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A High-Content, Multiplexed Screen in Human Breast Cancer Cells Identifies Profilin-1 Inducers with Anti-Migratory Activities

Figure 2

HTS assay development.

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 (pre-migration) or after 2 days in culture (2-day migration), and imaged on the ArrayScan II. A. Seeding density. Optimal gap closure with minimal background was obtained at 15,000 cells/well. B. DMSO tolerance. 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. C. Three-day variability. 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 (Table). 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. D. Control inhibitor studies. Using optimized assay conditions, identical IC50 curves for cytochalasin D were obtained in three independent runs (left panel). 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 (right panel).

Figure 2

doi: https://doi.org/10.1371/journal.pone.0088350.g002