Primary non‐transformed fibroblasts were cultured in standard media and plated in Seahorse XF24 Analyzer plates (Agilent) the day before each experiment (10,000–20,000 cells per well depending on the growth speed). On the day of the experiment, cells were washed and switched to the XF Base Medium one hour before experiment (2 mM glutamine was added for the ECAR assay per manufacturer's recommendation). Both ECAR and OCR were measured following manufacturer's protocol. After the experiment, cells were washed, and live cells were counted as a normalization factor.
The relative population of cells with active mitochondria was measured using a JC‐1 dye following manufacturer's protocol (ThermoFisher Scientific). Briefly, both control and PKAN lymphoblasts were washed and incubated with JC‐1, and the emission fluorescent intensities at 595 nm (red) and 535 nm (green) were measured. The red/green ratio from the control cells was set at 100%, and the PKAN cells were plotted as a relative percentage.
The relative population of cells with active mitochondria was measured using a JC‐1 dye following manufacturer's protocol (ThermoFisher Scientific). Briefly, both control and PKAN lymphoblasts were washed and incubated with JC‐1, and the emission fluorescent intensities at 595 nm (red) and 535 nm (green) were measured. The red/green ratio from the control cells was set at 100%, and the PKAN cells were plotted as a relative percentage.