2.4. Measurement of pHi

Cell suspensions in serum-free RPMI 1640 were washed and labeled with 10 μM BCECF-AM for 15 min at 37°C. After loading, the chamber was flushed for 5 min with HEPES-buffered Ringer solution to remove any deesterified dye. The perfusion chamber was mounted on the stage of an inverted microscope (Leica), which was used in the epifluorescence mode with a ×40 oil immersion objective. BCECF was successively excited at 490/10 and 440/10 nm, and the resultant fluorescent signal was monitored at 535/10 nm using an intensified charge-coupled device camera and specialized computer software (MetaFluor). Between 10 and 20 cells were outlined and monitored during the course of the measurements. The results from each cell were averaged and taken for final analysis. Intensity ratio (490/440) data was converted into pH_i values using the high-K⁺/nigericin calibration technique. To this end, the cells were perfused at the end of each experiment for 5 min with standard high-K⁺/nigericin (10 μg/mL) solution (pH 7.4). The intensity ratio data thus obtained was converted into pH values using the r_max, r_min, and pKa values previously generated from calibration experiments to obtain a standard nonlinear curve.