Jc 1 mitochondrial potential assay kit

Effect of nanoengineering on mitochondrial function was determined using JC-1 mitochondrial potential assay kit (Cayman Chemicals). MSCs were nanoengineered, washed with PBS and stained immediately or incubated with complete or serum free media for 16 ​h. Cells were then incubated with JC-1 staining solution for 15–20 ​min and then washed at 400×g for 5 ​min. After washing, J-aggregates were monitored via fluorescence measurements at excitation and emission wavelengths of 535 ​nm and 595 ​nm, respectively. J-monomers were monitored via fluorescence measurements at excitation and emission wavelengths of 485 ​nm and 535 ​nm, respectively. The ratio of the fluorescence intensity of J-aggregates to fluorescence intensity of monomers was used as an indicator of cell health.

CCA cells (3 ×
10⁴ cells/well in 200 μL)
were seeded into 24-well culture plates in triplicate. After culturing
for 16 h, cells were treated with different concentrations of CA (15,
20, and 25 μM) in complete medium containing 0.25% DMSO for
24 h. Control-untreated cells were cultured in complete medium containing
0.25% DMSO. Subsequently, cells were stained with JC-1 dye, a component
of the JC-1 mitochondrial potential assay kit (Cayman Chemical, MI,
USA). Changes in ΔΨm were assessed under a confocal laser
scanning microscope (Zeiss LSM 980 with Airyscan 2, Jena, Germany)
at an excitation/emission wavelength of 485/535 nm. Their red/green
fluorescence intensities were quantified by ImageJ software.