MIGR-hKCTD11-infected prostate cancer cell lines were cultured onto a 12 mm diameter glass. After 24 hrs, BrdU was added (BrdU labeling and detection kit, Boehringer Mannheim) according to the manufacturer's instructions. Cells were incubated for 8 and for 24 hours and then fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.25% Triton X in PBS, washed, and incubated with primary antibody (anti-GFP, Santa Cruz Biotechnology). After 1 hr, cells were washed and incubated with goat anti-rabbit Alexa Flour 488 (Santa Cruz Biotech) secondary antibody for 45 min. Cells were treated with 4% paraformaldehyde and then 2 N HCl. After three washes, cells were incubated with 1 : 10 primary anti-BrdU (BrdU labeling and detection kit, Boehringer Mannheim) and, afterward, with goat anti-mouse IgG TRITC (Sigma-Aldrich) secondary antibody. Hoechst was used for nuclear staining. Cells were analyzed by using a Zeiss Axioplan 2 (Carl Zeiss) fluorescence microscope.
Brdu labeling and detection kit
Manufactured by Roche
Sourced in United States, Germany
The BrdU labeling and detection kit is a laboratory tool used for the detection and visualization of cells that have incorporated the synthetic thymidine analog bromodeoxyuridine (BrdU) during DNA synthesis. The kit provides the necessary reagents and protocols to label, detect, and quantify BrdU-positive cells, which is a common technique in cell proliferation and cell cycle studies.
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Lab products found in correlation
In situ cell death detection kit, by Roche (2 mentions)
5 bromo 2 deoxyuridine brdu labeling reagent, by Roche (2 mentions)
Spectramax 340pc, by Molecular Devices (2 mentions)
Glutamine synthetase antibody, by BD (2 mentions)
β catenin antibody, by BD (2 mentions)
Sa β gal staining kit, by Cell Signaling Technology (2 mentions)
Axioplan 2, by Zeiss (1 mentions)
Goat anti mouse igg tritc, by Merck Group (1 mentions)
Anti gfp, by Santa Cruz Biotechnology (1 mentions)
Ks300, by Zeiss (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Axiovert 2, by Zeiss (1 mentions)
Fluorescein fitc conjugated secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Epoch gen5, by Agilent Technologies (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Dulbecco modified eagle medium (dmem), by Nacalai Tesque (1 mentions)
Epics xl 4 color cytometer, by Beckman Coulter (1 mentions)
Zen software, by Zeiss (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (1 mentions)
40 protocols using brdu labeling and detection kit
1
Prostate Cancer Cell Proliferation Assay
2
BrdU Labeling and Immunofluorescence Assay
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We used a bromodeoxyuridine (BrdU) labeling and detection kit from Boehringer Mannheim (Germany). Cells were seeded on glass slides, incubated for 1 h with BrdUrd (final concentration of 10 µmol/L), fixed, and permeabilized with ethanol-glycine solution. Slides were incubated with anti-BrdU mouse monoclonal antibody and with a fluorescein (FITC)-conjugated secondary antibody (Boehringer Mannheim, Mannheim, Germany) and mounted in PBS 1X-Glycerol on specimen holder slide.
For immunofluorescence, MDM2 was revealed with an anti-MDM2 primary antibody and fluorescein (FITC)-conjugated secondary antibodies (Jackson Immuno Research Laboratories, Inc., Philadelphia, PA, USA). Cell nuclei were identified by Hoechst 33258 (final concentration of 1 µg/mL; Sigma ChemicalCo.) staining. The fluorescent signal was visualized with an epifluorescent microscope (Axiovert 2, Zeiss; equipped with a ×100 lens) interfaced with the image analyzer software KS300 (Zeiss). At least 100 GFP-positive cells were counted in five different microscopic fields.
For immunofluorescence, MDM2 was revealed with an anti-MDM2 primary antibody and fluorescein (FITC)-conjugated secondary antibodies (Jackson Immuno Research Laboratories, Inc., Philadelphia, PA, USA). Cell nuclei were identified by Hoechst 33258 (final concentration of 1 µg/mL; Sigma ChemicalCo.) staining. The fluorescent signal was visualized with an epifluorescent microscope (Axiovert 2, Zeiss; equipped with a ×100 lens) interfaced with the image analyzer software KS300 (Zeiss). At least 100 GFP-positive cells were counted in five different microscopic fields.
3
C2C12 Cell Proliferation Assay
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C2C12 MBs (1×104 cells/well, 100μl) were seeded into 96-well plates for 24 hours. Fresh serum free medium containing the indicated concentrations of CCL2 was added. Cell proliferation was measured by monitoring BrdU incorporation. Cells were incubated with BrdU for 3 hours, and then cell proliferation was assayed using a BrdU labeling and detection kit (Roche, Mannheim, Germany). Absorbance was measured at 450 nm, with a reference wavelength of 650 nm, using a microplate reader (BioTek Epoch Gen5).
4
PTEN Regulation of MRC-5 Cell Proliferation
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MRC-5 cells were seeded on glass slides in 24-well plates and transfected with PTEN siRNA or NC siRNA for 24 h without FBS, followed by incubation with MEM-α containing 10 % FBS and antibodies for another 24 h. BrdU incorporation was measured using a BrdU labeling and detection kit (Roche, Mannheim, Germany) in accordance with the manufacturer’s protocol. Briefly, MRC-5 cells were labeled with 10 μM BrdU during an incubation of 50 min. After labeling, the cells were washed three times, fixed, and treated with mouse anti-BrdU monoclonal antibody for 30 min at 37 °C, followed by an anti-mouse-Ig-fluorescein working solution. Cells were washed three times and mounted with DAPI-containing mounting media (ZSGB-BIO, Beijing, China). Cellular proliferation was assessed by the percent of BrdU-staining cells in nuclear-positive cells in the same microscopic visual field. Experiments were performed three independent times. The BrdU positive rate was evaluated by counting five different microscopic fields per time point.
5
Quantifying Bovine Hyalocyte Proliferation
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In order to determine the extent of cellular proliferation of bovine hyalocytes, the incorporation of BrdU was quantified using a BrdU labeling and detection kit (11296736001; Roche, Indianapolis, IN), following the manufacturer's instructions. Briefly, 1 × 10 4 cells were cultured in DMEM (Nacalai Tesque) in 96-well mL plates at 37 C under 5% CO 2 . Cells were washed 24 h after plating, and the media were replaced with 100 mL vitreous from ERM or PDR patients. At 48 h after stimulation, 10 mmol/L BrdU was added to each well. The cells were continuously cultured under the same conditions for 2 h, during which BrdU was incorporated into freshly synthesized DNA. Following fixation of cells, cellular DNA was partially digested by nuclease treatment. The incorporated BrdU was detected and quantified using a peroxidase-labeled antibody for BrdU. Peroxidase catalyzes the cleavage of the substrate, which produces a color reaction. The absorbance at 450 nm was directly correlated to the level of BrdU incorporated into cellular DNA and was measured using a microplate reader (Bio-Rad) to assess the extent of hyalocyte proliferation.
6
BrdU Labeling for Kidney Analysis
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BrdU was administered to the mice (i.p., 1.5 mg/day) for 3 or 7 days before kidney collection. BrdU immunofluorescent staining was performed according to the manufacturer’s instructions (BrdU Labeling and Detection Kit, Roche, Indianapolis, IN, USA).
7
Quantifying Cell Proliferation in Mouse Embryos
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Cell proliferation activity was evaluated by 5-bromodeoxyuridine (BrdU) labeling and immunofluorescence staining (37 (link)). Briefly, timed pregnant mice were injected intraperitoneally with BrdU solution (3 mg/100 g of body weight) from a BrdU labeling and detection kit (Roche) 30 min before embryo harvesting. The collected embryos were fixed in 4% paraformaldehyde solution at 4 °C for 1 h and embedded in paraffin. Then embryos were processed for paraffin sectioning at 5 μm for immunohistochemical staining. BrdU-labeled cells were detected immunohistochemically in paraffin sections according to the manufacturer’s instructions. Apoptosis was assayed by TUNEL staining using the In Situ Cell Death Detection Kit (Roche) according to the manufacturer’s protocol. The cell proliferation rate was counted (n = 3–6 samples) and calculated as the percentage of BrdU-labeled cells among total nuclear stained cells (4′,6-diamidino-2-phenylindole [DAPI] positive) within a defined arbitrary area. The proliferation rate of control (ShhCre) embryos was set to 1, and then the relative proliferation rate of Isl1ShhCre mutant embryos was calculated.
8
Platelet-Mediated Cell Proliferation Assay
9
BrdU and Ki67 Imaging of Panc-1 Cells
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The BrdU labeling and detection kit (Roche) was used to detect the BrdU incorporation into cellular DNA as per the manufacturer’s instructions. Cells were fixed and visualized with a research fluorescence microscope (AX10 Observer SD; Carl Zeiss) at 200× magnification at room temperature. Ki67 protein was detected by immunostaining of Panc-1 cells using a rabbit antibody directed against Ki67 (1:50 dilution; Abcam) and secondary goat anti–rabbit conjugated Alexa Fluor 488 (1:200 dilution; Molecular Probe). Hoechst 33258 nuclear dye was used to visualize the nucleus. Cells were fixed and imaged with a research fluorescence microscope (AX10 Observer SD) under the magnification of 630× at room temperature. Image acquisition was performed using ZEN software (Carl Zeiss).
10
Cardiomyocyte Differentiation and Cell Cycle Analysis
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