Ccl11

Whole blood (500 μl/tube) was exposed to a stimulus known to induce piecemeal degranulation—100 ng/ml exotoxin (CCL11)(BioLegend, San Diego, CA), for 2 or 5 h at 37°C. Untreated samples were also included. After incubation, 200 μl of blood was removed, red blood cells were lysed, and the sample was then fixed (BD CellFix™, Becton Dickinson, Belgium).

Transwell migration assay of eosinophils was performed using 5 μm pore-sized cell culture inserts (Corning Costar Corporation, Cambridge, MA). Briefly, B16 melanoma cells were seeded (2 × 10⁴ in 600 µL) in the bottom compartment with or without IL-33 (100 ng/mL). Alternatively, the bottom compartment was filled with 600 µL of 2% FCS DMEM medium alone or containing IL-33 (100 ng/mL) or CCL11 (100 ng/mL, Biolegend). Eosinophils (IL-5 EO) were seeded in the upper insert (2 × 10⁵ in 100 µL). After 18 h incubation at 37 °C, 5% CO₂ the eosinophils migrated in the bottom compartment were enumerated by means of Neubauer chambers.

Eosinophils were enriched from mouse spleen by negative selection by staining with biotin-conjugated antibodies as above and using the EasySep Mouse Streptavidin RapidSpheres Isolation Kit (STEMCELL Technologies). Enriched cell populations were then stained and sort purified (CD45⁺SiglecF⁺CD11c⁻CD11b⁺). 1–1.5 × 10⁴ purified eosinophils in 80 μl pre-warmed RPMI-1640 media containing 5% bovine serum albumin were placed in the upper compartment of wells containing HTS 96-well Transwell Permeable Supports (Corning). The lower compartment contained various concentrations of CCL11 (BioLegend) in the absence or presence of IL-2c (0.2 μg IL-2 equivalent) in 235 μl. Cells were allowed to migrate at 37 °C in a humidified 5% CO₂ chamber for 2 h, the plate placed on ice for 10 min, and then centrifuged at 200 × g for 10 min to detach cells from the membranes. The transwells were then removed and the media containing cells in the bottom compartment was collected. The supernatant was centrifuged for 5 min at 200 × g, decanted, and the cell pellets were resuspended in 20 μl of AO/PI staining solution and counted on the Cellometer Auto 2000 cell viability counter (Nexcelom). Data are expressed as the average percentage of input cells that migrated to the bottom chamber, from duplicate wells.