Fig. 5

THP-1 macrophages were exposed for 16 h to MVs released during bacterial culture (bMVs), infection (MVs), vesicles from THP-1 macrophages that were stimulated with heat-inactivated bacteria (EVs), and to whole heat-inactivated bacteria at an MOI of 0.1 (hi-bacteria). Then the release of the pro-inflammatory cyto- or chemokines TNF-α (a), IL-8 (b) and IL-1β (c) was determined by ELISA. The number of MVs used for these exposures represents 2-times the physiological MV-concentration cells are theoretically exposed to when cells are infected with the bacteria at an MOI of 10. To calculate the amount of MVs to be used for these experiments we determined the vesicle recovery following vesicle-enrichment using ultrafiltration and vesicle purification by SEC using TRPS on MVs from infection experiments (MVs-SEC; based on NTHi and Mrc) and calibration beads (beads-SEC; based on 114 nm-sized Izon® calibration beads) (d). Data are represented as the mean ± SEM (n ≥ 4). *p < 0.05, **p < 0.01