MccΔ/Δ and wild-type siblings (Mcc-WT) were obtained from heterozygous crossings. Twenty-week-old mice were injected intraperitoneally with 100 mg/kg BrdU (Sigma-Aldrich, St. Louis, MO) and killed 2 hours after injection. The ileum and colon were harvested and fixed in 10% formalin for immunohistochemistry. Epitope retrieval was performed in a pressure cooker at 125ºC with DAKO (Glostrup, Denmark) buffer S2367, pH 9.0 for 2 minutes. The primary antibody for BrdU (1:50, DAKO) was incubated for 60 minutes. The number of BrdU-positive cells in each crypt were counted and divided by the total number of cells in each crypt to obtain the percentage of BrdU positivity. Twenty crypts were counted for each individual mouse and colon region.
Bromodeoxyuridine (brdu)
Manufactured by Agilent Technologies
Sourced in United States, United Kingdom
BrdU is a chemical compound that is structurally similar to the DNA base thymidine. It can be incorporated into dividing cells in place of thymidine during DNA synthesis, allowing for the identification and quantification of proliferating cells.
Automatically generated - may contain errors
Lab products found in correlation
Bromodeoxyuridine (brdu), by Merck Group (7 mentions)
Cyclin d1, by Cell Signaling Technology (2 mentions)
Bromodeoxyuridine (brdu), by Nacalai Tesque (2 mentions)
Biorevo software, by Keyence (2 mentions)
Bz 9000 microscope, by Keyence (2 mentions)
P stat3 tyr705, by Cell Signaling Technology (2 mentions)
Cell death detection kit, by Roche (2 mentions)
Cortisol, by Merck Group (2 mentions)
Powerwave xs, by Agilent Technologies (2 mentions)
Phytohemagglutinin (pha), by Merck Group (2 mentions)
Eutasil, by Sanofi (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Fv1000, by Olympus (1 mentions)
Cyclin d2, by Santa Cruz Biotechnology (1 mentions)
Phospho histone h3 p hh3, by Merck Group (1 mentions)
Click it edu alexa fluor 488 flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions)
F0313, by Agilent Technologies (1 mentions)
Mouse anti brdu antibody, by Agilent Technologies (1 mentions)
Fluorescein conjugated donkey anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
22 protocols using bromodeoxyuridine (brdu)
1
Proliferation Assay in Mouse Intestine
2
Quantifying Neuronal Proliferation in Dentate Gyrus
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In the last day of behavioral testing, animals were given single intraperitoneal injection of Bromo-deoxyuridine (BrdU, Sigma-Aldrich, 100 mg.kg−1; thymidine analog that incorporates into DNA during the S-phase of the mitotic process). Twenty-four hours after the injection, animals were deeply anaesthetized with sodium pentobarbital (20%; Eutasil, Sanofi) and were transcardially perfused with cold 4% paraformaldehyde (PFA). Brains were removed and post-fixed in 4% PFA. Serial coronal cryosections (20 μm) were cut and stained for BrdU (1:50; Dako). Secondary antibody Alexa Fluor® 488 (Molecular Probes) was used for detection. Nuclei were counterstained using DAPI. Proliferation densities were estimated in the subgranular zone (SGZ; defined as a two-cell layer-thick zone on the inner side of the granule cell layer of the dentate gyrus), using a confocal microscope (Olympus FV1000).
3
Quantifying Pancreatic Beta Cell Dynamics
4
Cell Cycle Analysis by BrdU and DNA Staining
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Cells were seeded in a T25 flask and when 50% confluence was reached, treated for 24 h with Adavosertib. Before harvest, cells were pulsed with 4nmol/L 5-bromo-20-deoxyurdine (BrdU, Sigma-Aldrich, 19–160) for 15 min, then harvested and fixed in 75% ice-cold EtOH overnight. Next, cells were incubated with 0.5 mg/mL RNAse A, permeabilized with 5 mol/L HCl:0.5% Triton X-100 for 20 min and HCl neutralized with 0.1 mol/L Na2B4O7. BrdU was stained overnight at 4 °C with mouse-anti-BrdU antibody (clone BU20a, M0744, Dako), and fluorescein isothiocyanate (FITC)-labeled with a conjugated rabbit anti-mouse antibody (F0313, Dako). DNA content was stained with propidiumiodide (PI) (5 µg PI per 106 cells, Sigma-Aldrich, P4170).
For VU-preSCC-M3, the Click-iT™ EdU Alexa Fluor™ 488 Flow Cytometry Assay Kit (ThermoFisher Scientific, C10420) was used according to the protocol of the manufacturer, and DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI).
BD LSR II FortessaTM and BD FACSDivaTM software V8.0.1 (BD Biosciences) were used for flow cytometry and data analysis.
For VU-preSCC-M3, the Click-iT™ EdU Alexa Fluor™ 488 Flow Cytometry Assay Kit (ThermoFisher Scientific, C10420) was used according to the protocol of the manufacturer, and DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI).
BD LSR II FortessaTM and BD FACSDivaTM software V8.0.1 (BD Biosciences) were used for flow cytometry and data analysis.
5
Cell Proliferation Analysis by BrdU Incorporation
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Cells were incubated in culture medium treated with or without HU for 72 h at 37 °C, and 5-bromo-2-deoxyuridine (BrdU, Sigma) was added to the culture media at a final concentration of 10 µM for 30 min. Pelleted cells were detached with trypsin, fixed with 80% ethanol, and resuspended in 30 mM HCl/0.5 mg/ml pepsin. BrdU was immunofluorescently labeled with a mouse anti-BrdU antibody (DAKO, clone Bu20a) and a fluorescein-conjugated donkey anti-mouse antibody (Life Technologies), and the cells were stained with propidium iodide (PI; 25 µg/ml) in the presence of ribonuclease A (50 µg/ml). Flow cytometry analyses were performed using an Accuri C6 flow cytometer (BD Biosciences).
6
Quantifying Pancreatic Beta Cell Proliferation
7
Cardiac Cell Proliferation Assessment
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BrdU (Sigma-Aldrich) was injected daily into the peritoneum (100 mg per kg body weight in PBS) for 3 days, with the last injection 2 h before harvesting the hearts. The hearts were fixed in 4% PFA, paraffin-embedded and sectioned. BrdU-positive nuclei were detected with mouse monoclonal anti-BrdU primary antibody (Dako; 1:250) and immunofluorescent staining as described above. BrdU- or Ki67-labelled cells and 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei were counted in nonmyocyte cells from 5 to 10 fields ( × 400 magnification) with focal fibrosis and/or expanded interstitium (identified with wheatgerm agglutinin staining) and in
areas with preserved myocardial architecture (for example, absence of both focal fibrosis and expanded interstitium) in 9–15 sections per heart. Myocytes were identified with fluorescent microscopy visualization of sarcomeres. Nuclei of sarcomere-negative cells within interstitial regions were designated as nonmyocyte nuclei. Nuclei were quantified using the ImageJ nucleus counter software. The percentage of proliferating cells was calculated as the number of positive BrdU- or Ki67-labelled nuclei divided by the number of DAPI-stained nuclei.
areas with preserved myocardial architecture (for example, absence of both focal fibrosis and expanded interstitium) in 9–15 sections per heart. Myocytes were identified with fluorescent microscopy visualization of sarcomeres. Nuclei of sarcomere-negative cells within interstitial regions were designated as nonmyocyte nuclei. Nuclei were quantified using the ImageJ nucleus counter software. The percentage of proliferating cells was calculated as the number of positive BrdU- or Ki67-labelled nuclei divided by the number of DAPI-stained nuclei.
8
Histological and Immunohistochemical Analysis of Colonic Specimens
9
Histological and Immunohistochemical Analysis of Colonic Specimens
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Colonic specimens were prepared and analyzed by pathologists at the City of Hope Pathology Core Lab. Histology and immunohistochemical staining procedures were described previously29 (link). The antibodies used were as follows: BrdU (DAKO, Cat#: M0744), Ki67 (DAKO, Cat#: M7204), and P-Stat3 Tyr705 (Cell Signaling Technology, Cat#: 9145). TUNEL staining was performed using the Cell Death Detection Kit from Roche (Cat#: 11684795910), according to the manufacturer’s instructions. Standards for tumor grading, epithelial damage score and inflammatory cell infiltration score are described in Tables S1 , S2 and S3 66 (link).
10
Quantifying Pancreatic Beta-cell Proliferation
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Mice were intraperitoneally injected with BrdU (100 µg/g; Nacalai Tesque, Inc) for 3 days and sacrificed. Three pancreatic tissue sections (100 µm apart) from each animal were prepared by fixation and paraffin-embedding. The sections were immunostained with antibodies to insulin, muscarinic acetylcholine receptor M1 (Santa Cruz), BrdU (Dako), and muscarinic acetylcholine receptor M3 (Bioss). Biotinylated secondary antibodies, a Vectastain elite ABC kit and a diaminobenzidine substrate kit (Vector) were used to examine the sections under bright-field microscopy to determine the β-cell mass. Alexa Fluor 488- and 555-conjugated secondary antibodies (Invitrogen) were used for fluorescence microscopy analysis. All images were captured using a BZ-9000 microscope (Keyence). The percentage area of the pancreatic tissues occupied by β cells was calculated using BIOREVO software (Keyence)31 (link). In the BrdU staining experiment, 22–78 islets in each mouse were analysed to assess the frequency of BrdU-positive cells among the insulin-positive cells.
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