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 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 hr. Finally, 10 μM BrdU was added to the plate and cells were incubated for 4 hr.
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 for 2 hr. Finally, 10 μM BrdU was added to the plate and cells were incubated for 4 hr.
|Product Includes||Quantity (with Count)||Solution Color|
|BrdU||1 x 150 µl|
|Fixing/denaturing Solution||2 x 25 ml|
|BrdU Mouse Detection mAb||1 x 500 µl||Green|
|Anti-mouse IgG, HRP-Linked Antibody||1 x 500 µl||Red|
|Detection Antibody Diluent||1 x 50 ml||Green|
|HRP Diluent||1 x 50 ml||Red|
|20X Wash Buffer||1 x 50 ml|
|TMB Substrate 7004||1 x 50 ml|
|STOP Solution 7002||2 x 25 ml|
Incubate for 30 min at room temperature.
NOTE: Watch the color change as it may be necessary to stop the reaction prior to the standard development time of 30 min.
posted October 2012
Protocol Id: 60
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.
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.
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).
Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.
Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.