4.9. MitoTracker Red CMXRos, Calcein Staining and Imaging

Cells were pre-loaded with AA and treated for 3 min (37 °C) in saline A with peroxynitrite in 35 mm tissue culture dishes containing an uncoated coverslip. Subsequently, the cells were post-incubated for a further 7 min with various additions and 50 nM MitoTracker Red CMXRos.

In other experiments the cells were incubated for 15 min (37 °C) in saline A with 1 μM calcein-acetoxymethyl ester and 1 mM CoCl₂, washed twice with saline A and treated with peroxynitrite (10 min), as detailed in the legend to the figures. After treatments, the cells were washed three times and analyzed with a fluorescence microscope. The resulting images were taken and processed as described above. The excitation and emission wavelengths were 488 and 515 nm (calcein) and 545 and 610 nm (MitoTracker Red CMXRos), with a 5-nm slit width for both emission and excitation. Images were collected with exposure times of 100–400 ms, digitally acquired and processed for fluorescence determination at the single cell level on a personal computer using the J-Image software. Mean fluorescence values were determined by averaging the fluorescence values of at least 50 cells/treatment condition/experiment.