Released granules were secretion competent in response to CCL11. (A) Culture medium of A23187-stimulated cells (2 μM, 90 minutes) was collected, and granule-rich subcellular structures were isolated. The cell-free structures were stimulated with 2 μM A23187 or the indicated concentrations of CCL11 for 60 minutes. Secreted ECP levels, assessed by enzyme-linked immunosorbent assay, were normalized with spontaneous ECP release (100%), and data are expressed as means ± SD, from 3 different donors. *P < .05 vs nonstimulated controls. The spontaneous secretion levels were 10.0 ± 4.4% of the total ECP in the structures. (B) AO-loaded eosinophils were stimulated with 2 μM A23187 to induce EETosis, followed by the isolation of subcellular granule structures. Among the 80 single granules or groups of granules, following CCL11 stimulation, significant responses with intense transient fluorescent flashes indicative of the release of monomeric AO were observed from 14 nonenveloped granules (17.5%). The secretory response was not observed by likely PM-bound clusters of granules (lower panels). Images were obtained with a Hamamatsu Orca-AG fire-wire cooled digital camera coupled to a BX62 Olympus microscope using a 60× PlanApo objective with a numerical aperture of 1.42. Fluorescence intensity was analyzed by iVision software and pseudocolored with red to represent the greatest intensity as indicated by the scale color. Experiments were repeated with eosinophils from 8 independent donors. The scale bar represents 3 μm (B).