Upright bright field microscope

The CGTH W-2 cells were grown to confluence as monolayer. The cells were scratched using a P200 pipette tip and imaged as previously described (22 (link)). Following 24 h treatment with DMSO or 30 mM 15d-PGJ₂ at 37°C, the wound healing was imaged using an upright bright-field microscope (Nikon Corporation) and analyzed by ImageJ. Images are representative of three separate experiments.

To determine migratory ability, 1×10⁵ CGTH W-2 cells were seeded in the upper chamber of a Transwell apparatus with an 8 mm pore polycarbonate membrane (Costar^®; Corning Incorporated, Corning, NY, USA). Following cell attachment, the cells were cultured in growth medium, with or without 30 mM 15d-PGJ₂ (Enzo Life Sciences, Inc., Farmingdale, NY, USA), for 24 h at 37°C. Following removal of the cells on the upper side of the polycarbonate membrane using a cotton swab, the cells on the lower side were fixed for 10 min in 10% formalin and stained for 5 min at room temperature with Coomassie brilliant blue G250 (Sigma-Aldrich; Merck Millipore). The numbers of migrated cells were imaged using an upright bright-field microscope (Nikon Corporation) and analyzed by ImageJ (version 1.64r; National Institutes of Health, Bethesda, MD, USA) in 3 randomly selected fields for each membrane and triplicate membranes examined for each experimental group.