A BrdU pulse double-labelling method for studying adaptive response

We describe a pulse BrdU double-labelling method which we have used for a cohort-analysis study of a conventional adaptive response experiment in stimulated human lymphocytes. This method allows us to identify the cycle position occupied by each scored metaphase at the time of primer and at the time of challenge. Responses in cells at defined developmental stages can then be compared irrespective of mitotic perturbation. Using a primer X-ray dose of 1.0 cGy, and a challenge dose of 1.5 Gy 6 h later, we found a transitory depression in frequency of total aberrations present in cells sampled at 6 h but not at 9 h after challenge. When defined cohorts were summed for both sample times, no significant adaptive response was observed. A repeat experiment where terminal BrdU labelling was used after the challenge failed to provide any positive response at either 6 or 9 h. The technique presented not only ensures that similar cell mixtures from a heterogeneous, asynchronous population can be scored and compared, but it serves also to demonstrate the kinetic complexity of stimulated lymphocyte cultures.