Trypan blue dye viability assay

Cells were incubated in 60-mm dishes with 4 ml of culture medium, at 1 × 10⁶ cells/dish under either normoxic conditions, or hypoxic conditions (1% O₂) for 0, 24, 48 and 72 h. For the trypan blue dye exclusion test, cells were stained using phosphate-buffered saline (PBS, Sigma-Aldrich, Saint Louis, MO) containing 0.5% trypan blue (Nacalai Tesque, Inc., Kyoto, Japan). Cell viability was assessed by counting the number of both stained and unstained cells using cell-count slides.