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.