X chromosome territories in human female lymphocytes

We developed an improved bromodeoxyuridine (BrdU)-DNA assay procedure for flow cytometric analysis. Which makes possible cytochemical investigation against cells after BrdU-DNA assay, such as immunochemical staining or in situ hybridization. Using this method, we performed fluorescence in situ hybridization (FISH) technique using DNA probes that recognize whole X chromosome of human lymphocytes in peripheral blood (resting cells) and cultured cells stimulated by phytohemagglutinin (PHA) (proliferative cells). Cultured cells were fractionated precisely into each stage of cell cycle by cell sorter after flow cytometric analysis. Following FISH procedure, the cells were examined for the volumes of two X chromosome territories in the nuclei using the optical section images on a confocal laser scanning microscope (CLSM). And we analyzed the variation in the volume ratio (R) of two X chromosome territories at different stages in cell cycle. Our data indicated that there were no significant difference between the volume of two X chromosome territories in female human lymphocytes at resting (G0) stage. Furthermore, they also showed no significant alternation throughout the cell cycle. These results may challenge the traditional concepts for inactivated chromosome in two points. First, the inactivated X chromosome is more decondensed throughout the cell cycle than previously thought. Second, although the inactivated X chromosome appears more decondensed during its self replication it dose not bring a great change in the volume of its territory.