NIH-3T3 cells (100 μL) were seeded at a density of 10,000 cells per well in a 96-well plate and incubated in a cell culture incubator at 37 °C with 5% CO2 for 24 hours. Subsequently, the cells were carefully washed with DPBS to eliminate any dead or unattached cells. Following the wash, the cells were treated with Mito-pH2 dye at various concentrations (0, 100, 300, 500, 2000, 5000 nM) and incubated for an additional 24 hours in the cell culture incubator. After this incubation period, the culture medium containing different concentrations of the dye was aspirated, and a mixture of 100 μL of fresh culture media and 10 μL of CCK-8 reagent was added to each well. The cells were then incubated for an additional two hours at 37 °C. After this incubation, absorbance at 450 nm was measured using a multimode microplate reader. pKa determination using absorbance spectroscopy: