Fig. (6) Assay development. (A): Time of starvation of Clone 10 cells in the presence of wortmannin. Points show Mean ± SD for 4 replicates. The signal was stable by 2 hr starvation and was not improved at 3 hr (Table). (B): Titration of number of Clone 10 cells/well, in the presence of wortmannin under starvation conditions for 2 hr. Bars show Mean ± SD for 4 replicates. The response to wortmannin was similar at all cell densities and the Z’ was stable at 0.80 ± 0.04 across the range. A cell concentration of 4,000/well was selected. (C): DMSO titration for Clone 10 cells ± 33 nM wortmannin under starvation conditions for 2 hr. Points are Mean ± SD for 4 replicates. Table: for each DMSO %, the Z’ was calculated from the 33 nM vs. 0 nM wortmannin value; Signal Window was the ratio of the same values. No effect of DMSO was seen ≤ 0.3% DMSO. (D): Effect of binning images of Clone 10 cells, 4000 cells/well in the presence of wortmannin under starvation conditions for 2 hr. The same cell images were used with and without applying 2x2 binning. Points are Mean ± SD for 4 replicates. No effect of binning on the Z’ (0.82 vs. 0.79) or wortmann in IC50 values was observed.