pH Measurement

Intracellular pH was quantified by flow cytometry with the pHRodo® Red AM Intracellular pH Indicator (Life Technologies) according to the manufacturer’s protocol. pHRodo® is weakly fluorescent at neutral pH and is increasingly fluorescent in acidic pH with a detection range between 4 and 9. Ku70^–/– cells were seeded in 100-mm dishes at a density of 1 × 10⁶ cells/dish and were then treated as described above with BPA or KBrO₃ or were co-exposed to both agents. Additionally, for every experiment, a calibration curve was prepared using an Intracellular pH Calibration Buffer Kit (Life Technologies). Four or 24 hr after the initiation of KBrO₃ treatment, cells were harvested using 0.25% trypsin, washed in 4 mL of PBS, and stained with pHRodo® Red at 37°C for 30 min. Cells were then washed twice in PBS, and the calibration curve samples were resuspended in valinomycin and nigericin with pH calibration buffers of pH 5.5, 6.5, and 7.5 for 5 min before analysis, according to the manufacturer’s protocol. The addition of valinomycin and nigericin assists in the equilibration of the intracellular space with the pH buffer. Samples were then analyzed by flow cytometry on an LSRII flow cytometer, and the mean fluorescence intensity was recorded for pHRodo® Red. A standard curve was prepared using the calibration buffer intensities, and the pH values were calculated for the control and treated samples. The mean pH values ± SEM calculated for four experiments are reported.