Chemotaxis Assay for Eosinophils and Neutrophils

Eosinophil chemotaxis was performed with purified human eosinophils, whereas human PMNL preparations were used to assess the migratory responsiveness of neutrophils. For all experiments, technical triplicates have been performed. Cells were resuspended in assay buffer, pretreated with KPR-6 (20 µM) or ISO-1 (20 µM; Merck) for 30 min at 37 °C, and allowed to migrate towards MIF (3 nM; Peprotech; eosinophils: n = 11, neutrophils: n = 12), IL-8 (10 nM; Immunotools; neutrophils: n = 10) or CCL11 (10 nM; Immunotools; eosinophils: n = 12) for another 60 min at 37 °C in a 48-well micro-Boyden chamber using PVP-free polycarbonate filters with a pore size of 5 µm (eosinophils) or 3 µm (neutrophils) (Sterlitech). Migrated cells were enumerated by flow cytometry on a BD Canto II flow cytometer (acquisition set for 30 s at medium flow rate). Therefore, eosinophils and neutrophils were gated by their forward and side scatter properties and by autofluorescence (Figure S1) [53 (link),54 (link)].