Calcium aceto methyl ester

For neutrophil isolation, the layer containing granulocytes was separated and placed in Hanks’s balanced salt solution with 2 mmol/L ethylenediaminetetraacetic acid and 2% dextran for 20 min. After the removal of red blood cells by hypotonic lysis, neutrophils were isolated using a neutrophil isolation kit (Miltenyi Biotec). For neutrophil stimulation, the cells were seeded (1 × 10⁶) and treated with 1 µM Dex or 1 μg/mL MlEVs in the presence of 10 μg/mL LPS for 24 h. For transfection, 50 nM synthetic miRNA mimics were transfected into neutrophils treated with 10 ng/mL IL-1β (R&D Systems). The levels of MPO in the supernatants were evaluated using an ELISA kit (R&D Systems). For the migration assay, the cells were stained with 2 μmol of calcium aceto-methyl ester (Life Technologies, Eugene, OR, USA) for 30 min at 37 °C and washed with 1× HBSS. Then, the cells (1 × 10⁵) were seeded in the upper chamber with a pore size of 3.0 μm (Neuro Probe, Gaithersburg, MD, USA). Phenol red-free RPMI medium containing ILCs (1 × 10³) was added to the lower chamber, and the Transwell plate was incubated for 2 hours at 37 °C. The signal was measured at excitation and emission wavelengths of 480 and 520 nm, respectively, with a fluorescence microplate reader (BioTek).

Isolated PBNs were stained with 2 μmol of calcium aceto-methyl ester (Life Technologies, Eugene, OR, USA) for the migration assay and 2′,7′-dichlorofluorescein diacetate (H2DCFDA) (Life Technologies) for the ROS assay for 30 min at 37 °C, after which the cells were washed once with 1× HBSS. For the migration assay, the cells were pretreated with anti-S100A9 for 30 min and then seeded on the upper chamber with a pore size of 3.0 μm (Neuro Probe, Gaithersburg, MD, USA). Phenol red-free RPMI medium containing S100A9 was added to the lower chamber, and the Transwell plate was incubated for 2 h at 37 °C. For the ROS assay, the cells were pretreated with anti-S100A9 for 30 min in phenol red-free RPMI and then treated with S100A9 for 30 min. The signal was measured at excitation and emission wavelengths of 480 and 520 nm, respectively, with a fluorescence microplate reader (Synergy HT; BioTek Instrument).