Primesurface96u plates

Expanded NSCs were seeded as a single-cell suspension of 1.0 × 10⁴ cells/well into non-cell-adhesive round-bottomed 96-well PrimeSurface96U plates (Sumitomo Bakelite, Tokyo, Japan) with 200 μL of Neural Expansion Medium containing Y-27632 before being cultured for 5 days. For routine maintenance, half the volume of medium without Y-27632 was changed on even-numbered days. Phase-contrast images of cultured cells were acquired using an IX71 microscope with CellSens software (OLYMPUS, Tokyo, Japan).

A549 and RERF-LC-MS cells derived from human lung adenocarcinoma were obtained from the RIKEN BRC through the National Bio-Resource Project of the MEXT (Ibaraki, Japan). The cells were grown in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, USA) as described previously [43 (link)]. In a two-dimensional (2D) model, the cells were grown on flat-bottomed 96-well plates, 6 cm and 10 cm dishes. In a three-dimensional (3D) model, the cells were grown on PrimeSurface 96U plates (Sumitomo Bakelite, Tokyo, Japan). After culturing for 72 h, the cells were incubated in the absence or presence of coffee ingredients for 6 h (real-time PCR) or 24 h (Western blotting and viability assays).

G636C-R4/BaF cells (1000 cells per well) were plated in RPMI-1640 supplemented with 10% heat-inactivated FBS with or without IL-3 (10 ng/mL) in 96-well plates. G636C-R4/3T3 cells (2000 cells per well) were plated in DMEM supplemented with 10% heat-inactivated FBS in PrimeSurface96U plates (Sumitomo Bakelite). The cells were treated with ASP5878 at 0–10,000 nM (3-fold serial dilutions, 10 concentration points) for 4 or 5 days, and the number of viable cells was determined using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). The IC₅₀ value of ASP5878 was calculated using nonlinear regression analysis with the Sigmoid–Emax model, and geometric mean IC₅₀ values and 95% confidence intervals (CIs) were calculated from three individual experiments. Microsoft Excel (Microsoft) and GraphPad Prism (GraphPad Software) were used for data analysis. Experiments were performed in accordance with Astellas’ guidelines.