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A spectrophotometric method using 6-carboxyfluorescein (CF) was developed to determine intracellular pH in anchorage-dependent monolayers of control cells of rat hepatic origin. Until now, such studies have been carried out with ascites cells in suspension, which lack specific controls for comparative studies. The rat cell line is grown on plastic Leighton tube slides which fit directly into 3 cm spectrophotometer cuvettes. One sample, without CF, serves as a control for the light-scattering properties of the cell monolayers. Steady-state determinations show a decline in intracellular pH from 7.3 to 6.8 ten minutes after the addition of glucose and quercetin. Kinetic determinations show that with the addition of glucose to substrate-free cells the rate of acid formation is -0.02 pH units/min; the addition of quercetin results in a further acceleration of the kinetic rate to -0.10 pH units/min. In both types of analyses, the change in intracellular pH is standardized with nigericin and external buffers, based on the decrease in the maximum absorption of CF at 492 nm. The results demonstrate that even with anchorage-dependent monolayers of a control hepatocyte line which produces very little acid, this spectrophotometric method permits determinations sufficiently sensitive for analysis of intracellular pH.

Spectrophotometric determination of intracellular pH with cultured rat liver epithelial cells.