Cd11bhi

To obtain human neutrophils, 10 mL of blood was drawn from healthy donors into heparinized Vacutainer tubes and mixed 1:1 with sterile 1X phosphate buffered saline (PBS) (approval UTK IRB-18-04604-XP) (Akhtar, Chaudhary, & He, 2010 (link)). After mixing, 10 mL of lymphocyte separation medium (Corning, Cat. 25-072-CV) was underlaid. Following centrifugation at 1400 rpm for 30 minutes, the top layers were aspirated off, leaving the red blood cell and neutrophil pellet. Pellets were resuspended in 20 mL Hanks’ Balanced Salt Solution 1x (Gibco, Cat. 14025076) and 20 mL 3% dextran in 0.9% NaCl. After incubating at room temperature for 20 minutes, the upper layer was transferred to a new tube. Following centrifugation at 400 × g for 5 minutes and aspiration of the supernatant, the pellet was washed with 20 mL ice-cold 0.2% NaCl and 20 mL ice-cold 1.6% NaCl two times. Following the final aspiration, the neutrophil pellet was resuspended in 10 mL RPMI 1640 1x (Corning, Cat. 10-040-CV). Neutrophil viability and counts were performed through Trypan blue stain (Lonza, Cat. 17–942E). Neutrophil purity was determined to be approximately 95% by flow cytometry analysis of CD11b^hi (Biolegend, Cat. 101212) and CD66b⁺ (Biolegend, Cat. 305104).