Figure 1. Confluent 661W cells incubated with dose range of staurosporine (Stsp).

Two 48-well plates of confluent 661W cells were incubated for 24 h with a concentration range of Stsp from 2.5 to 1000 nM, or DMSO alone as VC, and viability of the cells then was assessed by the sequential application of the CaAM and SO assays. In one plate probenecid was added to the CaAM assay medium. A: CaAM assay performed with and without probenecid. Without probenecid (gray bars), the maximum fluorescence signal, expressed as relative fluorescence units (RFU) achieved was ca. 2500 for VC, and the dynamic range between VC and the most toxic dose of Stsp (1000 nM) only amounted to a 61.8% decrease in RFU. In contrast, the inclusion of probenecid apparently conserved the intracellular calcein signal, resulting in a maximum mean value of ca. 7000 RFU for VC, and extending the dynamic range to a decrease of 82.4% as a result of incubation with 1000 nM Stsp. *P≤0.05 vs. VC for the CaAM assay with probenecid; §P≤0.05 vs. VC for only one concentration of Stsp (1000 nM) for the CaAM assay run without probenecid; #P≤0.05 for the CaAM assay RFU values in the absence of probenecid vs. the matching dose of Stsp when probenecid was present. B: Lack of dependency of the SO assay on the presence of probenecid. The SO assay values for the identical wells measured for Fig. 1A, showing no significant differences between SO assay results for matching doses of Stsp regardless of the presence of probenecid (remaining in the wells after the CaAM assay was performed). From 2.5 to 125 nM Stsp there was increasing toxicity as determined by this assay; however, at Stsp concentrations from 250 to 1000 nM there was a drop-off in RFU signal (see Results text, also Discussion). *P≤0.05 vs. VC values for SO assays run both with and without probenecid vs. matching VC plus or minus probenecid. Error bars are [1.96 × standard error (SE)].