References BrdU


DNA synthesis by (sub)populations of cells can be tracked by making use of radioactive form of thymidine, but it can also be done by the use of its analogue 5-bromo-2-deoxyuridine (BrdU). In the newly synthesized DNA thymidine is (partly) replaced by BrdU, which is in high concentration added to the culture. 
After fixation and permeabilization of the cells the BrdU can be shown by making use of a fluorochrome labelled antibody against the BrdU. Using DNAse during the staining period the BrdU becomes more available to the antibody without influencing the fluorescence or the structure of the cell. One company is bringing a kit that makes it possible to have a balanced presence of antibody and DNAse in the sample.
Several publications are available comparing the method to thymidine or using BrdU together with surface markers. Both in polyclonal and antigen specific stimulation.

We tried to enhance the protocol for the detection of cytokines together with the BrdU by adding an extra step. In the figure below a comparison is made between the standard protocol and ours. After the 'regular' 48 hrs we wash away the BrdU, that was added after 42 hours, and incubated the cells for an other 16 hrs (over night) and stimulated them with PMA/ionomycin for 6 hrs (total time is 70 hours now). During the last 4 hrs Brefeldin A (BFA) was present (table.1.). The idea is that the initial stimulation shows the reactive antigen cells in the population. These will be BrdU positive. And the PMA/ionomycin will show the cytokine profile of these cells. The number of cytokine producing cells is clearly enhanced. And also the total number of BrdU positive cells is larger (fig.1.). 
Before implementing this protocol further checks are necessary. Is the increase in BrdU positive cells due to the initial stimulation and only larger because of the additional 24 hrs incubation? Or is the increase due to the PMA/ionomycin stimulation? We didn't check this!

Figure.1. Protocol as described in the text and used to generate results for fig.1.

Figure.1. Comparison between protocol A and B as described in text and in table.1. Shown is the BrdU incorporation by CD4+ lymphocytes in PBMC after stimulation with SEB. And the production of IL-2 and IL-4 by these lymphocytes after stimulation with SEB with (B) and without (A) an extra a 4 hrs PMA/ionomycin stimulation.

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Page was last updated: 05-03-2005