Measurement of cytokine protein levels in tissue homogenates

The protein concentrations for tissue extracts were determined using the Pierce 660nm Protein Assay Kit (Thermo Scientific Inc., NY, USA). Samples were thawed at room temperature and vortexed to ensure well-mixed sample. Multiple cytokine analysis kits (IL-5, IL-17A, IL-23, IFN-γ, CXCL-8, and CCL-11) were obtained from R&D systems (Cat. No. LMSAHM) and data were collected using Luminex 100 (Luminex, Austin, TX, USA). Data analysis was performed using the MasterPlex QT version 2.0 (MiraiBio, Alameda, CA). All assays were run in duplicate according to the manufacturers’ protocol. Sensitivity of each cytokine is as follows: IL-5 (0.5 pg/mL), IL-17A (1.8 pg/mL), IL-23 (11.4 pg/mL), IFN-γ (0.4 pg/mL), CXCL-8 (1.8 pg/mL), and CCL-11 (14.6 pg/mL), All the protein levels in tissue homogenate were normalized to the concentration of total protein (mg/mL) [17].