Preparation of Cytokine and Inhibitor Solutions

Acetyl-beta-methacholine-chloride (Sigma) was dissolved in 0.9% saline for flexiVent studies. Mouse IL-1B (R&D Systems, MN, US) was dissolved in 0.9% sterile saline containing 0.1% BSA carrier to a concentration of 10 mg/mL, aliquoted and stored at −20°C until use. A new aliquot of mouse IL-1B (10 mg/mL) stock was diluted 1:1,000 into 0.9% sterile saline for intranasal delivery each day. Recombinant human IL-1B (R&D Systems) was dissolved in PBS to a concentration of 100 mg/mL, aliquoted and stored at −20°C until use. A new aliquot of human IL-1B (100 mg/mL) stock was diluted 1:10,000 into complete growth media for cell culture experiments each day. The CREB inhibitor 666-15 (Li et al., 2016 (link)) (R&D Systems) was initially dissolved in 100% DMSO at a concentration of 1 mM. The stock was diluted 1:10,000 into complete growth media for cell culture experiments.