[The measurement of the macrophage membrane potential by using an oxonol fluorescent probe]

Technical questions of macrophage (MP) membrane potential measuring with a probe bis(1,3-dibutyl barbiturate) trimethineoxonol (diBA-C4 (3)) have been elaborated. Measurements were made of single adherent cells. It was shown that at a high concentration of probe in the medium (900 nM) the fluorescent signal well traces the depolarization of membrane, whereas at a low concentration of probe (110 nM) the hyperpolarization is detected more effectively. To find out the reasons for this difference, measurements were made of dye distribution between the cell and the medium measured as well as of the kinetics of probe efflux from MP in the dye-free medium. The gradient of dye concentration on the cell-medium interface appeared to depend on the concentration of diBA-C4 (3) in the medium. Using gramicidin D and Na- and Cl-free solutions, the calibration of fluorescent signal was done; the value of K+ equilibrium potential of MP was -66 - -71 mV. The effect of quinidine and the binding of intracellular calcium result in a significant depolarization of MP membrane; a conclusion is made of the significant contribution of Ca(+)-dependent K(+)-channels to the maintenance of the MP resting potential.