Quenching of R18 in unroofed PC-12 cells by Co²⁺ binding to endogenous metal-binding sites. (A) Exemplar epifluorescent images of unroofed, R18-labeled cells initially (top), in the presence of 1 mM Co²⁺ (middle), and after subsequent washing with EDTA (bottom). Scale bar in top image applies to all images and is 20 µm. (B) Compiled data from exposing many unroofed, R18-labled cells to various concentrations of Co²⁺ in the bath. All cells from eight independent fields were averaged together, and the means from eight fields were then averaged to produce the points and SEM shown. The smooth curve is a fit with the Hill equation, with a K_1/2 of 40 µM and a slope of −0.53. We did not apply Co²⁺ concentrations >1 mM and allowed the F_Co/F value at high concentrations to float during the fit, giving a value of 0.42.