FIG. 6.

Outcomes of the primary assay optimization for the 1,536-well format. (A) NR2E3 concentration optimization. The assay was run at different concentrations of the NR2E3 protein. For each concentration, S/B ratio (•) and Z′-values (○) were calculated. The star indicates the optimal NR2E3 concentration. (B) Biotin titration. A 20-point, 2:3 serial dilution of biotin was tested in the NR2E3 assay. The IC₅₀ was calculated at 57±3 nM (n=4). (C) Dimethyl sulfoxide (DMSO) tolerance determination. Dose–responses of biotin were tested in the NR2E3 assay at different final DMSO concentrations ranging from 0.4% to 1.6%. The left Y-axis indicates the raw values of the positive (gray) and negative controls (black). Associated biotin IC₅₀ values (⋄), calculated S/B ratios (•), and Z′-values (○) are reported on the right Y-axis.