Fluorescence measurement of intracellular pH on proximal tubule suspensions. The need for a BCECF sink

BCECF is used for intracellular pH (pHi) measurement in microperfused tubules. In this case, the perfusate washes out all BCECF leaking from the cells away from the optical light path. We have explored the use of BCECF for pHi determination on suspensions of dog renal proximal tubules (Percoll). This raises specific problems due to the accumulation of BCECF in the extracellular compartment generated by desesterification of BCECF-AM during loading and to leaking of BCECF into the extracellular medium occurring during the waiting time and during the measurement procedure. Repeated washing of the suspension reduced in part this contamination but did not eliminate the continuous leakage of BCECF: the specific intracellular signal is progressively reduced. We have examined the use of anion exchange resin (Dowex 1X-8, 200-400 mesh, Cl- form) to bind the extracellular BCECF (negatively charged). Dowex beads glued to one wall of the cuvette out of the optical path constitute an optically neutral sink removing BCECF as it leaks out of the cells. Using this technique, we had estimated the pHi of dog proximal tubule to 7.374 +/- 0.032 at extracellular pH of 7.325 +/- 0.021. The cellular pH is acutely, but transiently, alkalinized by NH4Cl and acidified by Na acetate. The BCECF signal was calibrated using nigericin. This technique improves significantly the measurement of pHi by BCECF fluorescence in tissue suspensions.