Primesurface96u multiwell plates

We used a Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) for cell proliferation, a wound-healing assay for cell migration, BioCoat Matrigel invasion chambers (BD Biosciences, Bedford, MA, USA) for cell invasiveness, and the CytoSelect 48-Well Cell Adhesion Assay (Cell Biolabs, San Diego, CA, USA) for cell adhesion to extracellular matrix components. These assays were performed as previously described.² (link)^,40 (link) To evaluate total caspase activity, a Muse MultiCaspase Kit (Merck Millipore, Billerica, MA, USA) was used. The activities of caspase-3, -8, -9, and -12 were measured using the Caspase Colorimetric Assay Kit (BioVision, Milpitas, CA, USA). Mitochondrial membrane potential and cell-cycle distribution were assessed using a Muse MitoPotential Kit (Merck Millipore) and a Muse Cell Cycle Kit (Merck Millipore), respectively. ALDH, a surrogate marker of stem/progenitor cells, was estimated using the ALDEFLOUR fluorescent reagent system (STEMCELL Technologies, Vancouver, BC, Canada). ALDH-positive cells were determined using a FACSCalibur system (BD Biosciences, Franklin Lakes, NJ, USA). The three-dimensional spheroid cultures were analyzed using PrimeSurface96U multiwell plates (Sumitomo Bakelite, Tokyo, Japan).

SCAP (P3) were seeded on low cell attachment PrimeSurface 96 U multiwell plates (Sumitomo Bakelite, Tokyo, Japan) at different densities (1 × 10⁴, 5 × 10⁴, 1 × 10⁵, 2 × 10⁵, 3 × 10⁵, 4 × 10⁵, 5 × 10⁵, and 1 × 10⁶ per well) and for different periods (1, 2, 3, 7, 14, 21, and 28 days) in the growth medium to form spheroids. The spheroids were subsequently cultured for 4 weeks in the osteogenic/dentinogenic medium. The medium was changed twice a week. As a control, some spheroids were cultured in the growth medium. At each period, the cultured spheroids were imaged with a microscopy. Only aggregated area was measured by ImageJ software (NIH), and non-aggregated area was ignored from the measurement. The surface of the spherical structures was observed under a stereo microscopy SteREO Discovery.V12 (Carl Zeiss Microscopy, Jena, Germany). The spherical structures were also used for microcomputed tomographic (micro-CT) analysis.