CLC formation in EETosis in vitro. (A) Cytoplasmic CLC formation in an eosinophil undergoing ETosis (arrow). Eosinophils were observed with time-lapse, phase-contrast imaging following IL-5 and platelet activating factor stimulation. Images were captured from supplemental video 1. (B) CLCs were associated with EETosis. Eosinophils were stimulated with PMA (10 ng/mL) with/without DPI for 120 minutes. CLCs were counted using an inverted microscope (40× objective, Eclipse TE300, Nikon). A total of 240 fields from 4 independent donors were studied. The bar graph represents the mean ± standard deviation. (C) Cellular galectin-10 localization during EETosis. PMA stimulated cells were fixed at 15, 45, and 120 minutes and stained with anti-galectin-10 Ab (green). At 15 minutes (i), galectin-10 was eccentrically located in adherent eosinophils. Galectin-10 was homogeneously distributed in cytoplasm at 45 minutes (ii). Note the loss of the typical bilobed nuclear shape. At 120 minutes (iii), cytoplasmic galectin-10 had disappeared and CLCs (arrows) could be recognized. ETs (filamentous DNA in blue) were also evident. EVs (supplemental Figure 7B) were out of focus. (D) Extracellular CLC formation. (i) Stacked EETosis cells form varied sizes of CLCs. Eosinophils (5 × 10⁶ cells in 1.5 mL) were stimulated with PMA (10 ng/mL) in round-bottomed microtubes for 3 hours, followed by fixation and processing for TEM. Sectioned CLCs of variable size (arrowheads) were observed. (ii) Free galectin-10 levels in culture supernatants. Eosinophils were stimulated with PMA with/without DPI for 3 hours, and culture supernatants were recovered by centrifugation at 10 000g for 10 minutes to remove vesicles, free granules, and CLCs. The graph represents the mean ± standard deviation from 6 different donors. ***P < .001. cont, nonstimulated control.