Brdu uptake assay

Proliferation of SCs was evaluated using the BrdU uptake assay (Roche Diagnostics) according to the manufacturer’s protocol. Briefly, SCs were cultured at a density of 1.0 × 10⁴ cells/well in 96-well plates in growth medium with or without OUBs for 24 h under normoxic (20% O₂) or hypoxic (1% O₂) conditions as previously described with minor modification [64 (link)]. After the addition of 10 μM BrdU, cells were incubated for 2 h. The BrdU incorporation rate was measured using an automatic microplate reader at 370 nm.

Proliferation of SCs was evaluated using a BrdU uptake assay (Roche Diagnostics). Briefly, SCs were seeded into poly-l-lysine-coated 96 well plates (IWAKI) at a density of 1.0 × 10⁴ cells/well. Cells were left to adhere for 24 h prior to stimulation with NTP. Subsequently, the medium was changed to medium containing 10 μM BrdU, and cells were incubated for 2 h under standard cell culture conditions (37 °C and 5% CO₂). The culture plates were fixated with FixDenat solution and incubated with anti-BrdU POD antibody solution for 90 min at room temperature. After washing the plate with phosphate-buffered saline (PBS), substrate solution (tetramethyl benzidine) was added for 30 min. Absorption was measured at 370 nm, with 492 nm as the reference wavelength, on an automatic microplate reader.