4 6 diamidino 2 phenylinodole dapi

Cells were seeded on the GO scaffolds in 48-well plates at a density of 5 × 10⁴ cells per well and incubated for 1–7 days. The adhered cells were fixed with a 4% paraformaldehyde solution (Sigma-Aldrich, St. Louis, MO, USA) for 60 min, permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min, and stained with tetramethylrhodamine(TRITC)-conjugated phalloidin (Millipore, Burlington, MA, USA) and 4,6-diamidino-2-phenylinodole (DAPI; Millipore, Burlington, MA, USA) for 30 min. A fluorescence microscope (Nikon, Tokyo, Japan) was used for acquiring the images of the stained cells.

Cell viability was measured using a WST-1 assay (EZ-Cytox cell viability assay kit, Daeillab Service Co., Ltd., Seoul, Korea). Water-soluble formazan was quantified by a multiwell spectrophotometer (Victor 3, Perkin Elmer, Waltham, MA, USA), measured at 450 nm. For ICC, DPSCs (1 × 10⁴ cells sample-1) were seeded on the substrates and allowed to spread for 7 days in culture media at 37 °C in a humidified atmosphere containing 5% CO₂. The adhered cells were fixed with a 4% paraformaldehyde solution (Sigma-Aldrich, St. Louis, MO, USA) for 20 min, permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min, and stained with TRITC-conjugated phalloidin (Millipore, Burlington, MA, USA) and 4,6-diamidino-2-phenylinodole (DAPI; Millipore, Burlington, MA, USA) for 1 h. Focal adhesions (FAs) were stained with a monoclonal anti-vinculin antibody (1:100; Millipore, Burlington, MA, USA) and an FITC-conjugated goat anti-mouse secondary antibody (1:500; Millipore, Burlington, MA, USA). Images were taken using a confocal laser scanning microscope (LSM710, Carl Zeiss, Oberkochen, Germany).

Cells were seeded on the GO scaffolds in 48-well plates at a density of 1 × 10⁵ cells per well and incubated in osteogenic media for 2 weeks. The adhered cells were fixed with a 4% paraformaldehyde solution (Sigma-Aldrich, St. Louis, MO, USA) for 60 min, permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min, a primary antibody (polyclonal anti-human osteopontin goat IgG, R&D system, Minneapolis, MN, USA) for 1 h, and a secondary antibody (anti-goat IgG fluorescein isothiocyanate (FITC) conjugate) for 1 h, and stained with TRITC-conjugated phalloidin (Millipore, Burlington, MA, USA) and 4,6-diamidino-2-phenylinodole (DAPI; Millipore, Burlington, MA, USA) for 30 min. A fluorescence microscope (Nikon, Tokyo, Japan) was used for acquiring the images of the stained cells.