PINK1 Q456X homozygote PD patient neural cells were treated with low concentrations of valinomycin and mitochondrial reactive oxygen species (mROS) and GSH concentrations were measured. (A–C) After 24 hours of incubation in vehicle, 0.1 μM or 1 μM valinomycin, patient specific neural cells were labeled with a fluorescent indicator of mitochondrial reactive oxygen species and analyzed by fluorescence-activated cell sorting (FACS). (A) Few fluorescent cellular events were recorded from healthy subject neural cells exposed to 0.1 μM valinomycin relative to unstained cultures. In contrast, many fluorescent cellular events representing increased mROS production were recorded from PINK1 patient neural cells exposed to 0.1 μM valinomycin. (B) Quantification of fluorescent events revealed that 0.1 μM valinomycin increased the percentage of fluorescent PINK1 patient specific neural cells. (C) Parallel incubations of healthy subject and patient-specific neural cells with 1 nM or 10 nM hydrogen peroxide (H2O2) showed dose-dependent increases in the percentage of fluorescent cellular events independent of genotype, confirming the specificity of the assay for ROS levels. (D–I) After exposure to valinomycin (0.1 or 1 μM, D), concanamycin A (10 nM, E), MPP+ (5 μM, F), or H2O2 (10 μM, G), PINK1 patient neural cells (black bars) showed reduced GSH levels relative to healthy subject neural cells (white bars). In contrast, low concentrations of 6OHDA (1 or 10 μM, H) or MG132 (1 or 10 μM, I) did not change GSH levels in PINK1 patient or healthy subject neural cells. Data are represented as Mean ± SEM, N=3, * p<0.05 ANOVA PINK1 versus Healthy subject, δ p<0.05 ANOVA chemical stressor versus vehicle.