Rodhamine phalloidin

To analyze cell morphology, the osteoblasts were seeded on 8 samples (2 per type), which were washed in PBS after 24 hours. The blocks were then fixed with 4% paraformaldehyde (PFA) in PBS for 15min. After two washes with PBS, cells were permeabilized with 0,1% Triton X-100 (Sigma-Aldrich, St.Louis, MI, USA) in PBS (Avanzato et al. 2016; F. Mussano et al. 2018) . Following the manufacturer's protocol, cells were stained with Rodhamine-Phalloidin (Life Technologies Monza, Italy) and 1uM Dapi (Life Technologies) to detect the cytoskeleton and the present nuclei (Mussano, Genova, Rivolo, et al. 2017) (link) . Image acquisition was performed using a Nikon Eclipse Ti-E microscope with both a 20X and 40X objective (Plan Fluor Nikon, Tokyo, Japan) connected to it. Image analysis was performed by means of ImageJ software (ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/).

Cells were seeded on titanium disks in both control and plasma APDBD treatment condition (120'') at a concentration of 10 4 cells/well in a 24-well plate (BD, Milan Italy) and then kept in growth condition. After 30 minutes and 8 hours (36 disks for control group and 36 for APDBD treatment for each time-point) the titanium specimens were washed in PBS and then the cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15min. 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 1uM Dapi (Life Technologies) to respectively detect the cytoskeleton and the nuclei. Image acquisition was made recurring to a Nikon Eclipse Ti-E microscope with a 20X and 40X objective (Plan Fluor Nikon). Image analysis was performed by means of ImageJ software (ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/).