Calibration methods and avoidance of errors in measurement of intracellular pH (pHcyt) using the indicator bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in human platelets.

Determinations of pHcyt in suspensions of human platelets using BCECF [bis(carboxyethyl)-5(6)-carboxyfluorescein] can be seriously biased by leakage of the fluorescent indicator. Two methods ("pH jump" and "Mn(2+)") are presented for determining the fraction of external indicator (B ext) and eliminating this error. Both methods rely on rapid perturbations (pH jump or Mn(2+) addition), which affect the fluorescence of the external dye immediately and the intracellular dye more slowly. Identical values ofB ext are reported. Failure to correct for dye leakage can result in overestimation of pHcyt by as much as 0.4 unit at physiological external pH (pHext). Two methods of calibration of the cytoplasmic signal were compared after correcting forB ext: the "digitonin lysis" method and the "nigericin calibration" method. In the digitonin method the dye is released at the end of the experiment and the dependence of its fluorescence is determined as a function of pH. The method assumes that the fluorescence and titration characteristics of the dye in the cytoplasm are not different from those in solution. It gives pHcyt=6.75±0.07 for pHext=7.3. In the nigericin method, 150 mM external K(+) and 10 μM nigericin are used for the purpose of setting pHcyt=pHext to accomplish anin situ calibration. The method was complicated by extra leakage induced by nigericin. Assuming that the ionophore could equilibrate pH in the alkaline range, the fluorescence of the anionic form of BCECF in the cytoplasm would be 15% lower than in solution and pHcyt would be 0.3 unit higher than presented above. A number of observations favor the digitonin lysis method of calibration. The fluorescence polarization of BCECF in platelets is small and indistinguishable from that in solution (0.000±0.022). The spectrofluorimetric characteristics of the intracellular dye are identical to those in solution (150 mM NaCl or KCl). There was no evidence for self-quenching or binding to cellular elements for cytoplasmic BCECF concentrations up to 1.8 mM. The following agents are capable of introducing error: (1) the Na(+) substituteN-methyl-D-glucamine doubles theK d and decreases by 13% the ΔF max of BCECF; (2) the Na(+)/H(+) exchange inhibitor amiloride quenches BCECF fluorescence and is intrinsically fluorescent; and (3) bovine serum albumin (used to remove nigericin) quenches external BCECF with kinetics mimicking acidification of the cytoplasm.