C18-NTA adds high-affinity Co²⁺-binding sites to unroofed cells. (A) Epifluorescent images represent a field of unroofed, R18-labeled PC-12 cells, with the left-hand column showing the initial fluorescence intensity from 0 to 100 µM Co²⁺ in the bath, as indicated. The unroofed cells were then treated with C18-NTA (see Materials and methods), the C18-NTA was removed from the bath, and cells were then washed with EDTA and then again bathed in 0–100 µM Co²⁺ (middle column). Finally, the unroofed cells were washed with EDTA to recover the fluorescence intensity (right column). Scale bar shown in the top left image applies to all images and is 50 µm. (B) Concentration dependence for coverslip from which the example in A is shown. Points shown are means of the cells, and the bars represent the SEM.