Flow cytometric analysis of membrane potential in embryonic rat spinal cord cells

Flow cytometric analysis of membrane potential in suspensions of embryonic rat spinal cord cells was carried out in a fluorescence-activated cell sorter (FACS) using anionic voltage-sensitive, fluorescent dyes (oxonols). The FACS or flow cytometer is an analytical instrument that measures optical properties of large cell populations at a single cell level of resolution. The incorporation of oxonol allows relative measurements of membrane potential, since the partition of oxonol within the plasmalemma is directly related to the degree of cell depolarization. Incubation of cells in elevated K+ concentrations or with the Na+ channel agonist batrachotoxin (BTX) changed the fluorescence intensity distribution pattern of the live-cell population; these changes were consistent with the depolarizing effects of these manipulations. Fluorescence shifts were either undetectable or minimal in the dead-cell population. The BTX-induced shift was blocked by tetrodotoxin (TTX) and was reversed in Na+-free medium, indicating embryonic expression of functional Na+ channels. Fluorescence microscopy of sorted cells showed that live cells typically exhibited circumferential ring-like patterns, whose intensities were enhanced under depolarizing conditions. The results show that flow cytometry combined with oxonol dyes can be used to measure the relative membrane potential of large numbers of individual central nervous system cells. The analysis of the changes in the distributions of these membrane potentials can be used to reveal the development of functional ion conductance mechanisms.