Cell adhesion assay

Adhesion assay was performed as described in.⁵⁹ 96-well plates (Thermo Fischer Scientific) were coated either with a thin layer of Matrigel diluted with a serum-free medium (BD Bioscience), collagen type I (Life Technologies), Fibronectin, Collagen type IV (both from Sigma-Aldrich) at indicated concentrations, or 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) (negative control), or left uncoated. Cells were starved for 24 h without serum, trypsinized, washed twice with serum-free medium, and 2.5x10⁴ cells/well were resuspended in a serum-free medium and plated in 4 replicas for each condition. Cells were allowed to attach for 30 min (unless otherwise stated) at 37°C, then unattached cells were removed by washing 3 times with a serum-free medium. The remaining cells were fixed with 4% PFA for 20 min at room temperature. The cells were stained with 0.1% Crystal Violet for 30 min at room temperature, and then the plates were washed 10 times with PBS. Plates were air dried overnight, and the crystal violet was then extracted using 0.5% Triton-X100 in PBS for 30 min on a shaker. Absorbance at 540 nm was measured on PheraStar instrument (BioTek). Background absorbance (from blank wells) was subtracted from all test wells and the signal was normalized to the input (cells fixed 6 h after plating without washing). The assay was always performed in 5 technical and 3 biological replicas.