Transfected HCC827 and A549 cells (1 × 105 cells/ml) were seeded in 35 mm diameter dishes (containing a slide inside) and cultured for 1 day and synchronized with a medium containing 0.4% FBS for 3 days. Subsequently, 1.0 mg/ml BrdU reagent (BD Pharmingen, San Diego, CA, USA) was loaded and subsequently incubated at 37°C for 4 h. Next, the medium was discarded, and the slides were washed three times with phosphate buffer saline (PBS) and subsequently fixed with methanol for 10 min, then blocked with 5% normal rabbit serum, and DNA was denatured by formalin. Next, the cells were rinsed in PBS and then incubated with an anti-BrdU antibody (1:500, Beyotime, Shanghai, China). After incubation for 2 h at ambient temperature, the cells were incubated with secondary antibody (1:500, Beyotime) for 1 h at room temperature and then rinsed three times in PBS. After that, the cells were stained with DAPI staining solution (Beyotime, Haimen, China). Finally, the total numbers of cells and BrdU-positive cells in random five high-power visual fields were counted under a fluorescence microscope.
Anti brdu antibody
Manufactured by Beyotime
Sourced in China
The Anti-BrdU antibody is a laboratory reagent used to detect the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA during cell proliferation. It binds specifically to BrdU, allowing researchers to visualize and quantify cell division processes.
Automatically generated - may contain errors
Lab products found in correlation
Dapi staining solution, by Beyotime (3 mentions)
Brdu solution, by Beyotime (3 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Hoechest staining solution, by Beyotime (2 mentions)
Brdu reagent, by BD (1 mentions)
Brdu staining reagent, by Beyotime (1 mentions)
Dapi solution, by Beyotime (1 mentions)
8 protocols using anti brdu antibody
1
Cell Proliferation Assay in Lung Cancer
2
Cell Proliferation Rate Quantification
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Huh-7 and SMMC-7721 cells in the logarithmic growth phase were harvested to prepare the single-cell suspension, and the cells were then inoculated into a 24-well plate (1×105 cells/well) before BrdU staining reagent (Beyotime, Shanghai, China) was added. After the incubation for 12 h, the cells were fixed with 4% paraformaldehyde, incubated with anti-BrdU antibody (Beyotime, Shanghai, China) and then were stained with the DAPI staining solution (Beyotime, Shanghai, China) to label the nuclei. The BrdU positive cells and the DAPI positive cells in three visual fields were counted under a fluorescence microscope (Olympus, Tokyo, China). Cell proliferation rate=number of BrdU-positive cells/number of DAPI-positive cells.
3
Quantifying Proliferative Capacity of GC Cells
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Following transfection, GC cells in each group were moved onto coverslips (Beyotime, Shanghai, China) for 12 hours of culture. Later, the cells were incubated with BrdU solution (Beyotime, Shanghai, China) for 6 hours. The culture medium was discarded. GC cells were immobilized with 4% paraformaldehyde for 30 minutes, incubated with anti-BrdU antibody (Beyotime, Shanghai, China) for an hour at room temperature (RT), and flushed with phosphate buffer saline (PBS). The number of positive BrdU cells was calculated [25 (link)].
4
Cell Proliferation Assay Protocol
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The single-cell suspension was prepared with A549 cells and H1299 cells in the logarithmic growth phase. Subsequently, the cells were inoculated into a 24-well plate at the density of 1×105 cells/well. BrdU reagent (Beyotime Biotechnology, Shanghai, China) was supplemented into each well, and after 12 h of culture, the cells were incubated with anti-BrdU antibody (Beyotime Biotechnology, Shanghai, China) at room temperature for 2 h. After that, the cells were counterstained with DAPI staining solution (Beyotime Biotechnology, Shanghai, China). Then, the number of BrdU positive cells and the total number of DAPI positive cells were counted under a fluorescence microscope (Olympus, Tokyo, Japan). Cell proliferation rate=BrdU positive cells/DAPI positive cells × 100%.
5
BrdU Assay for VSMC Proliferation
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VSMCs in the logarithmic growth phase were harvested to make single-cell suspensions, and 1×105 cells/well were seeded into 24-well plates. After the cells adhered to the bottom of the wells, BrdU labeling reagent (WuHan AmyJet Scientific Inc., Wuhan, China) was added to each well according to instructions, and the plates were maintained in 5% CO2 at 37°C. After 8 h of culture, the medium was discarded, and the cells were fixed with 4% paraformaldehyde. Then 2 mol/L HCl was added to denature the DNA of the cells. The cells were then washed with PBS, and anti-BrdU antibody (Beyotime Biotechnology) was added prior to incubation of the cells at 4°C for 12 h. Next, DAPI solution (Beyotime Biotechnology) was added to stain the nuclei for 30 min. Under the fluorescence microscope, 3 fields were randomly selected and the number of the cells in each field was counted. The cell proliferation was calculated as follows: cell proliferation rate=number of BrdU-positive cells/number of DAPI-positive cells. The mean value of the cell proliferation rates in 3 fields was adopted as the proliferation rate for the cells in each group.
6
Proliferation Assay Using BrdU Kit
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After CC cells were seeded in 96-well plates at a density of 2 × 103 cells/well and cultured for 24 h, the BrdU kit (BD Pharmingen, San Diego, CA, USA) was added into each well (final concentration: 10 µM), and the culture was continued for 8 h. Subsequently, the medium was removed, and cells were fixed with 4% paraformaldehyde for 30 min and then incubated with anti-BrdU antibody (Beyotime, Shanghai, China) for 1 h at room temperature. After the cells were rinsed with phosphate buffer saline (PBS) for 3 times, Hoechest staining solution (Beyotime, Shanghai, China) was used for marking cell nuclei. Three fields were randomly selected under a fluorescence microscope. Cell proliferation rate = BrdU positive cells / Hoechst positive cells × 100%.
7
BrdU Cell Proliferation Assay
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The transfected GC cells in each group were transferred onto coverslips (Beyotime, Shanghai, China) and then cultured for 12 h. Subsequently, the cells were incubated BrdU solution (Beyotime, Shanghai, China) for 6 h. The GC cells, after the medium was discarded, were fixed with 4% paraformaldehyde for 30 min, and incubated with anti-BrdU antibody (Beyotime, Shanghai, China) at ambient temperature for 1 h, and then rinsed with phosphate buffer saline (PBS). Subsequently, the nuclei were stained employing Hoechest staining solution (Beyotime, Shanghai, China). After the cells were washed by PBS again, the numbers of BrdU and Hoechst positive cells were counted.
8
Cell Proliferation Assay via BrdU
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MDA-MB-231 and MCF-7 cells were transferred into 24-well plates (with a cover glass in each well, 2.5 × 105 cells/well), respectively. After 24 h, the cells were incubated with BrdU solution (Beyotime Biotechnology, Shanghai, China) for another 4 h and then fixed for 30 min with 4% paraformaldehyde. After the supernatant was discarded, the cells were incubated with anti-BrdU antibody (1: 500, Beyotime Biotechnology, Shanghai, China) for 30 min at room temperature and then with 100 μL of 1× Hoechst 33,342 reaction solution at room temperature in the dark for 20 min, followed by being rinsed with PBS. In 10 random high-power fields under the microscope, the number of BrdU-positive cells and the total number of cells were calculated, and the average was taken.
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