The proliferation of cells is a complex, multistep process that is regulated by a vast array of extracellular and intracellular signals. Most assays that measure cell proliferation involve the measurement of the rate of DNA replication in cells. The conventional method of measuring DNA synthesis involves the incorporation of tritiated thymidine into cellular DNA. Although, the thymidine incorporation assay is extremely sensitive, it suffers from several disadvantages. Tritiated thymidine has a relatively short shelf life and degrades quickly. On the other hand radioactive tritium has a half-life of 12 years, which makes disposal of such radioactive waste, a cumbersome and complicated process.
The 5-Bromo-2' deoxy-uridine (BRDU) Labeling and Detection Kit from Roche is an alternate method to measure cell proliferation. In this kit, the incorporation of BRDU into the DNA of cells is monitored using a specific monoclonal antibody to BRDU. The newer version of the kit incorporates nucleases in the BRDU solution, which permits direct labeling of the DNA in the cells (no additional denaturation steps required). The assay is as sensitive as the [3H] thymidine uptake assay. But in contrast to the 3H thymidine uptake assay, the BRDU kit is not associated with any of the hazards of handling and disposing of radiochemicals. The assay kit is supplied with detailed protocols to be followed for adherent or suspension cells as well as tissue sections. Out lab has used the kit extensively for measuring the percentage of cells in S-phase both in cell lines and tissues and we have been extremely satisfied with the results. In addition, the kit is compatible with double labeling protocols for immunohistochemistry.
The primary down-side of the kit is its cost – it is expensive. This is primarily because the BRDU antibody is used at a 1: 10 dilution and it cannot be re-used. Since most of the cell proliferation experiments are set up in duplicates, triplicates or quadruplicates; the kit contents get used quickly.
In conclusion, the BRDU kit is an accurate and sensitive method to measure cell proliferation. The kit is highly specific and gives very low background. It does not generate any radioactive contamination or waste, which makes it much less tedious to use. Since, the kit is expensive, it is difficult to optimize it for mass-scale screening assays. However, if money is not a consideration, the kit has no major drawbacks; it is worth its price. I would highly recommend this kit for proliferation assays, especially involving cell cycle-analysis.
Piyali Dasgupta, Ph.D.
Postdoctoral Fellow
H.Lee Moffitt Cancer Research Center
Tampa , Florida