Transwell chemotaxis assay.

Neutrophils isolated from human subjects were incubated for 20 min at RT in control or EVE-infused HBSS. Neutrophils exposed to EVE were assessed for viability relative to unexposed neutrophils via annexin V and propidium iodide staining and FACS (Supplemental Fig. SA; all Supplemental material is available at https://doi.org/10.6084/m9.figshare.9922601.v1). Cells were also assessed for viability by trypan blue staining before seeding in 6-mm Transwell permeable supports (3-μm pore size; Corning) in 24-well plates. The lower chambers contained HBSS alone or 100 nM of the chemoattractant fMLP. Neutrophils were allowed to migrate for 45 min at 37°C with 5% CO₂, after which inserts were removed, and cells that migrated in the lower well were lysed by addition of Triton X-100 (0.1% final, 10 min, 24°C). Samples of cell lysates (180 µL) were transferred to a fresh 96-well plate containing 20 µL of 100 mM N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide, a colorimetric elastase substrate. Following a 30-min incubation at 24°C, absorbance at 405 nm was measured using a SpectraMAX Gemini EM fluorescence reader (Molecular Devices, Sunnyvale, CA).