Cell surface pH calibration of WGA-pHRho and WGA-fluorescein. (A) CHO cells were labeled with either WGA-fluorescein or WGA-pHRho, and the change of fluorescence (ΔF/F₀%) was plotted using pH standards (HEPES; 1 mM, open symbols; 10 mM, closed symbols); F_o was chosen to be at the approximate pKa of each sensor (pH 7.0 for WGA-pHRho and pH 6.5 for WGA-fluorescein). The linear fits for WGA-pHRho were pH = 6.99 − 0.06 × [ΔF/F₀] and pH = 6.91 − 0.07 × [ΔF/F₀] for 1 and 10 mM, respectively; the linear fits for WGA-fluorescein were pH = 6.51 + 0.05 × [ΔF/F0] and pH = 6.52 + 0.05 × [ΔF/F₀] for 1 and 10 mM, respectively. (B) Conversion of ΔF/F₀ (%) to CHO cells expressing hHv-1 were held at −80 mV, and changes in fluorescence were elicited from 4-s depolarizations from 0 to 100 mV in 20-mV increments and converted into ΔpH using the calibration curves in A; WGA-pHRho, red; WGA-fluorescein, green. (C) ΔF/F₀ images of cells expressing hHv-1 taken at 4 s shown in B. Top: WGA-pHRho; bottom: WGA-fluorescein. Dotted white circles indicate the voltage-clamped cell. White scale bars represent 10 μm.