Isolation and Flow Cytometric Characterization of Plant Protoplasts

Cell structural properties measured by flow cytometry (FCM) have been used to discriminate the subpopulations of white blood cells, to resolve subpopulations in tumors, to separate mitotic cells from interphase cells, and to evaluate chromatin conformational changes in isolated nuclei. The molecular basis for the observed alterations in cytoplasm and chromatin conformation and structure is unknown but could involve the nuclear matrix and the cytomatrix. A new FCM method combining light-scattering measurements and detection of bromodeoxyuridine (BrdUrd) incorporation through fluorescent antibodies and the quantitation of cellular DNA content by propidium iodide (PI) allows the identification of additional compartments in the cell cycle. The thermal denaturation appears to enhance the differences in the chromatin structure of cells in the various phases of the cell cycle to the extent that cells could be separated based on the 90º scatter, which is mainly dependent on reflective and refractive components in the nucleus. Light scattering is correlated with chromatin condensation, as judged by the microscopic evaluation of cells sorted based on the light scatter. The method has the advantage over the parental BrdUrd/DNA bivariate analysis in allowing the G₂ and M phases of the cell cycle to be separated and the G₁ phase to be analyzed in detail.