ROS production assay

Neutrophils were stained in HBSS without calcium and magnesium containing 20 μM 2′, 7′-dichlorofluorescein diacetate (Sigma Aldrich) and were incubated with THP (50 μg/mL) for 30 min at 37°C with 5% CO₂. Where indicated, neutrophils were incubated with 1μg/mL mouse anti-human Siglec-9 monoclonal antibody α-Sig9(Sia), or mouse anti-human Siglec-9 monoclonal antibody α-Sig9(HA) for 15 min prior to incubation with THP. Cells were then added to a 96-well plate (5 × 10⁵ cells/well) and mixed at a 1:1 ratio with 25 nM PMA (Sigma Aldrich) to stimulate ROS release. Plates were incubated at 37°C with 5% CO₂ for 30 min in the dark. Fluorescence intensity (485nm excitation, 530nm emission) was measured in a SpectraMAX Gemini EM fluorescence plate reader (Molecular Devices, Sunnyvale, CA).