Endothelial cell proliferation assay

HUVEC proliferation was evaluated by 5-bromo-2′-deoxyuridine (BrdU) incorporation using an ELISA kit (Cell Proliferation ELISA, BrdU, Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. HUVECs (1 × 10⁴) were seeded into a 96-well plate in M199 medium containing 1% FBS for serum starvation. After 24 h, the HUVECs were treated with rIL-33 (20 ng ml⁻¹) alone or in combination with rsST2 for 36 h and then labelled with BrdU for 2 h. The cells were fixed and incubated with a peroxidase-conjugated anti-BrdU antibody. Then, the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine was added, and BrdU incorporation was quantified by measuring the light emission of the samples using a luminometer.