Figure 3.

Intracellular pH (pH_i) response of pHerry with NH₄Cl pulse in renal epithelia. pHerry is a genetically encoded and ratiometric pH sensor expressed in anterior Malpighian tubules (MTs) of Drosophila.¹⁰⁵ (A) Fluorescent images of pHerry (super ecliptic pHluorin [SEpH] [470/510 nm Ex/Em] and mCherry] 556/630 nm ex/em]) of UAS-pHerry driven by the capaR-GAL4 (principle cells of MT) in healthy anterior MTs. The region of interest (ROI) is marked. The background (BG) region is indicated. Scale bar: 50 μm. (B) Relative fluorescence changes of pHerry (SEpH and mCherry signals) of pHerry after 20 seconds of 40 mmol/L NH₄Cl. The mCherry signal does not vary, it is stable, yet the SEpH signal increases fluorescence with alkalization (ie, increased pH_i) and decreases fluorescence with NH₄Cl washout (acidification; ie, decreased pH_i). (C) The ratio of fluorescent signals (SEpH/mCherry) is calculated from data in panel B after calibration (30-min incubation in calibration iPBS: 10 μmol/L nigericin, 130 mmol/L K⁺, pH 7.4 and 9.0). (D) Calibration curve of the absolute pHerry ratio (SEpH/mCherry) after setting pH_i during exposure to calibration insect PBS (iPBS) at eight pH values. Gray circles are individual values from 8 preparations, and the black squares and bars are means ± SD. The curve is fit to Boltzmann distribution. (E) Same data as in panel D but normalized so that pH 7.0 has a ratio of 1.0. Reprinted with permission from Rossano and Romero.¹⁰³