Cell Proliferation ELISA, BrdU (colorimetric) From Roche Applied Science

Detection and quantification of cell proliferation is an important part of studying inhibitory or stimulatory substances. The term ‘cell proliferation’ actually includes many cellular activities that result in increased cell number, metabolic rate, total biomass and DNA synthesis, etc., so some indirect measurements of cell proliferation may quantify the increase of cell metabolic rate or total biomass instead of the cell division. However, as metabolic rate can be affected by other factors, it may not reflect the real picture. On the other hand, directly counting cells would be labor-intensive and impractical if the sample number is very large.

The Roche Cell Proliferation ELISA Kit quantifies cell proliferation by measuring the BrdU incorporation during DNA synthesis as the cells replicate. Replacement of the traditionally used [³H]-thymidine with 5-bromo-2’-deoxyuridine (BrdU) as the pyrimidine analog for DNA incorporation in dividing cells allows investigators to get rid of radioactive materials, thus reducing our exposure to toxic substances (and there are already many of them in a lab). Incorporated BrdU is then detected by colorimetric immunoassay.

I like this kit since its protocol is flexible and very convenient to use. The working BrdU labeling solution can be added directly to the treated cell culture. A wide range of initial seeding densities, drug treatment durations and BrdU labeling times can be accommodated. The kit is sensitive enough to clearly show the difference. After BrdU labeling, medium can be removed by tapping off or suction. At this stage, the cells can be stored at 4°C if you want to stop. Otherwise fix the cells with the FixDenat provided with the kit. Thorough removal of this solution is a little difficult, however. Vigorous tapping is required to get rid of this exceptionally adhesive liquid. After removal of the fixative, the procedures are more straightforward. Incubate the cells with the anti-BrdU-POD antibody provided, wash and then add the substrate for color development. Finally, absorbance can be measured with an ELISA reader.

Since BrdU only incorporates into newly synthesized DNA, the presence of non-proliferating cells and dead cell debris will not affect the results as they would in methods that involve quantifying total biomass or total viable cells. This kit is quite handy to use as it provides all the reagents required for the assay. In addition, the high flexibility of the protocol and wide dynamic range of the assay let me design my experiment easily.