Since the production of the first monoclonal antibodies directed against the thymidine analogue bromodeoxyuridine (BrdU), flow cytometry has been used for the measurement of the labeling index of both normal and tumor cell populations. Recently Li et al. (1994) have described a new method for the measurement of BrdU uptake, in which DNA denaturation steps are eliminated and the detection of BrdU is based on the selective production of DNA breaks at points where the BrdU has been incorporated by short time exposure to UV light. One of the most attractive features of this method is that it would allow the combined assessment of BrdU incorporation and staining for cell surface antigens. However, in such a case if propidium iodide staining is not used, this technique would lack information about G0/G1 and G2/M phase cells.

In the present study a new method for the simultaneous assessment of the distribution of a cell population in the G1/G1, S and G2/M cell cycle phases by using single parameter flow cytometry based on BrdU incorporation is described.

For that purpose both K562 cell line and peripheral blood lymphocytes stimulated with 1% PHA for 48 hours were used. Cells were cultured in the presence of BrdU during 30 min. prior to cell harvesting. Once collected, cells were exposed to UV light for 5 min. and immediately after, they were fixed in 70% ethanol for at least 30 min, following the method of Li et al (1994). Once cells were fixed, they were placed for 30 min. at 37º in the presence of terminal deoxynucleotidyl transferase and dUTP labeled with digoxigenin. After resuspending the cells in a blocking buffer, they were incubated for 30 min. with a monoclonal antibody against digoxigenin labeled with FITC.

Data acquisition was performed in a FACScan flow cytometer and for the analysis, the Attractors software was used.

Our results show that by using multiparameter flow cytometry, Go/G1, S and G2/M cell populations can be clearly resolved on the basis of FITC fluorescence and light scatter parameters. Accordingly, S-phase cells can be identified by their FITC staining. From the cells which are negative for anti-digoxigenin-FITC antibody, two clear populations can be resolved in a FSC, SSC, pulse-width tridimensional plot, the values obtained for G0/G1 cells being lower than those G2/M cells for all three parameters.