Rapid endothelial cell activation in vitro follows targeted laser pulse. HUVECs were loaded with Fluo-4 AM (3μM) and observed using fluorescence microscopy. (A) Representative images of a cell before and after a direct laser pulse to the point the indicated (X). An increase in Fluo-4 fluorescence (green) reflects a rise in intracellular Ca²⁺. (B) Quantification of this signal is plotted against time showing 1 representative trace (solid line) and the mean trace of laser-induced activation of 41 cells (dotted line). (C) Similarly prepared HUVECs loaded with Fluo-4 AM were stimulated with ADP (10μM), thrombin (1 U/mL), or histamine (10μM) as a comparison to the laser-induced activation. Agonists or vehicle were added after 10 seconds of image acquisition. The graph shows median curves as a comparison of the kinetics of these modes of activation. ADP, solid line; thrombin, – – –; histamine, -.-.; laser, ---; vehicle ⋯. (D) The peak cell activation was extracted from the kinetic data for each agonist and the laser. The mean ± SEM is plotted for each group; ADP stimulation, n = 82 cells from 7 experiments, histamine n = 45 cells from 3 experiments; thrombin n = 85 cells from 6 experiments; vehicle n = 10 cells from 2 experiments.