4 6 diamidino 2 phenylindole dihydrochloride dapi

BM-MSCs (2 × 10³ cells per mL) were seeded onto SLA or SLA-DOPA titanium discs in 24-well plates with proliferation medium (α-MEM; containing 10% fetal bovine serum and 1% penicillin/streptomycin). These were cultured for 1, 3 or 24 h, fixed in 4% paraformaldehyde solution for 10 min, permeabilized in 0.1% Triton X-100/phosphate buffered saline (PBS), and incubated in 200 μL of 1 μg mL⁻¹ phalloidin-FITC (Sigma, P5282) in PBS for 1 h to label the actin cytoskeleton. The nuclei were labeled with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Cell Signaling Technology, Danvers, MA, USA). Samples were examined by confocal laser scanning microscopy (CLSM, Carl Zeiss, Jena, Germany) at 488 nm wavelength or under UV light. To quantify cell adhesion, BM-MSCs (6 × 10³ cells per mL) were seeded onto titanium discs, cultured for 1, 3 or 24 h, washed in PBS three times to remove non-adherent cells, fixed, permeabilized, and nuclei were labeled using DAPI. Cell numbers in three randomly selected light microscope fields (Olympus, Tokyo, Japan; ×10 objective) were calculated using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, Maryland, USA).