The DIMSCAN system uses digital image fluorescence microscopy to quantify live cells, which selectively accumulate FDA. It is capable of measuring cytotoxicity over a 4-log dynamic range, after digital-image thresholding and eosin-Y quenching [7 (link), 30 (link)]. Cell lines were seeded in 150 μl of complete medium at 700 – 8,000 cells per well (dependent on cell size and doubling time) in 96-well plates. After overnight incubation, drugs were added as single agents in 100 μl of complete medium to the cells, at the following concentrations: 0.003–10 nM for vincristine, 0.01–100 μM for melphalan and etoposide, and 0.01–100 nM for rapamycin. Stock solutions of melphalan and etoposide were prepared at 10 mM in DMSO; vincristine at 1 mM in normal saline; rapamycin 1 mM in DMSO. Controls were treated with the appropriate drug vehicles (DMSO for etoposide, melphalan and rapamycin: final DMSO content of ≤0.1% at the highest concentration tested). Six replicate wells were tested per concentration as well as per control. Following 4 days (96 h) of incubation, 50 μl of 0.5% eosin-Y + 10 μg/ml of FDA were added to the wells. After 20 minutes of incubation in the dark, the fluorescence of viable cells in each well was measured using the DIMSCAN system. The results were expressed as the survival fraction of treated cells for each concentration relative to the control cells. The standard deviations (sd) for control wells were less than 15%, and for any plates in which the sd exceeded 15%, the assay was repeated.