For calcium imaging, iMGLs were plated on poly-L-lysine-coated coverslips and 1 hour later were incubated with Fura-2-AM (Molecular Probes) calcium dye diluted in Ringer solution containing (in mM): NaCl 140, KCl 4.5, CaCl2 2, MgCl2 1, HEPES 10, glucose 10, sucrose 5, pH=7.4. After 1-hour incubation, the dye was washed out 3 times using Ringer solution and treated for 1 hour with either P2ry12 inhibitor PSB0739 (50 μM, Tocris) or Vehicle (DMSO) and used for experiments. Baseline Ca2+ signal (I340/I380) were measured for more than 100 s and then ADP (10 μM) was introduced under steady flow after baseline measurement. Ca2+ recordings were performed on Zeiss (Axiovert 35)-based imaging setup and data acquisition was conducted with Metafluor software (Molecular Devices). Data analysis was performed using Metafluor, Origin Pro, and Prism 6.0.
