Flow cytometric analysis of experimental parameters for the immunofluorescent labeling of BrdUrd in various tumour...

This report describes the results of the comparison of three different methods and three monoclonal antibodies to stain cells in suspension for incorporated bromodeoxyuridine and total DNA content. The procedures were tested in three different experimental tumour cell lines. The sensitivity of the different procedures was expressed as the ratio of the anti-BrdUrd fluorescence intensities of the S and G1 phase cells (FS/FG1 ratio). There were remarkable differences in sensitivity between the different procedures. With the heat denaturation the most favourable FS/FG1 ratio's were obtained but substantial cell loss occurred during this procedure which is a disadvantage for clinical application. With the pepsin digestion + acid denaturation procedure cell loss was negligible. The standard acid denaturation procedure was inferior to the other two methods. Using the pepsin digestion + acid denaturation procedure we examined the variations in sensitivity for the different monoclonal antibodies and cell lines and the influence of BrdUrd concentration, labelingtime and cell concentration. The binding characteristics for the various antibodies differed considerably in our hands. Only with the IU4 antibody we obtained FS/FG1 ratio's comparable with those described in the literature. No difference was observed between the cell lines. Variation in cell concentration between 1 x 10(4) to 1 x 10(6) ml nor BrdUrd concentration appeared to influence the sensitivity of the procedure. A labelingtime of 1 h or even 30 min seems to be more than sufficient for an optimal FS/FG1 ratio. Our results indicate that using the appropriate antibody and immunofluorescence BrdUrd can be detected by flow cytometry, after incorporation into the DNA of tumour cells under a wide range of culture conditions.(ABSTRACT TRUNCATED AT 250 WORDS)