Blood sampling and analysis
TNF-α
BLOOD
All participants were instructed to fast for ≥ 8 h before sample collection. In the morning, 8 − 10 a.m., 10 mL of blood was collected from the antebrachial vein by a clinical pathologist. The blood was centrifuged at 3000 rpm for 10 min in Combi-514R (Hanil, Seoul, Korea) for further analysis. An enzyme-linked immunosorbent assay (ELISA) was used to measure circulating plasma levels of TNF-α and IL-6 (Quantikine HS Human TNF-α and Quantikine HS Human IL-6 kits, respectively; R&D Systems, Minneapolis, USA). For data acquisition and analysis of the ELISA assays, we used a VersaMax absorbance microplate reader (Molecular Devices, Sunnyvale, CA, USA). Blood CRP was analyzed by turbidimetric immunoassay using a CRP4 kit (Roche, Basel, Switzerland) Cobas 8000 equipment (Roche). Serum VEGF was analyzed using a Human VEGF Quantikine ELISA kit (R&D Systems) and the VersaMax absorbance microplate (Molecular Devices).