EETosis releases weblike chromatin structures from lytic cells. (A) Eosinophils were stimulated with the indicated stimuli in 0.1% BSA medium for 120 minutes or in A23187 for 60 minutes and fixed. EETosis was detected using extracellular histone staining without permeabilization, as described in “Materials and methods” and supplemental Figure 3. Green indicates histones, and red indicates DNA. Images were obtained using a BX62 microscope (20×, equipped with a Qimaging Rolera EM-C2 cooled digital camera (Surrey, BC, Canada) in conjunction with SlideBook 5.0 image analysis software (Intelligent Imaging Innovations, Denver, CO), or equipped with a Qimaging Retiga EXi cooled digital camera in conjunction with iVision image analysis software). Experiments were repeated with eosinophils from 3 independent donors with similar results. (B) Eosinophils were stimulated with 2 μM A23187 for 60 minutes and processed for SEM. Large DNA nets were released from lytic cells and often were associated with originating cells (outlined by arrows, left). Weblike DNA nets consisted of 25- to 35-nm diameter fibers aggregated into larger fibers (right). Scale bars represent 20 μm (A), 10 μm (B, left panel), and 500 nm (B, right panel).
