Intact granules and limited granule protein localization at extracellular DNA nets released through EETosis-mediated cell lysis. (A) Eosinophil EETosis was induced using 1 mg/mL immobilized IgG (120 minutes) or 2 μM A23187 (60 minutes) and fixed and permeabilized, then stained for MBP (green) and DNA (PI, red). Most released DNA nets did not contain free granule MBP protein and instead showed punctate or cell-associated staining. Images were obtained with a BX62 Olympus upright microscope, 20× UPlanApo objective with a numerical aperture of 0.70, coupled to a Hamamatsu Orca-AG fire-wire cooled digital camera (Hamamatsu Photonics, Hamamatsu, Japan; images were acquired using iVision software). Data are representative of >3 experiments from independent donors with similar results. (B) Cells were processed for TEM directly on slide surfaces. A23187-induced (2 μM, 60 minutes) EETosis eosinophils exhibit disrupted plasma membranes with clusters of released secretory granules (arrows) and DNA nets (Nets). Extracellular free granules (B, insert) show their typical morphologies with full crystalloid cores and matrices and intact granule-delimiting membranes. Some granules were entrapped in DNA nets. Scale bars represent 20 μm (A), 1 μm (B), and 700 nm (B, insert).