Endotoxin priming elicits intracellular ROS production.
A, intracellular ROS were measured by flow cytometry using the cell permeable DHR 123 fluorescent ROS-sensitive probe. LOS:sCD14 10 ng/ml, elicited a time-dependent increase in intracellular ROS as compared to control. Control cells at time 0, immediately following addition of the probe, are set to a value of 1, and data are presented as fold-increase with respect to this value, * = p < 0.05, n = 10. B, using Oxyburst to measure intracellular ROS in endocytic compartments, LOS:sCD14-primed PMNs displayed a significantly greater percentage of Oxyburst positive cells than did control PMN, * = p < 0.05, n = 6. C, representative histogram from compiled data in B. D–F, confocal microscopy demonstrated Oxyburst positive vesicular compartments in a subset of cells. Representative confocal image of PMN after 30 min of priming with LOS:sCD14 demonstrating Oxyburst positive, ROS-containing vesicles (D), colocalizing with TR-dextran containing vesicles (E), and merged image (F).