Cells were incubated with BrdU (BrdU Labeling Reagent, Ready-to-use, Invitrogen, 1:100 diluted in culture medium) for 6 h at 37 °C. Cells were washed with PBS and fixed in acid ethanol (90% ethanol, 5% acetic acid, 5% H2O) for 30 min at room temperature, followed by incubation in 2 M HCl for 20 min at room temperature. 0.1 M sodium borate (pH 8.5) was added to the cells and incubated for another 2 min. Cells were washed with PBS and incubated with BrdU antibody (Cell Signaling, 1:1 400) in 3% BSA-PBS at 4 °C overnight. After washed with PBS, the cells were incubated with Cy3-conjugated donkey anti-mouse IgG. Nuclei were stained with DAPI (Sigma). The BrdU-positive cells were counted using an Olympus microscope (IX71).
Brdu antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The BrdU antibody is a laboratory reagent used to detect and quantify cell proliferation. It recognizes and binds to 5-bromo-2'-deoxyuridine (BrdU), a synthetic nucleoside analog that is incorporated into the DNA of dividing cells during the S phase of the cell cycle. This antibody can be used in various immunoassay techniques, such as immunohistochemistry and flow cytometry, to identify and analyze proliferating cells.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Brdu labeling reagent, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342 dye, by Thermo Fisher Scientific (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Imagexpress micro microscope, by Molecular Devices (1 mentions)
5 bromo 2 deoxyuridine brdu, by Merck Group (1 mentions)
5 protocols using brdu antibody
1
BrdU Labeling and Quantification
2
Comprehensive Cell Proliferation and Survival Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the cell proliferation assay, ReNCell VM cells were seeded into laminin coated 96-well plates at a density of 4 × 103 cells/well (1.25 × 104 cells/cm2). Cells were treated with indicated compound or DMSO control for 48 hours before they are fixed and stained with Hoechst 33342 dye (Life Technologies, Grand Island, NY, USA). The plate was scanned with an ImageXpress Micro microscope (Molecular Devices, Sunnyvale, CA, USA) using a 4× objective to take an image covering the whole well for each well. Cell numbers were counted using the ImageJ software with an automatic nuclei counter plug-in. Cell proliferation was also measured by BrdU assay. Cells were grown in laminin coated 96-well plates, and treated with indicated compound for 48 hours. After that cells were pulsed with 10 μM BrdU for 2 hours, then stained with BrdU antibody (1:1000, Cell Signaling, Danvers, MA, USA). For the survival assay, cells were differentiated in the presence of indicated compounds. Then the cells were fixed, permeabilized and assayed with the Roche in situ Cell Death detection kit following the manufacturer’s instruction. Cells were also blocked and stained with additional markers as indicated.
3
BrdU Incorporation Immunofluorescence Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BrdU incorporation was analyzed according to the Immunofluorescence Protocol for Labeling with BrdU antibody from the Cell Signaling Technology (Danvers, MA, USA). Details are provided in the Supplementary Methods.
4
GH-induced Proliferation in Ba/F3 and BEAS-2B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ba/F3 cells transduced with WT and GHRP495T were starved overnight and seeded at a cell density of 1 × 104 cells/ml duplicate in growth media supplemented with GH at 4.5 nM instead of IL-3 (day 0). Cells were counted daily by Trypan blue exclusion using a hemocytometer. Proliferation in BEAS-2B was measured by BrdU incorporation. Briefly, cells were grown on coverslips at equal seeding densities and serum starved the following day to induce cell cycle synchronisation. Cells were then treated with GH at 4.5 nM for 24 and 48 h. Before fixing, cells were given 20 μM 5-bromo-2′-deoxyuridine (BrdU, Sigma-Aldrich) for 1 h. Cells were washed with PBS and fixed in 70% ethanol and coverslips were stained with BrdU antibody (#5292, Cell Signaling, Danvers, MA, USA) as per the manufacturer’s guidelines, followed by Alexa Fluor-488 antibody and counterstained with 10 μg/ml 4,6-diamidino-2-phenylindole (DAPI). Random fields of views were first imaged for DAPI and the same field of view was imaged for BrdU and quantified blind. Data are represented as percentage of BrdU-positive cells.
5
In vivo S-phase Entry Analysis
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!