Intracellular pH measurement with BCECF or pHrodo™ Red AM
Measurement of intracellular pH was performed using 5 µM of pHrodo™ Red AM Intracellular pH Indicator (Cat. No. P35372) or 5 µM of BCECF. pH clamping was carried out using a mixture of 10 mM nigericin and 10 mM valinomycin in Hepes-based buffers at different pHs. The fluorescence was measured using a high content screening (HCS) instrument. This data clearly indicates that while the signal of BCECF AM drops with drop in pH, signal of pHrodo™ Red AM increases with drop in pH. Further, while the signal of BCECF gets weaker due to photobleaching or other factors, the signal of pHrodo™ Red AM is consistent during this experiment.