Cellular Cytoskeletal Visualization on Titanium

Cells were seeded on titanium discs (n = 648) at a concentration of 10⁴ cells/well in a 48-well plate (BD, Milan Italy) and then kept in growth condition. After 20 min, 24 h and 72 h (T0, T1, T2), the titanium specimens were washed in Phosphate Buffer Saline (PBS) and then the cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min. After two washes with PBS, cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS. Following the manufacturer’s protocol, cells were stained with Rodhamine-Phalloidin (Life Technologies) and 1 uM 4′,6-diamidino-2-phenylindole (Dapi, Life Technologies) to respectively detect the cytoskeleton and the nuclei [45 (link),46 (link)]. Image acquisition was made recurring to a Nikon Eclipse Ti-E microscope with 40X objective (Plan Fluor Nikon) [47 (link)]. Image analysis [48 (link),49 (link),50 (link)] was performed using ImageJ software (ImageJ, U.S. National Institutes of Health, Bethesda, MA, USA, http://imagej.nih.gov/ij/).