Chemotx microplates

A microchemotaxis chamber with polyvinylpyrrolidone-free polycarbonate filter (5 μm pore size) was used for the chemotaxis assay. THP-1 monocytic cells (8 × 10⁶ cells/mL) were plated in the upper wells of ChemoTx microplates (Neuro Probe Inc. Gaithersburg, MD, USA). The culture medium derived from untreated or IL-33-treated cells was added to the lower wells. The number of THP-1 cells migrated to the lower chamber was counted by a hemocytometer. The culture medium from the untreated cells supplemented with recombinant human MCP-1 at 100 nmol/L (PeproTech, Rocky Hill, NT, USA) served as a positive control. Normal goat IgG (R&D Systems, Minneapolis, MN, USA) was used as a negative control. To evaluate MCP-1 specific chemotaxis, anti-human MCP-1 polyclonal antibody (R&D Systems, Minneapolis, MN, USA) was added at 80 μg/mL to neutralize the secreted MCP-1.

Freshly isolated neutrophils (n = 5 horses) were loaded with ng/μL of calcein AM (Molecular Probes, Life Technologies, Burlington, ON) during 30 min at 37°C with gentle agitation, centrifuged at 300 × g for 10 min, and re-suspended in a fresh solution of PBS/FBS 10%(v/v). Neutrophils were then pre-treated for 30 min at 37°C with 0, 250, 500, or 1000 ng/mL of SCGB 1A1 or SCGB 1A1A. Twenty μL of sample was plated onto the ChemoTx microplates (Neuro Probe, Maryland, MD). The lower chambers were loaded with 29 µL of a PBS/FBS 10%(v/v) solution supplemented with 0.3 µg/mL of recombinant human IL-8 (R&D Systems, Minneapolis, MN) as chemoattractant. Serial dilutions of the cell suspension (1 × 10⁵, 7.5 × 10⁴, 5 × 10⁴, 2.5 × 10⁴, 1.25 × 10⁴, and 0 cells) were added to the chambers for a calibration curve, and all samples were analyzed in triplicate. Each microplate was incubated at 37°C (5% CO₂) for 45 min to allow migration. The filter membrane was then rinsed with PBS to remove non-migrating neutrophils, and fluorescence in wells was measured with a Synergy HT microplate reader using Gen5 analysis software (BioTek, Vermont, USA).