BrdU (5-bromo-2’-deoxyuridine) is a thymidine analog used in DNA synthesis–based cell proliferation assays. The BrdU assay principle involves the incorporation of BrdU into newly synthesized DNA during cell division. BrdU labeling and detection are achieved by using an anti-BrdU antibody, which binds to the incorporated BrdU, allowing for the imaging and quantification of proliferating cells. This process, often referred to as BrdU staining/labeling, is a critical component of the BrdU cell proliferation assay protocol and is detailed here.

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Materials

This protocol can be used for:

  • Detecting DNA synthesis using a fluorescence microscope

This protocol should not be used for:

You will need the following for this protocol:

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Protocol steps

Prepare stock solutions

  1. Dissolve 100 mg BrdU in 32.5 mL anhydrous DMSO (10 mM stock solution).
  2. Dilute 10 µL of this stock solution in 10 mL of 37°C tissue culture medium to make a 10 µM labeling solution.


Label cells with BrdU

  1. Culture cells in appropriate vessel for microscopy.
  2. Remove culture medium from cells and replace with BrdU labeling solution.
  3. Incubate cells at 37°C for 2 hours.
  4. Remove labeling solution and wash two times with PBS.
  5. Wash with PBS (3 times, 2 minutes each).


Fix, permeabilize, and acid-wash

  1. Remove PBS and add 1 mL of 3.7% formaldehyde in PBS to each well.
  2. Incubate for 15 minutes at room temperature.
  3. Wash with PBS (3 times, 2 minutes each).
  4. Remove PBS and add 1 mL of Triton X-100 permeabilization buffer to each well.
  5. Incubate for 20 minutes at room temperature.
  6. Remove permeabilization buffer and add 1 mL of 1 N HCl.
  7. Incubate 10 minutes on ice.
  8. Remove this solution and add 1 mL of 2 N HCl.
  9. Incubate 10 minutes at room temperature.
  10. Add 1 mL phosphate/citric acid buffer, pH 7.4.
  11. Incubate 10 minutes at room temperature.
  12. Wash with Triton X-100 permeabilization buffer (3 times, 2 minutes each).


Detect incorporated BrdU

  1. Remove this solution and add 1 mL of antibody staining buffer.
  2. Add anti-BrdU primary antibody.
  3. Incubate overnight at room temperature.
  4. Wash with Triton X-100 permeabilization buffer (3 times, 2 minutes each).
  5. Add fluorescently labeled secondary antibody.
  6. Incubate one hour at room temperature.


Imaging

  1. Add PBS to each well.
  2. Image cells with appropriate filters.

 

Protocol tips

  • Leftover BrdU stock solution can be stored frozen for up to one year.
  • Use short incubation time for rapidly proliferating cells, and longer incubation for slow-growing cells.
  • Acid treatment separates DNA into single strands so the primary antibody can access the incorporated BrdU.

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Ordering information

BrdU cell proliferation materials

Frequently asked questions

What is BrdU labeling?

BrdU labeling is a method used to detect cell proliferation by incorporating bromodeoxyuridine (BrdU), a thymidine analog, into newly synthesized DNA during the S-phase of the cell cycle. This allows researchers to measure DNA synthesis and study cell cycle dynamics.

What is the principle of BrdU assay?

The BrdU assay is based on the incorporation of bromodeoxyuridine (BrdU), a thymidine analog, into newly synthesized DNA during the S-phase of the cell cycle. After incorporation, cells are fixed, and the DNA is denatured to expose the BrdU. Anti-BrdU antibodies are then used to detect and quantify the incorporated BrdU, allowing measurement of cell proliferation and DNA synthesis.

How to detect BrdU?

To detect BrdU, incubate cells with BrdU, fix them, denature the DNA, stain with anti-BrdU antibodies, and analyze using flow cytometry or fluorescence microscopy.

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For Research Use Only. Not for use in diagnostic procedures.

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