Hil 33

The concentrations of cytokines hIL-33, hIL-1α, hCCL-2 (R&D systems), hTSLP, mIL-33, mTSLP, mIL-4 and mIL-1α (eBiosciences) in the cell supernatant, Lung homogenate and in the BALF were estimated using paired antibodies following manufacturer’s instructions.

We collected fresh blood in EDTA anticoagulant tubes at Shanghai Ruijin Hospital, Shanghai Jiaotong University School of Medicine. Human PBMCs were obtained by using Ficoll-Paque PLUS (GE Healthcare). The isolated PBMCs were washed and resuspended in PBS containing 2% fetal bovine serum (FBS). The cells were cultured in RPMI-1640 medium supplemented with hIL-2 (R&D Systems), hIL-7 (R&D Systems), and hIL-33 (R&D Systems). The IL-13 and IL-5 levels in the supernatant were then measured.

Tissue and in vitro cell samples lysed with a lysis buffer (Cat. No. 3228, Sigma), followed by 20-min centrifugation to remove cellular debris. Plasma samples were obtained from whole blood processed by collection into anti-coagulant-containing plasma tubes followed by 15-min centrifugation. Conditional media were collected at 48 or 72 h of confluent monolayer cells and were centrifuged to remove cellular debris before use. Four different assays were performed according to manufacturer's protocol to detect hPDGF-BB (Cat. No. DY220, R&D Systems), mPDGF-BB (Cat. No. MBB00, R&D Systems), mIL-33 (Cat. No. M3300, R&D Systems) or hIL-33 (Cat. No. 435907, BioLegend). Optimal standard curves were applied to individual assays and the absorbance values were detected at 450 nm using a microplate reader.