MG-63 and Saos-2 cells were seeded in 24-well plates. After 24 h, they were added with 10 μmol/L BrdU (Beyotime, Shanghai, China) during the logarithmic proliferation phase for 2 h. After denaturing the cells, BrdU primary antibody (ab8152, 1: 100, Abcam, Shanghai, China) was added and incubated for 2 h at room temperature. Next, with the addition of fluorescent secondary antibody, the cells underwent another 2-hour incubation at room temperature. Finally, we labeled the nucleus with 10 μmol/L Hoechst33342 (Beyotime, Shanghai, China) and took photographs using a fluorescent inverted microscope and performed statistical analysis.
Brdu primary antibody
Manufactured by Abcam
Sourced in United States
The BrdU primary antibody is a laboratory reagent used for the detection and quantification of cells undergoing DNA synthesis during the S-phase of the cell cycle. It binds specifically to the thymidine analog bromodeoxyuridine (BrdU), which is incorporated into newly synthesized DNA during cell division.
Automatically generated - may contain errors
Lab products found in correlation
Bromodeoxyuridine (brdu), by Merck Group (4 mentions)
Bromodeoxyuridine (brdu), by Beyotime (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Dnase, by Tiangen Biotech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fluorescence microscopy, by Nikon (1 mentions)
Dylight 594 griffona simplicifolia lectin b4, by Vector Laboratories (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Goat serum, by ZSGB-BIO (1 mentions)
Aqueous one solution cell proliferation assay kit, by Promega (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
9 protocols using brdu primary antibody
1
BrdU Incorporation Assay for Cell Proliferation
2
Quantifying Cell Proliferation with BrdU
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According to previous experimental methods [29 (link)], Cells plated 24-well plates were fixed in paraformaldehyde, BrdU primary antibody was incubated (Abcam, Cambridge, MA, USA) with cells overnight. Finally, nuclei were stained by DAPI, and followed by imaging to select the field of vision.
3
TBMS1 Cytotoxicity in Melanoma Cells
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2 × 104 melanoma cells per well were cultured in 24-well plates overnight in a 37°C incubator and then were treated with TBMS1 alone or combined with other drugs for another 48 h. DMSO was used as control. After 48 h, 10 μg/ml BrdU (Sigma Aldrich) was added into cells for 2 h and then fixed with 4% paraformaldehyde for 15 min. Then cells was treated with 2 M HCl for 30 min and then followed by 0.3% Triton X-100 treatment for 15 min. Subsequently, cells were blocked with 10% goat serum in the PBST buffer (ZSGB-Bio, Beijing, China). Cells were then incubated with BrdU primary antibody (Abcam, Cambridge, MA, United States) and next with secondary antibody (Life Sciences, New York, NY, United States). Before being observed by microscopy, cells were stained with Hoechst 33258 (Life Sciences). BrdU-positive melanoma cells in random fields were counted and calculated.
4
BrdU Incorporation Assay Protocol
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1.5 × 103 cells/well cells were cultured in 96‐well dish in the medium with a final concentration of 10μM BrdU. After incubation for 3 h, cells were fixed by 4% paraformaldehyde (PFA) for 15 min, then washed by PBS solution and treated with 0.2 TrtonX‐100, after which they were further washed and treated with DNase (Tiangen, China) for 20 min at room temperature. Then washed the cells with PBS and added BrdU primary antibody (Abcam, Cambridge, MA, USA) and incubated the cells at 4°C for 12 h. After washing the cell with PBS, cells were incubated with secondary antibody for 60 min. Washed the cells with PBS and detected the BrdU positive cells by fluorescence microscope.
5
BrdU Incorporation Assay for Cell Proliferation
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A BrdU incorporation assay was used to measure the cell proliferation. H9c2 cells were seeded into a μ-Slide 8-well glass bottom plate at a total number of 7500 per well. After treatment, the cells were incubated with medium containing 10 μM BrdU (Sigma, USA) for 24 hours. Then, cells were fixed by 70% ethanol and incubated with 2 M HCl for 30 min at room temperature. 1% BSA was used for blocking, and the cells were incubated with a BrdU primary antibody (1 : 500, Abcam, UK) overnight and with a secondary antibody (1: 250) for 2 hours at room temperature. DAPI (Invitrogen, USA) was stained in the final step. A fluorescent image was detected using a confocal microscope equipped with a 63x oil immersion objective. Blue fluorescence represents DAPI staining, and red fluorescence represents BrdU. Images were analyzed by using the manufacturer's software. The percentage of positive cells was calculated by counting the double-stained cells and total cells (as a standard) in five randomized fields.
6
Quantifying Proliferation in CNV Mice
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Proliferation of vascular endothelial cells in CNV mice was assessed by immunofluorescence staining for 5-bromo-2-deoxyuridine (BrdU). At D0 and D7 after photocoagulation, the mice were given an intravitreous injection with ATN-161 (1 μL, 1 μg/μL) in one eye and PBS (1 μL) in the other eye. These mice were injected intraperitoneally with BrdU (Sigma, St. Louis, MO) 0.2 mg per gram body weight at D2, D5, D7, D10, D13, and D14 [23 (link),24 (link)]. Eyes were collected at D7 and D14 and fixed with 4% paraformaldehyde for 1 h and then cut into 10-μm-thick slices. After being washed and permeabilized in PBS-T (PBS containing 1% Triton X-100) for 30 min, these slices were placed in 2N HCl for 30 min at room temperature and then washed 3 times with PBS-T. Before incubated in the mixture of Alexa 488-conjugated anti-rat secondary antibody (Abcam) and Dylight 594 Griffona simplicifolia Lectin-B4 (Vector Laboratories), the slices were incubated in BrdU primary antibody (Abcam) overnight at 4°C. Prolong gold antifade reagent with DAPI (Invitrogen) was used to label nuclei. The choroidal membranes of the mice were carefully dissected and flat-mounted after performing the steps described above. The images were captured by fluorescence microscopy (Nikon).
7
BrdU Quantification of Endothelial Cells
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Mice at E10.0 of gestation were weighed and injected with 0.1 μg/g BrdU. Half an hour after injection, the mice were euthanized via cervical dislocation. Embryos were isolated immediately, fixed with 4% paraformaldehyde for 2 h at 4 °C and embedded in paraffin. BrdU was detected by treating tissue sections with 2 N HCl for 20 min at 37 °C and incubating BrdU primary antibody (Abcam, 1:100) overnight at 4 °C. After 3 washes for 2 min each in PBS, a corresponding HRP-conjugated secondary antibody (Zhongshan golden bridge) was applied for 30 min at RT. After 3 washes for 2 min each in PBS, sections were stained with TSA Plus Fluorescein Kit (Histova, NEON 5-color IHC Kit for FFPE, 1:100, 20–60 s). After all antigens were labeled, images were captured on an Akoya Vectra Polaris (PerkinElmer). The numbers of BrdU+ endothelial cells in each EP cluster were measured using ImageJ software from merged images.
8
Melanoma Cell Proliferation Assay by BrdU Staining
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Cell proliferation was monitored by BrdU staining.61 2 × 104 melanoma cells in logarithmic phase were seeded in 24-well plates and then attached overnight in 37 °C incubator. demethylzeylasteral (5 μM) was then added to medium and changed to melanoma cells. DMSO was used as control. After 48 h, 10 μg/ml BrdU (Sigma Aldrich, USA) was added into cells for 2 h and then fixed with 4% paraformaldehyde for 15 min. After treating with 2 M HCL and then treating with 0.3% TritonX-100, cells were blocking with 10% goat serum (ZSGB-Bio, Beijing, China). Cells were then successively incubated with BrdU primary antibody (Abcam, Cambridge, MA, USA) and then with secondary antibody (Life, New York, NY, USA). Before observing by microscopy, cells were stained with Hochest (Life, New York, NY, USA). BrdU-positive cells in random fields were counted.
9
Cell Proliferation and BrdU Incorporation Assays
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CellTiter 96 ® . A Queous One Solution Cell Proliferation Assay kit (Promega Corporation, Madison, WI, USA) was used to perform the 3-( 4
MTS) assay by following the instruction. The record absorbance at 490 nm was detected by microplate reader. We performed the detection every 24 h during 3 days.
Bromodeoxyuridine (BrdU) incorporation assay. Cells were seeded in 96-wells (2x10 3 cells/well) in the culture medium with BrdU incubation. After 2 h, phosphate-buffered saline (PBS) solution with paraformaldehyde (PFA) (4%) was used to fix cells for 20 min. Cells were washed by PBS and treated by DNase for 20 min at room temperature. Then, BrdU primary antibody (Abcam, Cambridge, MA, USA) was added and incubated at 4˚C for 12 h. After washing by PBS, cells were incubated with secondary antibody at room temperature for 60 min. Cell nucleus were dyed by 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich; Merck KGaA). BrdU-positive cells were counted by microscope.
MTS) assay by following the instruction. The record absorbance at 490 nm was detected by microplate reader. We performed the detection every 24 h during 3 days.
Bromodeoxyuridine (BrdU) incorporation assay. Cells were seeded in 96-wells (2x10 3 cells/well) in the culture medium with BrdU incubation. After 2 h, phosphate-buffered saline (PBS) solution with paraformaldehyde (PFA) (4%) was used to fix cells for 20 min. Cells were washed by PBS and treated by DNase for 20 min at room temperature. Then, BrdU primary antibody (Abcam, Cambridge, MA, USA) was added and incubated at 4˚C for 12 h. After washing by PBS, cells were incubated with secondary antibody at room temperature for 60 min. Cell nucleus were dyed by 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich; Merck KGaA). BrdU-positive cells were counted by microscope.
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