Identification of DNA-replicating lymphocyte subsets using a new method to label the bromo-deoxyuridine incorporated into the DNA

Abstract

A flow cytometric procedure measuring the 5-bromo-2-deoxyuridine incorporated into the DNA of cells in S phase was modified to make it compatible with the immunofluorescence labelling of cell surface antigens. The modifications were introduced at the stages of cell fixation and DNA denaturation. Ethanol was replaced by Tween 20-containing paraformaldehyde and hydrochloric acid was used for the denaturation of DNA by bovine pancreatic DNase-I. These two modifications permitted the preservation of immunofluorescence properties and the use of fluorochromes of the phycobiliprotein family such as phycoerythrin and allophycocyanin. This new procedure is suitable for evaluating leucocyte subsets proliferating in vitro following stimulation. As an illustration the immunosuppressive effect of cyclosporin A on PHA stimulated T4-lymphocytes was evaluated.