4.9. Mitochondrial Membrane Potential (MMP)

Fluorescence dye rhodamine-123 (R123) was used to investigate MMP. For this, cells were seeded into 24-well plates and incubated with 1 µM or 10 µM UA for 24 h. After incubation, 0.4 µM R123 were added to the cells at 37 °C and 5% CO₂ for 15 min. Subsequently, cells were centrifuged at 300× g for 5 min. before medium was removed and cells were washed with HBSS buffer (supplemented with Mg²⁺, Ca²⁺ and HEPES; pH 7.4; 37 °C) to remove excess dye. Cells were resuspended in fresh HBSS buffer and R123 fluorescence was determined with a ClarioStar plate reader (BMG Labtech, Ortenberg, Germany) at an excitation wavelength of 490 nm and an emission wavelength of 535 nm.