To evaluate mitochondrial toxicity (mitochondrial membrane potential and reactive oxygen species (ROS) production), confocal fluorescence microscopy was performed on HDF cells plated in 35 mm tissue culture dishes containing 14 mm glass bottom wells coated with poly-d-lysine (MatTek Corporation) at 10 000 cells/dish. The next day, the media was replaced with 2.0 mL of media containing 1 nM of gold nanoparticles (AuNPs). After 24 hours, the media was removed, and cells were washed with PBS before adding 2.0 mL of AuNP-free media to each dish.
To prepare the samples for imaging after 72 hours, the media was removed, and the cells were washed with 2.0 mL of HBSS. Then, 2.0 mL of the dye (JC-1 2.5 μg mL−1 or MitoSOX 5 μM) in OPTI-MEM were added to the dishes and incubated at 37 °C in the dark for 15 minutes. The cells were washed twice with 2.0 mL of HBSS followed by the addition of 2.0 mL of OPTI-MEM to the dishes. The cells were imaged with a Zeiss LSM 710 multiphoton confocal microscope with the laser power, gain, magnification, and all other parameters held constant. JC-1 and MitoSOX were evaluated as independent experiments.
To prepare the samples for imaging after 72 hours, the media was removed, and the cells were washed with 2.0 mL of HBSS. Then, 2.0 mL of the dye (JC-1 2.5 μg mL−1 or MitoSOX 5 μM) in OPTI-MEM were added to the dishes and incubated at 37 °C in the dark for 15 minutes. The cells were washed twice with 2.0 mL of HBSS followed by the addition of 2.0 mL of OPTI-MEM to the dishes. The cells were imaged with a Zeiss LSM 710 multiphoton confocal microscope with the laser power, gain, magnification, and all other parameters held constant. JC-1 and MitoSOX were evaluated as independent experiments.