Mitochondrial membrane potential measurement in rat cerebellar neurons by flow cytometry.

Abstract

Mitochondrial membrane potential (MMP) in dissociated rat cerebellar neurons was measured using rhodamine 123 (Rh 123) as fluorescent dye, and flow cytometry. Dye distribution was studied by confocal scanning microscopy. Propidium iodide (PI)-marked cells (dead cells) were not stained by Rh 123, while the green fluorescence of living cells was restricted to mitochondria. Incubation of cells with different ionophores resulted in a maximal inhibition of Rh 123 fluorescence of 27.0 +/- 5.9% (valinomycin), 55.6 +/- 7.2% (ionomycin), and 37.3 +/- 5.1% (gramicidin). Ionophores decreased cell viability at high concentrations, measured as the number of propidium iodide-marked cells. Exposure of cell suspensions to the mitochondrial specific uncoupling agent CCCP caused a decrease in Rh 123 fluorescence (40 +/- 6.1%). Conversely, oxidative stress induced by H2O2 did not affect Rh 123 fluorescence. Impairment of glucose bioavailability reduced Rh 123 fluorescence. 2-Deoxy-D-glucose decreased the MMP with a maximal inhibition of 24.0 +/- 4.4%. Lack of glucose in the incubation medium also resulted in a decrease in MMP. Moreover, application of L-glutamate and N-methyl-D-aspartate (NMDA) (the excitatory amino acids) decreased Rh 123 uptake in a dose-dependent manner, which suggests that the measurement of MMP in dissociated cerebellar neurons by flow cytometry is a suitable method to detect the activity of drugs acting on glutamate receptors.