Product Pathways - Screening Technologies

BrdU Cell Proliferation Assay Kit #6813

Kit Includes	Quantity
BrdU	0.15 ml
Fixing/denaturing Solution	25 ml
BrdU Detection Antibody	0.5 ml
Anti-mouse IgG, HRP-linked Antibody	11 ml
Detection Antibody Diluent	50 ml
HRP-linked Antibody Diluent	50 ml
20X Wash Buffer	50 ml
TMB Substrate #7004	50 ml
STOP Solution #7002	50 ml

Description

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. Then a BrdU mouse mAb is added to detect the incorporated BrdU (The denaturing of DNA is necessary to improve the accessibility of the incorporated BrdU to the detection antibody). Anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate TMB is added to develop color. The magnitude of the absorbance for the developed color is proportional to the quantity of BrdU incorporated into cells, which is a direct indication of cell proliferation.

Specificity / Sensitivity

BrdU Cell Proliferation Assay kit detects BrdU incorporation into cellular DNA during cell proliferation. The BrdU-labeled DNA has to 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 the best result, a cell number titration (Figure 1) is recommended.

ELISA

Figure 1: C2C12 cells were seeded at varying density (in serum free medium) as shown in the figure in a 96-well plate and incubated overnight. Serum was added to the plate at various concentration as shown in the figure and cells were incubated for 24 hours. Finally, 10 μM BrdU was added to the plate and cells were incubated with BrdU containing medium for 4 hours.

ELISA

Figure 2: Treatment of MCF 10A cells with Human Epidermal Growth Factor (hEGF) #8916 increases cell proliferation as detected by BrdU Cell Proliferation Assay Kit #6813. 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 hours. Finally, 10 μM BrdU was added to the plate and cells were incubated for 4 hours.

ELISA

Figure 3. Jurkat cells were seeded at 4x10⁴ cells/well in a 96-well plate and incubated overnight. Cells were then treated with various concentrations of doxorubicin (red) or staurosporine (blue) for 2 hours. Finally, 10 μM BrdU was added to the plate and cells were incubated for 4 hours.

Background

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 as well as denaturation of double stranded DNA for various immunodetection applications (1-4).

Application References

Canino, C. et al. (2011) Oncogene , . Applications: ELISA

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