A SPECTROSCOPIC METHOD FOR ASSESSING CONFLUENCE OF EPITHELIAL-CELL CULTURES

We describe a convenient nonelectrophysiological technique for assessing cell proliferation and subsequent tight junction formation for epithelial monolayers grown on permeable supports. The method involves the use of phenol red (PR), a standard pH indicator in most cell culture media. In addition, we report a systematic error in a commercially available system for measuring transepithelial electrical properties. Briefly, the flux of PR across the epithelium was measured from the serosal solution into the mucosal solution. The mucosal solution was first replaced with a PR-free solution and then collected at timed intervals. The PR concentration was measured using a spectrophotometer set at the isosbestic point for PR (479 nm). PR flux was then calculated and used as an index of the permeability of the epithelium to PR. This method was tested using the renal epithelial cell line A6. After cell seeding, PR flux decreased in two phases: an initial large decrease, associated with cell growth and monolayer confluence, and a second decrease associated with tight junction formation [assessed by measuring transepithelial conductance (G(t))]. In addition to monitoring tight junction formation, PR flux measurements were also used to estimate the net movement of solution by the epithelial cells between the mucosal and serosal compartments. For convenience, G(t) was initially measured in culture dishes using a commercially available "chopstick" electrode system. However, the chopstick system yielded G(t) values that were on average 51% lower than values for the same preparations when measured in standard Ussing-type chambers. The discrepancy was due to a nonuniform current field produced by the chopstick electrodes.