2.9. pH Measurements Using LysoSensor Yellow/Blue

Cells were grown in 12-well plates and treated with SFN or were serum-deprived. The cells were then collected, washed with warm Hank’s Balanced Salt Solution (HBSS), and stained for 5 min with 5 µM LysoSensor Yellow/Blue in HBSS. After additional washing, the cells were transferred to wells of a 96-well plate. Fluorescence intensities (I, λ_Ex/λ_Em = 340/520 nm, and II, λ_Ex/λ_Em = 380/520 nm) were measured using EnSpire Multimode plate reader (Perkin Elmer, Waltham, MA, USA) at 37 °C. Fluorescence intensity ratio (I/II) was then calculated and lysosomal pH was determined using a calibration curve. The experiments were performed in triplicate.