Eosinophil-Macrophage Interaction Assay

Eosinophils were isolated as described previously (25 (link)) from NJ.1638 or IL-13^−/−/NJ.1638 (23 (link), 24 (link)) mice. IL-33 (Peprotech) was added to activate the eosinophils to 50ng/mL (IL-33 was omitted from the resting eosinophil experimental group). Cells were incubated at 37°C, 5%CO₂ and 24 hours later were washed three times to remove added cytokines. Macrophages were isolated from C57BL/6J or MMP-12^−/− mice as described by Lasbury et al (26 (link)). Cells were plated at 150,000 cells per well in 24 well culture plates (Corning, Corning, NY) and cultured for 48 hours with the addition of either eosinophils (750,000 cells) or media alone in 500μl total volume. To assess contact dependency the above was performed in transwell plates (0.4μm pore) (Corning).