Voltage‐sensitive cyanine dye fluorescence signals in lymphocytes: Plasma membrane and mitochondrial components

Abstract

The origin of the cyanine dye fluorescence signal in murine and human peripheral blood leukocytes was investigated using the oxa- and indo-carbo-cyanines di-O-C₅(3). and di-l-C₅(3). Fluorescence signals from individual cells suspended with nanomolar concentrations of the dyes were measured in a flow cytometer modified to permit simultaneous four-parameter analysis (including two-color fluorescence or fluorescence polarization measurements). The contributions of mitochondrial membrane potential (Ψ_m) and plasma membrane potential (Ψ_pm) to the total voltage-sensitive fluorescence signal were found to depend on the equilibrium extracellular dye concentration, manipulated in these experiments by varying the ratio of dye to cell density. Hence, conditions could be chosen that amplified either the Ψ_m or the Ψ_pm component. Selective depolarization of lymphocytes or polymorphonuclear leukocytes (PMN) in mixed cell suspensions demonstrated that defining the partition of dye between cells and medium is requisite to assessing the heterogeneity of cell responses by cyanine dye fluorescence. At extracellular dye concentrations exceeding 5 nM in equilibrated cell suspensions, both mitochondrial and plasma membrane dye toxicity were observed. In murine splenic lymphocytes, plasma membrane toxicity (dye-induced depolarization) was selective for the B lymphocytes. Certain problems in calibration of Ψ_pm with valinomycin at low dye concentrations and perturbations of Ψ_pm by mitochondrial inhibitors are presented. These findings address the current controversy concerning Ψ_m and Ψ_pm measurement in intact cells by cyanine dye fluorescence. The finding of selective toxicity at low cyanine dye concentrations suggest that purported differences in resting Ψ_m among cells or changes in Ψ_pm with cell activation may reflect varible susceptibility to dye toxicity rather than intrinsic cell properties.