Pmw 10md camera

In vitro cell migration of H400 and BICR56 cells was assessed at 16 h using a Transwell migration assay (CLS3422, Sigma-Aldrich) as previously described^{41 (link)}. Kava constituents FKA (10 μg/ml), FKB (2.5 μg/ml) and yangonin (10 μg/ml) were used to treat the cells in the upper chambers of the assay. 1.5 × 10⁴ cells/well were seeded in serum-free media. A culture medium with 10% FBS without antibiotics was used in the lower chamber. After incubation in standard conditions (37 °C, 5% CO₂), the medium was removed. The cells in the upper chamber were fixed in 4% formalin for 2 min at room temperature (r/t), permeabilised with 100% ice-cold methanol for 20 min at room temperature and stained (UV protected) using 2% crystal violet solution for 20 min. After the removal of the non-migrated cells, the migrated cells (5 random fields/well) were observed under a BH2 Olympus microscope equipped with a PMW-10MD Sony camera. Captured images were analysed to count migrated cells using ImageJ Software (ImageJ v. 1.50i, National Institutes of Health). All the experiments were performed in triplicate.

In vitro cell invasion of H400 and BICR56 cells was assessed at 22 h using a Corning BioCoat Matrigel Invasion Chamber assay (Corning 354480) as previously described^{41 (link)}. Kava constituents FKA (10 μg/ml), FKB (2.5 μg/ml) and yangonin (10 μg/ml) were used to treat the cells in the upper chambers of the assay. Cells were seeded with a density of 3.0 × 10⁵ cells/well. A culture medium with 10% FBS without antibiotics was used in the lower chamber as chemoattractant. After incubation in standard conditions for 22 h, the medium was removed. The cells in the upper chamber were fixed with 100% ice-cold methanol for 2 min at room temperature and stained (UV protected) using 2% crystal violet solution for 2 min, as per manufacturer's instructions. After the removal of the non-migrated cells, the migrated cells (5 random fields/well) were observed under a BH2 Olympus microscope equipped with a PMW-10MD Sony camera. Captured images were analysed to count migrated cells using ImageJ Software (ImageJ v. 1.50i, National Institutes of Health). All the experiments were performed in triplicate.