Isolation of Neutrophils from Peripheral Blood

Peripheral blood was obtained from healthy donors using lithium-heparin vacutainers (Greiner Bio-One, Chonburi, Thailand). Neutrophils were isolated as previously described (31 (link)). In brief, whole blood was mixed with HetaSep solution (STEMCELL-Technologies, Vancouver, Canada) at a ratio of 5:1 and incubated at 37°C for 30 min. Then, the upper layer containing all nucleated blood cells was taken and gently layered on top of Ficoll-Paque solution (GE Healthcare, Little Chalfont, UK) at a ratio of 1:1, followed by centrifugation at 500 g for 30 min. After peripheral blood mononuclear cells (PBMC), plasma and Ficoll-Paque solution were carefully discarded, the neutrophil pellet was resuspended in 1 mL RPMI 1640 complete medium (RPMI 1640 (Gibco, Life Technologies, Paisley, UK) with 10% fetal bovine serum (FBS) (Gibco, Life Technologies, Paisley, UK)). Hypotonic ammonium chloride lysis buffer was applied to eliminate residual erythrocytes for 4 min, followed by centrifugation at 400 g for 3 min. the neutrophil pellet was then resuspended in RPMI 1640 complete medium and checked for neutrophil purity (> 95%) and viability (> 95% required for further investigations).