The BrdU assay is a standard laboratory technique used to measure cell proliferation (cell division). It relies on the incorporation of the synthetic compound BrdU into the DNA of dividing cells. By labeling newly synthesized DNA, the assay provides a quantifiable way to determine the percentage of cells actively progressing through their reproductive cycle. This non-radioactive technique is a precise alternative to older methods that relied on radioactive materials.
The Mechanism of BrdU Incorporation
The BrdU assay is based on the cell’s natural process of DNA replication. BrdU (5-bromo-2′-deoxyuridine) is a synthetic molecule that structurally mimics the nucleoside thymidine, one of the four building blocks used to construct new DNA strands. The difference is the substitution of a bromine atom for the methyl group found on the thymidine ring.
When BrdU is introduced, actively dividing cells absorb it. During the S-phase (synthesis phase) of the cell cycle, when the cell replicates its genome, the machinery mistakenly incorporates BrdU into the new DNA strand instead of thymidine. This incorporation serves as a permanent tag within the nuclear DNA.
Cells resting in the G0 or G1 phases will not incorporate the BrdU label. Only cells that have progressed through the S-phase during the incubation period will be labeled. This selective tagging allows researchers to distinguish between proliferating and quiescent cells, providing a direct measurement of DNA synthesis.
Research Applications of the BrdU Assay
The ability to precisely measure cell division makes the BrdU assay widely used across biological research. In cancer research, where uncontrolled proliferation is a defining characteristic, the assay measures the growth rate of tumor cells. It is used to assess how effectively a new drug or therapy inhibits growth; a reduction in BrdU incorporation indicates successful suppression of the proliferation rate.
The assay is also used in neurobiology for mapping neurogenesis, the creation of new neurons. Scientists administer BrdU to label dividing precursor cells in the brain and track their fate as they mature. This technique helps study how factors like aging or disease affect the brain’s ability to generate new cells.
Furthermore, the BrdU assay is employed in toxicology and drug screening studies. Researchers use it to determine if a specific compound or toxin has an inhibitory or stimulatory effect on cell growth. Quantifying the percentage of BrdU-positive cells after exposure allows scientists to assess the compound’s impact on cellular health.
Step-by-Step Guide to the BrdU Protocol
The BrdU protocol is a multi-step process designed to label dividing cells and make the incorporated BrdU visible for detection.
BrdU Labeling or Pulsing
The BrdU compound is added to the cell culture medium or injected into a living organism. Cells are incubated with the solution for a specific period, known as the pulse time, often 30 minutes to several hours. This duration is chosen to ensure that only cells actively synthesizing DNA during that window incorporate the label, providing a snapshot of the proliferation rate.
Cell Fixation and DNA Denaturation
After labeling, cells undergo fixation, typically with formaldehyde, to preserve cellular structures and halt biological processes. Denaturation is necessary because BrdU is tightly embedded within the double-stranded DNA helix, making it inaccessible to the detection antibody. DNA denaturation, usually achieved with a strong acid like hydrochloric acid (HCl) or a nuclease enzyme, separates the DNA strands. This exposes the incorporated BrdU molecules for antibody targeting.
Antibody Staining
This stage is the core detection mechanism. A specific anti-BrdU primary antibody is introduced, designed to bind selectively only to the exposed BrdU molecules in the DNA. This primary antibody is then detected by a secondary antibody that is chemically linked to a fluorescent dye or an enzyme. The secondary antibody binds to the primary antibody, marking the location of the incorporated BrdU with a visible signal.
Detection
The signal generated by the labeled cells is captured and quantified. If the secondary antibody is linked to a fluorescent dye, the signal is visualized using a fluorescence microscope. If the secondary antibody is linked to an enzyme, a substrate is added that reacts to produce a measurable color change, which can be quantified using a plate reader.
Analyzing and Quantifying BrdU Results
After the staining protocol, labeled cells are analyzed using two primary methods to generate quantifiable data.
Fluorescence Microscopy or Immunohistochemistry
This approach allows for the direct visualization of labeled cells within a tissue section or culture dish. The fluorescent signal pinpoints the location of proliferating cells, often appearing as bright nuclei against a counterstained background. Researchers manually count the number of BrdU-positive cells and express the result as a percentage of the total cell count, providing an index of cell proliferation.
Flow Cytometry
Flow cytometry rapidly and objectively quantifies BrdU incorporation in large populations of suspended cells. Cells flow past a laser beam, and a detector measures the fluorescent signal intensity from the anti-BrdU antibody in each cell. This technique can combine BrdU detection with total DNA staining, using dyes like DAPI, to determine the cell cycle phase of each BrdU-positive cell. The resulting data is displayed as a scatter plot, allowing calculation of the Proliferation Index.