*3.12. Cell Adhesion on Collagen Coatings*

Cell adhesion was performed on collagen coatings prepared from solutions with different concentrations. Briefly, collagen in 20 mM acetic acid solution was added to 6-well plates and let to dry overnight at room temperature in an orbital shaker, using the solution concentration and volume needed to achieve different collagen densities (0–1.5 μg/mm2). After evaporation at room temperature, collagen was crosslinked with 50 mM EDC/NHS (1:1) solution for 24 h at 4 ◦C. Coatings were washed 10× with 1× phosphate buffer solution (PBS) and sterilized by UV radiation, as described before. MRC-5 cells were seeded at a density of 5 × 104 cells/mL on coverslips with and without collagen coatings. After 16 h incubation at 37 ◦C and humidified 5% CO2 atmosphere, cell culture medium was removed, cells were washed with PBS and stained. Cell were permeated with 0.2% Triton-X solution for 5 min at room temperature and then incubated with Phalloidin/DAPI in PBS at 250 ng/mL and 1 μg/mL, respectively, for 45 min.

Live/dead stain was performed with calcein-AM (2 μg/mL) and propidium iodide (1 μg/mL), for 15 min at room temperature. Fluorescent images were acquired in Axio Imager Z1m (Zeiss, Göttingen, Germany), analysed in Zen lite 2.1 (Zeiss, Germany). Shape descriptor parameter aspect ratio was determined using phalloidin signal processing by ImageJ software. Aspect ratio is a shape descriptor corresponding to the ratio between the largest diameter and the smallest diameter perpendicular to it of a round-like particle or body; when the body has nearly a circular shape, the aspect ratio is close to 1, but if the body assumes an elongated shape, for instance caused by stretching, the aspect ratio increases.
