Plasma IL-33 Quantification Protocol

Plasma IL-33 level was measured using a technique described previously [12 (link)]. Briefly, the blood samples were centrifuged (10 min at 1,000×g) within 30 min after collection in tubes containing sodium ethylenediaminetetraacetic acid anticoagulant, and the collected plasma was stored at ≤ − 20 °C. Plasma samples were prepared for analysis in a 96-well plate utilizing a custom human cytokine. IL-33 level was measured by enzyme-linked immunosorbent assay (ELISA) using a Human IL-33 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) and 12 times concentrated with Amicon Ultra-0.5 mL Centrifugal Filter (Merck Millipore, Burlington, MA, USA) devices to improve the detection yield. We followed the kit-specific protocol provided with the BioTek’s PowerWave XS analyser (BioTek Instruments, Winooski, VT, USA). The result is described as 450 optical density (OD).