Cd11b a488 cr3

Cells were immunolabeled with TREM-1-APC (R&D Systems), CD11b-A488 (CR3) (Biolegend) and Ly6G-BV421 (BioLegend) (panel1) or CD11b-A488 (CR3) (Biolegend), CD120a-APC (TNFRI) (BioLegend), CD120b-PE (TNFRII) (BioLegend), CD16/32-APC-Cy7 (FcγRII/III) (BioLegend) and ICAM-1-PE-Cy7 (Biolegend) (panel 2). Neutrophils (0.1 million) were resuspended in 100 μL HBSS^+/+ containing 5.56mM glucose in 96-well round-bottomed plates and primed for 30 min at 37 °C with 2 μg/mL TNF-α. Unprimed control cells received buffer alone. After washing, resting or TNF-α primed neutrophils were washed with PBS^−/− and stained for 10 min with the cell viability marker, Zombie Aqua (BioLegend). After Zombie Aqua staining, cells were blocked for 10 min with PBS^−/− containing 2% FCS and 2% serum of the respective antibody host species. Cells were subsequently stained for 15 min in the continued presence of blocking buffer. After immunolabeling, all cells were washed twice, resuspended in MACS buffer (PBS^−/− + 2% FBS + 1 mM EDTA) and analyzed on Cytek Aurora spectral flow cytometer. Data were analyzed with FlowJo v10 software. All incubations were performed at room temperature, protected from light.