Snap 2058 zen pro 2012

Cellular morphology was evaluated after seeding 10 × 10⁴ 3T3 fibroblast on the surface of STHB formulations placed on 6-well culture plates (Corning Inc., Corning, NY). After being incubated for 24 h, cells were fixed using a 4% paraformaldehyde solution (Sigma-Aldrich, St. Louis, MO), followed by F-actin staining using phalloidin red (Thermo Fisher Scientific, Waltham, MA). Cellular fluorescence micrographs at different locations of the material surfaces were obtained using a fluorescence microscope (Zeiss, Oberkochen, Germany) and analyzed by Snap 2058-Zen Pro 2012 software (Zeiss, Oberkochen, Germany). Sixty individual cells per group were randomly selected in each micrograph for analysis. The maximum orthogonal length, width, and area of each cell were measured using ImageJ software (National Institutes of Health, Bethesda, MD), and the aspect ratio was calculated dividing the longer length by the shorter length of the cell.

THP-1 cells were pre-treated for 24 h with TNF-α to induce an inflammatory phenotype, another group of cells remained untreated for the purpose of this experiment. After treatment, THP-1 cells (10 × 10⁴) were cultured in 24-well plates (Corning Inc., Corning, NY) coated with 0.2 mL of each STHB formulation and Matrigel Matrix (Corning, Inc., Corning, NY) as a positive control. After 24 h of incubation, cells were washed with PBS to remove unattached cells, F-actin was stained using phalloidin red (Thermo Fisher Scientific, Waltham, MA) and the nucleus using DAPI (Sigma-Aldrich, St. Louis, MO). Fluorescence microscopy images (Zeiss, Oberkochen, Germany) were taken to analyze cell adherence and quantify numbers per mm² by Snap 2058-Zen Pro 2012 software (Zeiss, Oberkochen, Germany).