Intracellular pH measurement with BCECF

Cells were loaded with 10 μM (HEK293 cells) or 5 μM BCECF-AM (sperm) (Invitrogen) for 10 min at 37°C and 10% CO₂. Afterwards, HEK293 cells were washed three times in 1 ml ES before starting the measurement. For sperm, the dye was removed by single centrifugation (700 x g, 7 min, RT) and resuspension in TYH buffer. For HEK293 cells, fluorescence was measured in a plate reader (FLUOstar Omega; BMGLabtech) at 29°C. For calibrating the intracellular pH (pH_i) using the null-point method, cells were permeabilized with Triton‐X 100 (final concentration of 0.1% ) followed by addition of different pH buffer solutions (pH 5–8). BCECF was excited at 440 nm and 485 nm and the emission was detected at 520 nm. The fluorescence emission ratio at both excitation channels was normalized to the initial fluorescence ratio and plotted as a function of pH. A linear regression analysis (Origin; OriginLab) was performed to calculate the pH_i.

Changes in pH_i in sperm were either analysed in a rapid-mixing device (SFM-400; Biologic) in the stopped-flow mode or in a spectrofluorometer (Quantamaster 40, PTI) at 37°C. In the stopped-flow device, the sperm suspension (5·10⁶ sperm/ml) was rapidly mixed 1:1 (v/v) at a flow rate of 0.5 ml/s with the respective stimulants. Fluorescence was excited by a SpectraX Light Engine (Lumencor), whose intensity was modulated with a frequency of 10 kHz. The excitation light was passed through a BrightLine 452/45 nm filter (Semrock) onto the cuvette (FC-15, Biologic). Emission light was recorded in a dual-emission mode using BrightLine 494/20-nm and BrightLine 540/10-nm filters (Semrock) by photomultiplier modules (H10723-20; Hamamatsu Photonics). The signal was amplified and filtered through a lock-in amplifier (7230 DualPhase, Ameteky). Data acquisition was performed with a data acquisition pad (PCI-6221; National Instruments) and Bio-Kine software v. 4.49 (Bio-Logic, Illingen, Germany). The pH signal represent the ratio of F494/540 and is depicted as the percent of the relative change in ratio (ΔR/R) with respect to the mean of the first three data points at the onset of the signal. The control (TYH) signal was subtracted from the NH₄Cl or bicarbonate traces.

To analyze the effect of changing the extracellular Ca²⁺ on the intracellular pH, measurements were performed at the spectrofluorometer in respective TYH buffer containing 1 x 10⁴ sperm/ml. BAPTA (1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrapotassium salt, Sigma-Aldrich) was dissolved in the modified TYH buffer (pH 7.4) and added to the sperm samples at a final concentrations of 3 mM. BCECF was excited at 452 nm and the emission was detected at 494 nm and 540 nm. The fluorescence emission ratio of 540/494 was calculated and plotted against time. The ratio was corrected for a drift in fluorescence. The drift was determined by calculating the slope of the response after addition of buffer experiment, which was multiplied to the time axis to calculate the drift over time. The drift was subtracted from the sample data. The drift corrected data was finally normalized to the initial emission ratio.