gata2^hi eosinophils degranulate in response to H polygyrus helminth extracts. (A) IPEX from 3 WT fish was cultured in media containing either HpAg over a concentration gradient or in PBS alone for 4 hours. Cell-free supernatants were collected and assayed for peroxidase activity using the OPD assay. An average of the optical density (A₄₉₂) for each dose was taken from 3 independent experiments. Error bars represent SD. (B) Time course of degranulation as measured by OPD assay. IPEX was cultured in media with 10 μg HpAg; then cell-free supernatants were assayed for degranulation. (C) Purified gata2^hi cells (∼ 5 × 10⁴) from IPEX were lysed with 0.2% Triton-X or cultured for 2 hours in media with either 10 μg of HpAg or PBS. Cell-free supernatants were then collected and assayed for peroxidase activity using the OPD assay. Bars represent the mean optical density (A₄₉₂) measured from 3 independent experiments with SD. *P < .05. **P < .001. (D) TEM photomicrographs show purified gata2^hi cells (∼ 2.5 × 10⁵) cultured for 2 hours in media with either 12 μg of HpAg (iii-viii) or PBS (i-ii). Note heterogeneous populations of granules that display a range of electron lucency; granules are completely lucent (black arrowheads), marbled (white arrowheads), or variegated (white arrows). Electron-lucent granules were localized along cell peripheries (iii-vi). N indicates nucleus. Images are representative of 2 independent experiments.