HTS assay development.

<p>MDA-MB-231 cells were plated in Oris™ Pro 384 plates and allowed to attach for 2 h. Plates were stained with Hoechst 33342 immediately thereafter (<i>pre-migration</i>) or after 2 days in culture (<i>2-day migration</i>), and imaged on the ArrayScan II. <b>A. Seeding density.</b> Optimal gap closure with minimal background was obtained at 15,000 cells/well. <b>B. DMSO tolerance.</b> 16 wells each of minimum (pre-migration) and maximum (two-day migration) controls were treated with a ten-point, two-fold gradient of vehicle (DMSO) and numbers of cells that had migrated into the exclusion zone were enumerated. Assay performance decreased at concentrations above 0.6% DMSO due to toxicity. <b>C. Three-day variability.</b> Two full microplates of minimum and maximum controls were treated with vehicle (0.1% DMSO) on three consecutive days using equipment to be used in HTS. Intra-plate and inter-plate variability parameters were calculated (<i>Table</i>). SD, standard deviation; CV, coefficient of variance; PL to PL, plate to plate comparison; S:B ratio, signal-to-background ratio. Scatter plots illustrate day to day performance; the lower Z-factor on day 3 was a result of a partially obstructed dispense manifold. <b>D. Control inhibitor studies.</b> Using optimized assay conditions, identical IC₅₀ curves for cytochalasin D were obtained in three independent runs (<i>left panel</i>). Multiparametric profiling of cell migration, toxicity (cell density), and nuclear morphology (brightness and area) document selective inhibition of cell migration in the absence of overt cytotoxicity (<i>right panel</i>).