Peripheral blood samples were collected in tubes containing EDTA and processed within an hour after collection. Neutrophils were isolated by density centrifugation using the Histopaque gradient technique (Sigma-Aldrich) according to the manufacturer’s protocol. Contaminating erythrocytes were removed with FACS lysing solution and washed with PBS containing 1% BSA. Viability of neutrophils after purification, assessed by trypan blue exclusion and flow cytometry of propidium iodide-stained cells, was greater than 96%.
For priming of isolated neutrophils, cells (1.5 × 106 cells/mL) were cultured at 37°C in RPMI-1640 (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 100 U/mL of penicillin, 100 µg/mL of streptomycin in the absence or presence of 100 ng/mL LPS (Escherichia coli 0111:B4, Sigma-Aldrich) in a total volume of 500 µL for 4 hours. After stimulation, neutrophils were harvested, washed once with cold PBS and stained with anti-CD54, CD64, CD11b, CD15, TLR2 (clone TL2.1), TLR4 (clone HTA125) (all BioLegend) and analyzed with flow cytometer similarly to the analysis of surface antigens. Supernatants were stored at -80°C until analysis of cytokine production.
For priming of isolated neutrophils, cells (1.5 × 106 cells/mL) were cultured at 37°C in RPMI-1640 (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 100 U/mL of penicillin, 100 µg/mL of streptomycin in the absence or presence of 100 ng/mL LPS (Escherichia coli 0111:B4, Sigma-Aldrich) in a total volume of 500 µL for 4 hours. After stimulation, neutrophils were harvested, washed once with cold PBS and stained with anti-CD54, CD64, CD11b, CD15, TLR2 (clone TL2.1), TLR4 (clone HTA125) (all BioLegend) and analyzed with flow cytometer similarly to the analysis of surface antigens. Supernatants were stored at -80°C until analysis of cytokine production.