Cytoselect 48 well cell adhesion assay

WT and FAK^KO pericyte adhesion properties were measured using the CytoSelect™ 48-Well Cell Adhesion Assay (CBA-070, Cell Biolabs). Detached cells (25,000 cells/well) were seeded on each matrix for 45 min, washed and fixed following the manufacturer’s instructions. Cell Stain solution was applied and OD measured at 560 nm with a plate reader.

The cells were grown in a monolayer culture in the presence or absence of the various P. lutea leaf extracts. After 3 d, the cells were assayed. Adhesion and wound healing assays were carried out using commercial kits (CytoSelect 48-Well Cell Adhesion Assay, CytoSelect 24-Well Wound Healing Assay, Cell Biolabs) according to the manufacturer's protocol. To assay migration, a chemotaxis chamber containing a membrane with 8 mm pores (Chemotaxicell, Kurabo, Osaka Japan) was coated with 50 μg/mL bovine type I collagen (Koken, Tokyo, Japan) or 50 μg/mL bovine fibronectin (Sigma-Aldrich) according to the manufacturer's protocol and was set into each well of a 24-well culture plate. After drying the Chemotaxicell membrane, 500 μL of DMEM with or without 10% FBS was added to the bottom of the well as a chemoattractant, 200 μL of DMEM containing 2 × 10⁴ cells was added to the chamber, then the plate was incubated for 24 h at 37°C, 5% CO₂, and 100% humidity. The membranes were then washed, removed from the chamber, fixed with 4% formaldehyde (Wako, Osaka, Japan), and stained with crystal violet (Wako). The membranes were examined under an optical microscope and photographs (×400) were taken.