BrdU Cell Proliferation Assay From Calbiochem

This assay analyzes the proliferation of cells by utilizing bromodeoxyuridine (BrdU) as an analog of the DNA nucleotide, thymidine, which incorporates into the synthesized DNA of actively dividing cells. The extent of BrdU incorporation is reflected in the intensity of absorbance of the final reaction.

The BrdU Cell Proliferation Assay Kit contains all necessary reagents to perform 200 or 1000 tests. We used this kit to assess the proliferation of lymphocytes in response to each of several specific antigen, including selected allergens and bacterial antigens. Since the occurrence of allergen-specific T cells in peripheral blood is not high, cultures of cells were performed in 48-well plates in order to accommodate 1 million cells in 0,5 ml. Cells were stimulated, or not, with antigens for 6 days and on this day, the BrdU was added in an amount proportionally larger then indicated in protocol for the last 18 hours of the culture. For a negative control, cells without added BrdU were also cultured.

The first thing to do before continuing with the procedure is to remove the fixative/denaturating solution from refrigerator since it has to stay 4 hours at room temperature (RT) before it can be used. Near the end of the 4 hours, the culture plates can be centrifuged and the cells at the bottom of the plate fixed and the DNA denatured. Although the instructions say that the plates can be stored at 4ºC up to 1 week at this point, I have never used this opportunity. After the appropriate dilution of the anti-BrdU antibody (1:100), it was added to the wells and incubated for 1 hour at RT. After this, plates were manually washed and the secondary antibody (conjugated with horseradish peroxidase) was added and incubated for 30 min at RT. Next, plates were washed again and 200 ul of substrate was added and plates incubated for 15 min in the dark. After this, the same amount of stop solution (200 ul) was added. Two hundred aliquots from each well were transferred to wells of 96-well plate and read on spectrophotometer at the dual wavelengths, 450 nm and 540 nm (or 450 nm and 595 nm).

Since we needed to increase the number of cells per test, we used the kit for 1000 tests to perform 200 of them. Even though this is 5 times fewer tests, it was still good choice. This will be especially true for labs with no possibility of running radioisotope-based assays. In this kit, BrdU incorporates in the newly synthesized cells and is good replacement for ³H thymidine based assays, even though, in my experience, the latter shows better sensitivity. However, for mitogen or other multipotent factors of cell stimulation, the BrdU-based proliferation assay from Calbiochem is as good in sensitivity as a radioisotope-based assay and much safer and less expensive to use.

Another advantage of this kit is that it measures cell proliferation, not cell viability. Many kits are named as proliferation assays, but are based on the assessment of cell viability; this kit is truly referring to the actual process of proliferation. We plan to continue the use of this kit in our investigation of the inhibitory effect of T regulatory cells on the proliferation of responding cells.