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BrdU Cell Proliferation Chemiluminescent Assay Kit
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BrdU Cell Proliferation Chemiluminescent Assay Kit #5492

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BrdU Cell Proliferation Chemiluminescent Assay Kit: Image 1
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.
BrdU Cell Proliferation Chemiluminescent Assay Kit: Image 2
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.
BrdU Cell Proliferation Chemiluminescent Assay Kit: Image 3
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.
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To Purchase # 5492
Cat. # Size Qty. Price
5492S		 1 Kit  (500 assays (96 well format))
$ 670

Custom Formulation

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
Luminol/Enhancer Solution 1 x 25 ml
Stable Peroxide Buffer 1 x 25 ml
20X Wash Buffer 1 x 50 ml

Protocol

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BrdU Cell Proliferation Chemiluminescent Assay Kit Protocol

A. Reagent Preparation

  1. Prepare 1X Wash Buffer by diluting 20X Wash Buffer (included in each BrdU ELISA Kit) in purified water.
  2. Prepare 1X detection antibody solution by diluting BrdU Detection Antibody 1:100 with Detection Antibody Diluent (green).
  3. Prepare 1X HRP-conjugated secondary antibody solution by diluting Anti-mouse IgG, HRP-linked Antibody 1:100 with HRP-linked Antibody Diluent (red).
  4. Prepare 10X BrdU solution by diluting BrdU 1:100 with cell culture medium.

B. BrdU Incorporation

  1. Plate cells in 96-well plate and incubate with respective test substance. Typical seed cell number is 2500–100000 cells/well depending on cell growth rate. Typical incubation time is 1–72 hr.
  2. Add prepared 10X BrdU solution to plate wells, for a final 1X concentration. (Example: For 100 µl medium in the plate, add 10 µl of 10X BrdU solution per well.)
  3. Place cells in incubator. Typical incubation time is 1–24 hr.
  4. Remove medium. For suspension cells, centrifuge the plate at 300 g for 10 min, then remove medium.

C. BrdU Assay

  1. Add 100 µl/well of the Fixing/Denaturing Solution, keep the plate at room temperature for 30 min. Remove solution.
  2. Add 100 µl/well prepared 1X detection antibody solution, keep plate at room temperature for 1 hr. Remove solution and wash plate 3 times with 1X Wash Buffer.
  3. Add 100 µl/well prepared 1X HRP-conjugated secondary antibody solution, keep plate at room temperature for 30 min. Remove the solution and wash plate 3 times with 1X Wash Buffer.
  4. Prepare Working Solution by mixing equal parts Luminol/Enhancer Solution and Stable Peroxide Buffer.
  5. Add 100 µl of the Working Solution to each well. Use a plate-based luminometer to measure Relative Light Units (RLU) at 425 nM within 1–10 min following addition of the substrate. Optimal signal intensity is achieved when read within 10 min.

posted October 2012

Protocol Id: 8

Product 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. 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.

Species Reactivity:

All Species Expected

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 and denaturation of double stranded DNA for various immunodetection applications (1-4).
  1. Darzynkiewicz, Z. and Juan, G. (2001) Curr Protoc Cytom Chapter 7, Unit 7.7.
  2. Leif, R.C. et al. (2004) Cytometry A 58, 45-52.
  3. Staszkiewicz, J. et al. (2009) Biochem Biophys Res Commun 378, 539-44.
  4. Rothaeusler, K. and Baumgarth, N. (2007) Curr Protoc Cytom Chapter 7, Unit7.31.

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