Bromodeoxyuridine (brdu)

Manufactured by Thermo Fisher Scientific

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BrdU is a synthetic nucleoside that is an analog of the DNA base thymidine. It can be incorporated into the newly synthesized DNA of dividing cells, substituting for thymidine during the DNA replication process. This allows for the detection and quantification of cellular proliferation.

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Bromodeoxyuridine (BrdU, (2 citations)

216 protocols using bromodeoxyuridine (brdu)

Quantifying DNA Methylation and Proliferation

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Cells were fixed in 4% paraformaldehyde for 15 min and incubated overnight with the primary antibody (5mC, Active Motif, cat.no. 39649, lot 06116002, used 1:250; NESTIN, BD, cat.no 611658, lot 45333, used 1:500; SOX2, R&D, cat.no MAB2018, lot KGQ0200041, used 1:50; BrdU, Serotech, cat.no OBT0030, lot 0512, used 1:250), followed by a 2 h incubation with a fluorophore-conjugated secondary antibody (Jackson Laboratories) and DAPI. Cells stained for 5mC and BrdU were pre-treated with 0.9% Triton in PBS for 15 min followed by 2 N HCl for 15 min followed by 10 mM Tris-HCl, pH 8, for 10 min prior to incubation with the primary antibody. Imaging of cells was performed with a fluorescence microscope (Leica).
BrdU was incorporated by adding BrdU (10 µM, Life Technologies) to the culture media of different wells for 5 consecutive days, starting on day 10 after FACS, and fixing the cells 24 h after exposure. Cells were stained for Dapi, BrdU, and 5mC. The 5mC expression and BrdU incorporation was evaluated using a Cellomics Scan 6.6.0 from Thermo Scientific and HCS Studio. Cells were quantified as Dapi + /BrdU + /5mC + in the control group and Dapi + /BrdU + /5mC− in the DNMT1-KO group. On average 11,906 control cells and 7,774 DNMT1-KO cells were analyzed per time point.

Jönsson M.E., Ludvik Brattås P., Gustafsson C., Petri R., Yudovich D., Pircs K., Verschuere S., Madsen S., Hansson J., Larsson J., Månsson R., Meissner A, & Jakobsson J. (2019). Activation of neuronal genes via LINE-1 elements upon global DNA demethylation in human neural progenitors. Nature Communications, 10, 3182.

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BrdU Chasing in Chondrocyte Differentiation

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In the BrdU chase assay, 2-week-old mice received two intraperitoneal injections of BrdU (Invitrogen; 1 ml/100 g body weight) with an intervening interval of 6 h and were euthanatized 48 h after the first injection to ensure that BrdU-labelled chondrocytes had sufficient time to differentiate. Long bones were harvested, fixed in 4% paraformaldehyde, decalcified in EDTA, and embedded in paraffin. Visualization of BrdU was performed via immunohistochemistry or immunofluorescence with the anti-BrdU antibody.

Zhang Y., Wen J., Lai R., Zhang J., Li K., Zhang Y., Liu A, & Bai X. (2024). Rheb1 is required for limb growth through regulating chondrogenesis in growth plate. Cell and Tissue Research, 395(3), 261-269.

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Quantifying Intestinal Cell Migration

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Mice analyzed for bromodeoxyuridine (BrdU) label retention were given 50 mg/kg BrdU (Thermo Fisher, Waltham, MA) in PBS by intraperitoneal injection. Mice were sacrificed 24- and 48-hours post-injection, and intestines were harvested as described above. BrdU IHC was performed as stated above using mouse anti-BrdU antibodies at 1:100 dilution (ThermoFisher, Waltham, MA) and the tissues imaged using ImageScope software. To assess cell migration, the distance from the base of the crypt to the forefront-most BrdU-positive cell was measured. Three blind observers quantified the distance migrated from ~ 100 villi (n = 3 mice/genotype).

Robinson S.C., Chaudhary R., Jiménez-Saiz R., Rayner L.G., Bayer L., Jordana M, & Daniel J.M. (2019). Kaiso-induced intestinal inflammation is preceded by diminished E-cadherin expression and intestinal integrity. PLoS ONE, 14(6), e0217220.

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Comprehensive Cell Proliferation Analysis

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In cell proliferation assay, 1×10⁴ cells/well were seeded into a 96-well plate. At different experimental end time point, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was carried out using the MTS assay kit (Thermofisher, Shanghai, China) according to the manufacturer’s protocol.
In 5-Bromo-2′-deoxyuridine (BrdU) assay, the cells were cultured for three days according to designed experimental condition before labeling with (3 µg/mL) BrdU (Thermofisher) for 4 h. The analysis of BrdU was performed as previously described.9 (link)
In colony formation assay, 1 × 10⁴ cells/well were seed in the 6-well plate after designed experimental treatment. After 2 weeks, cell colonies were fixed in 10% formalin and stained with crystal violet (0.1% w/v).
The apoptosis of cell was analyzed by Hoechst-33,258 (Thermofisher) nuclear staining. After treatment, the cells were stained with Hoechst-33,258 staining medium (0.1% Hoechst, 0.5% NP-40, 4% formaldehyde in PBS). At least 400 cells in each group were count, and the cells with fragmented nuclear were considered as apoptosis cells.

Ni S., Wei Q, & Yang L. (2020). ADORA1 Promotes Hepatocellular Carcinoma Progression via PI3K/AKT Pathway. OncoTargets and therapy, 13, 12409-12419.

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MTS and BrdU Cell Growth Assays

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For the cell growth test at different time points, the MTS assay was carried out with an MTS test kit (Thermo Fisher Scientific). A Wallac Victor 1420 Multilabel Counter was used to measure the absorbance. Each test was repeated 3 times. For the 5-bromo-2′-deoxyuridine (BrdU) test, cells were cultured for 3 days and then labeled with BrdU (3 μg/mL) (Thermo Fisher Scientific) for 4 h. The BrdU results were analyzed according to a previously described method (DeWaal et al., 2018 (link)).

Lu Q., Ni Y., Wang W., Wang L., Jiang T, & Shang L. (2021). Dynamin 3 Inhibits the Proliferation of Non-small-Cell Lung Cancer Cells by Suppressing c-MET–GBR2–STAT3 Complex Formation. Frontiers in Cell and Developmental Biology, 9, 641403.

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Comprehensive Cell Growth and Apoptosis Evaluation

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For cell growth assay, 1 × 10 4 cells/well were seeded in 96-well plates. At different time points, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was performed using the MTS assay kit (Thermofisher, Shanghai, China) according to the manufacturer's instructions. Chemiluminescence was measured by using a Wallac Victor 1420 Multilabel Counter (Perkin Elmer, Waltham, MA, USA). Each assay was conducted in triplicate and repeated three times. In 5-Bromo-2′-deoxyuridine (BrdU) assay, the cells were cultured for 3 days before labelling with (3 µg/mL) BrdU (Thermofisher) for 4 h. The analysis of BrdU was performed as previously described [21] .
The apoptosis of cells was analysed by Hoechst-33258 (Thermofisher) nuclear staining. Briefly, the cells with supernatant were collected and stained with Hoechst-33258 staining medium (0.1% Hoechst, 0.5% NP-40, 4% formaldehyde in PBS). The fracted nuclear was counted in 400 cells in each treatment group. The percentage of fragmented cells was calculated as the percentage of apoptosis.
For clonogenic survival assay, 1 × 10 4 cells per well were seeded in the six-well plates. After 2 weeks, cell colonies were fixed in 10% formalin and stained with crystal violet (0.1% w/v).

Zhao Y., Li N., Zhao J., & Shi S. (2020). High expression of hexokinase 2 promotes lung cancer proliferation and metastasis. Archives of Medical Science.

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Cell Cycle Analysis by Flow Cytometry

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To evaluate the MMS-induced perturbations in cell cycle progression, cells were grown for 24h in culture medium, then pulse-labeled with 30 μM BrdU (Life Technologies Corporation) for 30 min. After extensive washing, cells were collected and fixed in 90% cold ethanol on ice for at least 1h. Cells were processed for flow cytometry as follows: after fixation, cells were exposed to acid denaturation (2 N HCl), neutralization buffer (0.1 M sodium tetraborate) and blocking solution (10% NGS/PBS). After that, cells were incubated with an anti-BrdU fluorescently-labeled antibody (eFluor® 450, eBioscience). Samples were resuspendend in 20 μg/ml propidium iodide (Sigma-Aldrich, St. Louis, MO, USA). Cytofluorimetric acquisition was done on a BD FACScalibur using CellQuest software (BD Biosciences, San Jose, CA, USA), and analyses were performed using FlowJo software v. 7.6.5 (Tree Star, Inc., Ashland, OR, USA).

Simonelli V., Leuzzi G., Basile G., D’Errico M., Fortini P., Franchitto A., Viti V., Brown A.R., Parlanti E., Pascucci B., Palli D., Giuliani A., Palombo F., Sobol R.W, & Dogliotti E. (2016). Crosstalk between mismatch repair and base excision repair in human gastric cancer. Oncotarget, 8(49), 84827-84840.

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In Vivo Leukocyte Proliferation Assay

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For measurement of the proliferation of leukocytes in vivo, mice received i.p. injection of BrdU (1 mg; eBioscience), and the detection of the incorporated BrdU content in cells was performed 24 hrs after the injection flow cytometry using a BrdU staining kit for flow cytometry (eBioscience).

Takagi H., Arimura K., Uto T., Fukaya T., Nakamura T., Choijookhuu N., Hishikawa Y, & Sato K. (2016). Plasmacytoid dendritic cells orchestrate TLR7-mediated innate and adaptive immunity for the initiation of autoimmune inflammation. Scientific Reports, 6, 24477.

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Cell Cycle Analysis of Influenza-Infected Cells

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Cell cycle profiles were analyzed by BrdU incorporation and DNA content staining. Briefly, presynchronized A549 cells were infected at an MOI of 2 with influenza A/WSN/33 strain or mock-infected. Immediately before collection, cells were incubated in 10 μm BrdU (Invitrogen) for 1 h and fixed overnight in 70% ethanol at −20 °C. BrdU-pulsed cells were rescued from ethanol and denatured by 2 m hydrochloric acid with 0.5% Triton X-100 for 30 min at room temperature, followed by neutralization with 100 mm borate buffer (pH 8.5). BrdU labeling was revealed with Alexa Fluor 647-conjugated BrdU mouse mAb (clone MoBU-1; Invitrogen). Total DNA content was measured by 7-aminoactinomycin D (BD PharMingen) staining. Cells were then immediately analyzed on a BD LSR II flow cytometer (BD Immunocytometry Systems). Approximately 30,000 cells were acquired for each sample, and data were analyzed using FlowJo software (Treestar).

Fan Y., Mok C.K., Chan M.C., Zhang Y., Nal B., Kien F., Bruzzone R, & Sanyal S. (2017). Cell Cycle-independent Role of Cyclin D3 in Host Restriction of Influenza Virus Infection. The Journal of Biological Chemistry, 292(12), 5070-5088.

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Quantifying Basal Cell Renewal Probability

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Basal cell renewal probability was quantified based on nucleoside incorporation and differentiation marker expression as previously described (Ying et al., 2018 (link)). Since we can uniquely label a population of epidermal progenitor cells (EdU+ BrdU⁻) that have undergone division within the initial 2 hours as described above, we can also assess the differentiation state of their daughter cells based on expression of differentiation marker keratin 10 (K10). 50mg/kg EdU (Invitrogen) was administered intraperitoneally, followed by 100mg/kg BrdU (Invitrogen) injection 2 hours later, before assessing nucleoside incorporation by immunofluorescence 6 hours post-BrdU injection. We calculated the probability of progenitor cell renewal during the first 2 hours using the following equation: Renewal probability= (number of EdU+ BrdU⁻ K10⁻ cells) / (total number of EdU⁺ BrdU⁻ cells).

Cai E.Y., Kufeld M.N., Schuster S., Arora S., Larkin M., Germanos A.A., Hsieh A.C, & Beronja S. (2020). Selective translation of cell fate regulators mediates tolerance to broad oncogenic stress. Cell stem cell, 27(2), 270-283.e7.

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