5-Bromo-2'-deoxy-uridine Labeling and Detection Kit I from Roche Applied Science

The ability to measure DNA synthesis or cell prolifera¬tion is important in cell biology research. It may be studied by monitoring the incorporation of a radioisotope, [³H]-thymidine, into cellular DNA and subsequent autoradiography. Because this assay is labor intensive and uses expensive and potentially hazardous materials, alternative assays have been developed. In one of these alternative experimental approaches, 5-bromo-2'-deoxy-uridine (BrdU) may be used instead of thymidine. Cells that have incorporated BrdU into DNA are easily detected using a monoclonal antibody against BrdU and an enzyme- or fluorochrome-conjugated second antibody.

The 5-Bromo-2'-deoxy-uridine Labeling and Detection Kit I from Roche comes with all the necessary reagents for up to 100 tests detecting BrdU incorporation into cellular DNA in vitro or in vivo, using immunofluorescence microscopy as detection method. All the vials are color coded for easy handing and include the sterile BrdU labeling reagent, wash buffer, incubation buffer, anti-BrdU antibody with nucleases for DNA denaturation, and a fluorescein labeled sheep anti-mouse antibody.

I used this kit for the detection of newly synthesized cellular DNA by in vitro labeling of cultured suspension cells which were subsequently fixed and spun down on slides using a Cytospin (Shadon, Thermo Scientific). The cells were visualized by performing confocal laser microscopy.

There are several advantages of this kit over other methods. The kit follows a normal immunofluorescence protocol; therefore, it is easy and standardized. It is safe, since no radioisotopes get used. Because the denaturation of DNA is done by using nucleases, it allows for a highly sensitive detection of BrdU. And lastely, a multiplex staining with reagents with different wavelengths than fluorescein can be perfomed.

The kit offers different protocols for the usage of adherent cells, suspension culture cells, and tissue sections. The first step is the labeling of cells with BrdU. After labeling, fixation of the cells is required and if using suspension cell cultures, the cells need to be spun down on a slide for further processing. The protocol recommends poly-L-lysine coated slides, which indeed work the best, since using fibrinogen coated glass slides, which I tried without reading the protocol in detail, resulted in an unacceptable, high background staining. The next step is already the detection of BrdU with the primary antibody. There is no explicit blocking step included in the protocol which I found quite irritating at first, but even without blocking, the staining still works. After some washes, the secondary fluorescent antibody gets applied to the sample and incubated for 30 minutes at 37ºC, as was the primary antibody, too. Afterwards, the samples get washed again and are now ready for either imaging or co-staining with an additional fluorescent stain. For co-staining I regularly used WGA-555 (Invitrogen) or one of the red mitotracker dyes (Invitrogen), which are added at the stage of cell culture, with very good success.

Overall, I think the kit is working very well, even without a blocking step.