Hoechst 33342 dye uptake as a probe of membrane permeability changes in mammalian cells

Abstract

Flow cytometric analysis of the uptake of the DNA-specific and fluorescent probe Hoechst 33342 (HO342) offers a simple and rapid method for measuring membrane transport rates in mammalian cells and identifying cellular subpopulations that differ in their membrane transport rates. In a Chinese hamster ovary wild-type cell line and in three colchicine-resistant lines derived from it, the rate of uptake of HO342 dye decreases as the stepwise resistance to colchicine increases. A colchicine-sensitive revertant cell line shows an increased rate of dye uptake. Drug resistance in these lines has previously been shown to be related to changes in transport rate. In a manner similar to colchicine, the rate of uptake of HO342 dye shows nonsaturation kinetics. The effects of KCN, a metabolic inhibitor, on HO342 dye uptake, both in the presence and in the absence of glucose, is similar to that previously observed for colchicine uptake. When murine spleen cells are stained with HO342 under appropriate conditions, one sees two populations of lymphocytes differing in HO342 fluorescence intensity, a difference not related to DNA content. The two subpopulations show the same relative difference in both colchicine and HO342 uptake. HO342 dye appears, therefore, to enter mammalian cells by the same mechanism as colchicine--i.e., by unmediated diffusion--and can be used as a probe of cytoplasmic membrane permeability. Potential applications of the dye in studies of drug resistance, detection of activated T cells, and recognition of lymphocyte subpopulations are discussed.