IP3 assay

IP₃ accumulation in macrophages was measured using the HitHunter IP₃ Fluorescence Polarization Assay (GE Healthcare, 90-0037-02). M0 macrophages were cultured in RPMI 1640 containing 10% FBS and M-CSF (100 ng/ml) and serum-starved overnight, and a series of different concentrations of potential endogenous agonists were added to designated wells in a 96-well plate. The reaction was quenched after 1 min, and the tracer was added, followed by an IP₃-binding protein. Fluorescence was excited with a wavelength of 483 nm, and emission was measured at 530 nm.