2.7. Intracellular cAMP assay

The cAMP concentration was measured using Cyclic AMP XP assay kit (Cell Signaling Technology). Briefly, 3 DIV cortical neurons (2.5 × 10⁵ cells) were treated with 50 ng/mL of BDNF or 20 μM forskolin (positive control) for 15 min with or without pretreatment with 100 nM K252a for 45 min. Then, neurons were lysed with 100 μl of lysis buffer. Fifty microliters of cell lysate was transferred into microtubes, and the cAMP concentration was measured according to the protocol provided in the kit. In each experiment, a standard curve was generated in parallel and used to calculate the cAMP concentration.