Product Pathways - Cell Cycle / Checkpoint
BrdU Cell Proliferation Chemiluminescent Assay Kit #5492
|Fixing/denaturing Solution||2 x 25 ml|
|BrdU Detection Antibody||0.5 ml|
|Anti-mouse IgG, HRP-linked Antibody||0.5 ml|
|Detection Antibody Diluent||50 ml|
|HRP-linked Antibody Diluent||50 ml|
|Luminol/Enhancer Solution||25 ml|
|Stable Peroxide Buffer||25 ml|
|20X Wash Buffer||50 ml|
Reactivity Key: All=All species expected
Species cross-reactivity is determined by western blot.
The BrdU Cell Proliferation Assay Kit detects 5-bromo-2’-deoxyuridine (BrdU) incorporated into cellular DNA during cell proliferation using an anti-BrdU antibody. When cells are cultured with labeling medium that contains BrdU, this pyrimidine analog is incorporated in place of thymidine into the newly synthesized DNA of proliferating cells. After removing labeling medium, cells are fixed and the DNA is denatured with our fixing/denaturing solution. Denaturing of DNA is necessary to improve the accessibility of the incorporated BrdU to the detection antibody. A BrdU mouse mAb is then added to detect the incorporated BrdU. Anti-mouse IgG, HRP-linked Antibody is used to recognize the bound detection antibody. Chemiluminescent reagent is added for signal development. The magnitude of light emission, measured in relative light units (RLU), is proportional to the quantity of BrdU incorporated into cells, which is a direct indication of cell proliferation.
Specificity / Sensitivity
BrdU Cell Proliferation Chemiluminescent Assay Kit detects BrdU incorporation into cellular DNA during cell proliferation. The BrdU-labeled DNA must be denatured to be detected by the BrdU mouse mAb used in this kit. This BrdU mouse mAb does not cross-react with endogenous DNA. Depending on the cell type and the incubation time applied in the assay, 0.2-2x104 cells/well are sufficient for most experimental setups. For best results, a cell number titration (Figure 1) is recommended.
Figure 1. C2C12 cells were seeded at varying density in serum free medium in a 96-well plate and incubated overnight. Serum was added to the plate at various concentrations and cells were incubated for 24 hr. Finally, 10 μM BrdU was added to the plate and cells were incubated for 4 hr.
Figure 2. Treatment of MCF 10A cells with Human Epidermal Growth Factor (hEGF) #8916 increases cell proliferation as detected by the BrdU Cell Proliferation Chemiluminescent Assay Kit #5492. MCF 10A cells were seeded at 1x104 cells/well in a 96-well plate and incubated overnight. Cells were then starved in serum free medium overnight. hEGF was added to the plate and cells were incubated for 24 hr. Finally, 10 μM BrdU was added to the plate and cells were incubated for 4 hr.
Figure 3. Jurkat cells were seeded at 5x104 cells/well in a 96-well plate and incubated overnight. Cells were then treated with various concentrations of doxorubicin for 2 hr. Finally, 10 μM BrdU was added to the plate and cells were incubated for 4 hr.
Halogenated nucleotides such as the pyrimidine analog bromodeoxyuridine (BrdU) are useful for labeling nascent DNA in living cells and tissues. BrdU becomes incorporated into replicating DNA in place of thymidine and subsequent immunodetection of BrdU using specific monoclonal antibodies allows labeling of cells in S phase of the cell cycle. After pulse-labeling cells or tissues with bromodeoxyuridine, BrdU (Bu20a) Mouse mAb can be used to detect BrdU incorporated into single stranded DNA. Please see our detailed protocol for information regarding the labeling procedure and denaturation of double stranded DNA for various immunodetection applications (1-4).
- Darzynkiewicz, Z. and Juan, G. (2001) Curr Protoc Cytom Chapter 7, Unit 7.7.
- Leif, R.C. et al. (2004) Cytometry A 58, 45-52.
- Staszkiewicz, J. et al. (2009) Biochem Biophys Res Commun 378, 539-44.
- Rothaeusler, K. and Baumgarth, N. (2007) Curr Protoc Cytom Chapter 7, Unit7.31.
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
* Product-specific protocol.
For Research Use Only. Not For Use In Diagnostic Procedures.