Mab275

The CXCL1 antibody (9µg/mL; cat. no. MAB275; R&D SYSTEMS, Minneapolis, MN, USA), IL-8/CXCL8 (0.4µg/mL; cat. no. MAB208; R&D SYSTEMS, Minneapolis, MN, USA), and IL-18 (1.2µg/mL; cat. no. AF2548; R&D SYSTEMS, Minneapolis, MN, USA) were used for the neutralization assay. The normal mouse IgG1 antibody (1 µg/mL; cat. no. sc-3877; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used as the control antibody. The experiment was conducted in the same manner as the cell culture environment, and the subsequent experiments or results were performed/obtained after 24–48 h.

Culture supernatants and BAL samples were assayed for IL-1β, MIP-1α, TNFα, IL-6, IL-10, IL-18, IL-17A and CXCL1 by ELISA. Antibody pairs for the IL-1β (MAB401 and BAF401; clone 30311 and polyclonal; R&D Systems; 8 and 4 μg ml⁻¹, respectively) and CXCL1 (mouse: MAB453 and BAF453; clone 48415 and polyclonal; R&D Systems; 8 and 0.4 μg ml⁻¹, respectively; human: MAB275, clone 20326, 4 μg ml⁻¹; BAF275, polyclonal, 40 ng ml⁻¹) and CCL3/ MIP-1α (Quantikine ELISA kit, Product #MMA00) ELISAs were from R&D Systems. Antibody pairs for IL-1α (capture: product #14-7011-85, clone ALF-161; detection: product #13-7111-85, polyclonal), TNFα (capture: product #14-7325-85, clone 1F3F3D4; detection: product #13-7326-85, clones MP6-XT22 and MP6-XT3), IL-6 (capture: product #14-7061-85, clone MP5-20F3; detection: product #13-7062-85, clone MP5-32C11), IL-10 (capture: product #14-7101-85, clone JES5-16E3; detection: product #13-7102-85, clone JES5-2A5) and IL-17A (capture: product #14-7175-85, clone eBio 17CK15A5; detection: product #13-7177-85, clone eBio17B7) were from eBiosciences and used at a 1:250 dilution. Antibody pairs for IL-18 were from MBL International (capture: product #D047-3, clone 74, 1:1,000; detection: product #D048-6, clone 93-10C, 1:2,000).