Live Cell Calcium Imaging of ATP Signaling

Live cell imaging and calcium mobilization experiments were performed as described previously [1 (link),19 (link)]. Cells were preincubated with ARL 67156 trisodium salt (ARL) (Sigma-Aldrich (St. Louis, MO, USA)) (10, 20, 50 nM) [25 (link),26 (link)], Fluo-4AM (Thermo Fisher, Waltham, MA, USA) (1:100) and the counterstain SirActin (Cytoskeleton, Inc., Denver, CO, USA) (1:1000) for 20 min at 37 °C. Images were collected every 3 s for 45 min on the Zeiss Axiovert LSM 880 confocal microscope (Zeiss, Thornwood, NY, USA) utilizing the 20× air objective. Scratch wounds or agonist stimulation by ATP, 2,3-O-(4-benzoylbenzoyl)-ATP (BzATP) (Sigma-Aldrich (St. Louis, MO, USA)), or ATP (Sigma-Aldrich (St. Louis, MO, USA)) were performed after 100 frames (5 min). All injuries were made using a 25-gauge needle.