Flow cytometry analysis
Flow cytometry was used to detect CD40 and ICOSL expression on purified eosinophils at 0h, 24h, or 48h. Every time when eosinophils were isolated, CD16 was used to access their purity. Briefly, harvested eosinophils were resuspended in PBS with 1% FBS. 100 µL cell suspension was incubated with the fluorescein-conjugated antibody at 4℃ in the dark for 20 min. All the antibodies were purchased from Biolegend, San Diego, Calif, the detailed information was as follows: CD16-FITC (360716, B73.1), PE anti-human CD40 (334308, 5C3), APC anti-human ICOSL (309407, 2D3).