cAMP Quantification in IL-13 Stimulated Cells

The cAMP-Glo Max Assay (Promega, cat. #V1681) was followed according to the manufacturer’s instructions and as previously described by our lab (Sponchiado et al., 2023 (link)). Briefly, NCI-H322 cells were seeded at a density of 10,000 cells/well in 96-well clear bottom assay plate per the manufacturer’s protocol (Corning, cat. #3610). The next day, cells were stimulated with IL-13 or vehicle control (as detailed above). A standard curve was constructed according to the manufacturer’s instructions. The plate was read on an Agilent Bio Tek Syngery LX multi-mode reader using endpoint luminescence protocol. The cAMP concentrations for vehicle and IL-13-treated NCI-H322 cells were calculated using the values from the standard curve and plotted in a sigmoidal 4PL curve (GraphPad Prism 9) as we previously described (Sponchiado et al., 2023 (link)).