96 pin woundmaker

The effect of treatment on cell migration was assessed in a scratch−wound assay using the IncuCyte^® Zoom Kinetic Imaging System (Essen BioScience, USA). UW-CSCC1 and UW-CSCC2 were seeded (n = 10) onto collagen 1−coated 96−well ImageLock plates (Essen BioScience, USA) and grown to near confluency in their respective growth media. To prevent proliferation and isolate migratory effects, cells were serum−starved prior to investigation of their motility. This was achieved by replacing media in the wells with a 1% FCS analogue of the relevant growth factor−free culture media. After 24 h incubation in this low serum containing media, the cells were scratched according to manufacturer’s instructions using the 96−pin Woundmaker™ (Essen BioScience, USA). The cells were subsequently washed with serum−free media, then incubated in low serum media containing at 37 °C, 5% CO₂, and imaged over 48 h at ×10 objective to track cell motility and wound width. IncuCyte™ ZOOM software (Essent BioScience, USA) was used to interpret wound width reduction over time. Output was analysed using GraphPad Prism (GraphPad Software, USA), applying a one−way ANOVA with Tukey’s multiple−comparison post−test.

Normal human dermal fibroblasts (NHDF) (2 × 10⁴ cells/well) were seeded in 96-well plates (Essen Bioscience, Newark, United Kingdom). Once fibroblast reached confluence, a scratch was made using the 96-pin WoundMaker (Essen Bioscience). Then, cell media was replaced by fresh serum-free DMEM supplemented with 1% (v/v) penicillin/streptomycin. At that point, CBD treatments were added in combination with either TGFβ1 or rhIL-4 (10 ng/ml) to induce cell proliferation. Images were taken every 3 h for 48 h, and data analysed using IncuCyte HD software.

Cell migration was evaluated with wound-healing and transwell assay. For the wound-healing assay, cells (3×10⁴cells/well) were seeded into 96-well culture plates (Corning-Costar, NY, USA). When cells reached >90% confluence, at 12 hours after seeding, wound scratches were made by a 96-pin WoundMaker (Essen BioScience), and relative wound densities were measured by the IncuCyte Zoom system over a 2-day period. The experiments were performed in triplicate.
In transwell chamber migration and invasion assays, 3×10⁴ cells were put into each chamber, providing with serum-free medium, and permitted to pass through a polycarbonate filter, which had been either precoated by 100 μg Matrigel (Becton Dickinson, San Jose, CA) for the invasion assay or left uncoated for the migration assay. The outside of chambers was filled with DMEM, containing 10% FBS. Cells on the upper surface of the filters were erased after 24 hours (SGC-7901) or 48 hours (HGC-27, BGC-823 and MGC-803) in migration assays. Cells on the upper surface of the filters were erased after 48 hours (SGC-7901) or 72 hours (HGC-27, BGC-823 and MGC-803) in invasion assays. The membranes were fixed with methanol for 15 minutes and stained with 0.5% crystal violet for 15 minutes. The cells on the underside of the filter were photographed and counted in five randomly selected microscopic views.

The modulation of cell migration was analyzed by wound-healing assays. Briefly, NHDFs were seeded in a 96-well Essen ImageLock plate (Essen BioScience) and were grown to confluence. After 24 h, the scratches were made using the 96-pin WoundMaker (Essen BioScience), followed by incubation of the cultures in media with 10 ng/ml of mitomycin C to block cell proliferation. VCE-004.8 and TGFβ1 or rhIL-4 (10 ng/ml) were added and wound images were taken every 60 min for 36 h, and the data analyzed by the integrated metric Relative Wound Density part of the live content cell imaging system IncuCyte HD (Essen BioScience).