Intracellular calcium mobilization assays

Ligand-induced intracellular calcium mobilization was assessed using Fluo-4 NW Calcium Assay kit (Thermo Fisher Scientific) following the manufacturer's instructions. Briefly, HMDMs were seeded (50,000/well) in black clear-bottom 96-well TC plates (Corning) for 24 h before the assay. The cells were first stained with the Fluo-4 dye in assay buffer (1× HBSS, 20 mm HEPES) for 45 min (37 °C, 5% CO₂). Respective ligands were prepared in assay buffer containing 0.5% BSA. On a Flexstation 3 platform, the fluorescence (excitation/emission, 494/516 nm) was continually monitored for a total of 100 s with ligand addition performed at 16 s.