Confocal microscopy of phagocytosis and phagocytosis independent anti-inflammatory activity of ectosomes. (A) HMDMs were incubated with fluorescently labeled ectosomes for 30 minutes, fixed, and analyzed by confocal laser microscopy (i-ii). Alternatively, macrophages were preincubated cytochalasin D (cytD) prior to the addition of ectosomes (iii-iv). The lens used was a Zeiss Plan-Neofluar 40× and 100× from Zeiss AG (Feldbach, Switzerland). The imaging medium was Vectashield fluorescence mounting medium (Vector Laboratories, Burlingame, CA). The camera was a Zeiss Axiovert Laser Scanning Microscope (LSM510) from Zeiss AG (Feldbach, Switzerland). The acquisition software was LSM510 Software (Zeiss). (B-D) HMDMs were incubated with medium alone or with medium supplemented with zymosan for 24 hours. Alternatively, ectosomes were added to the incubation medium with or without preincubating the macrophages with cytD. Error bars indicate standard deviation of the mean.