired on a Zeiss Axiovert 200M microscope. T-SCE was scored as a CO-FISH telomere signal split between the two chromatids of a metaphase chromosome.
Kim J., Sun C., Tran A.D., Chin P.J., Ruiz P.D., Wang K., Gibbons R.J., Gamble M.J., Liu Y, & Oberdoerffer P. (2019). The macroH2A1.2 histone variant links ATRX loss to alternative telomere lengthening.. Nature structural & molecular biology, 26(3), 213-219.
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