Intracellular cAMP assay

Intracellular cAMP levels were measured using Discoverx cAMP Hunter™ eXpress kits (Discoverx, Birmingham, UK) following the manufacturer’s protocol. Briefly, CHO-K1 cells stably expressing human C5aR1 receptor were plated into ½ area 96 well plates (15,000 cells/well) and incubated at 37°C, 5% CO₂ for 24 hours. Cells were then treated with either vehicle (Cell assay reagent) or 3 nM netrin-1 for 45 min at 37°C, 5% CO₂ before being stimulated with either vehicle or C5a at the indicated concentration for 30 min at 37°C, 5% CO₂. Cell lysis and cAMP detection were then performed as per the manufacturer’s protocol. Luminescence measurements were taken using a PHERAstar microplate reader.