Intracellular Calcium Mobilization

To detect intracellular calcium mobilization, the Fluo-8 No Wash Calcium Assay kit was used, following manufacturer’s protocol (ab112129, Abcam, Cambridge, Great Britain). Briefly, 1x10⁵ cells per well were seeded in black/flat clear bottom 96-well plates (Corning Incorporated, New York, NY, USA) and settled at 37°C with 5% CO₂. Cells were treated as described before. Fluo-8 stock solution was added together with PMA stimulation. Cells were incubated for 30 min at culture condition (37°C, 5% CO₂) and afterwards 30 min at room temperature. Fluorescence was measured at excitation/emission = 490/525nm continuously every 15 min for 2 h using a microplate reader (Flex Station^® 3). Peak results for Calcium influx at 2 h after stimulation are displayed as normalized data on PMA-stimulated controls.