Figure 2

Scheme of the experimental design modified from http://probes.invitrogen.com/media/pis/mp10016.pdf. A) Cells cultured in the 96 well plate were preincubated with the Tl⁺-sensitive FluxOR dye (Dye) in a Cl^--free loading buffer (−Cl^-) to promote c-NKCC cotransporter activation. The loading buffer was also K⁺-free (−K⁺) to rule out the competition of K⁺ with Tl⁺ at the binding site onto the c-NKCC cotransporter. FluxOR, loaded within the cytoplasm, is quenched in the absence of Tl⁺ ions. B) The assay is started with the following addition of Tl⁺ and Cl^- in the assay plate. Tl⁺ flows inside the cells along with Na⁺ and Cl^- through the c-NKCC cotransporter. Upon binding to cytosolic Tl⁺, the FluxOR dye exhibits a strong increase in fluorescence intensity.