Fig. 5
a Adhesion assay of NIH3T3 cells. NIH3T3 cells were plated at 1 × 105 cells per well of a 12-well plate, each well containing a titanium plate treated with either distilled water (DW), 10 mM CaCl2 (Ca) or 10 mM MgCl2 (Mg). Plates were then shaken three times for 5 min at 75 rpm to remove non-/weakly-adherent cells. Remaining cell numbers were determined using WST-8 reagent and measurement of absorbance at 450 nm. Data are mean ± SD. Statistical analysis was by ANOVA with Dunnett’s post-test. Asterisk denotes a statistically significant difference. b Localization of adhesive protein in NIH3T3 cells. Adherent cells were incubated firstly with rabbit anti-mouse vinculin polyclonal antibody followed by fluorescein isothiocyanate-conjugated anti-rabbit IgG secondary antibody (green). Actin filaments were labeled with tetramethylrhodamine isothiocyanate-conjugated phalloidin (red).Blue staining represents DAPI visualization of the nuclei