Opaque 96 well microplate

ROS production by HL-60 cells or primary neutrophils was measured using chemiluminescence. HL-60 cells were differentiated into neutrophil-like cells by incubation with RPMI 1640 medium (Thermo Fisher Scientific) containing 1.3% DMSO (Sigma), 15% fetal bovine serum (FBS, Thermo Fisher Scientific) and 25 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Thermo Fisher Scientific) for 5–6 days. Differentiated HL-60 cells or primary neutrophils were washed and resuspended in phenol red free RPMI 1640 containing 2% FBS and luminol (Sigma) was added (1 mM). Approximately 5 × 10⁴ HL-60 cells in 90 μL were seeded into an opaque 96-well microplate (Perkin-Elmer) and the basal luminescence was measured every 2 min for 5 min on a FilterMax F3 plate reader (Molecular Devices). HL-60 cells were infected with 10 μL of bacteria (MOI = 10) opsonized with 20% human serum (30 min at room temperature) or were mock treated, and ROS production was measured every 2 min using a plate reader. In some experiments, human serum was treated with Futhan, 10 mM EGTA, or 10 mM Mg²⁺ plus 10 mM EGTA (MgEGTA) before opsonizing bacteria.