Luminol-Based ROS Quantification Assay

A quantitative assay was performed to measure the generation of ROS in different conditions. ROS generated in response to glucose treatment at different time points was measured using Luminol/HRP chemiluminescence assay for ROS detection. Briefly, PMNs were seeded in 96-well black plate (Corning 3,991 polystyrene flat bottom) in triplicates and 50 μM Luminol (Sigma- Aldrich A4685) and 1.2 U/ml horseradish peroxidase (Sigma- Aldrich P8250) were added to each well. As a positive control, PMNs were stimulated with 600 nM PMA (Sigma P8139). Plates were gently tapped to ensure mixing of all reagents. Luminescence was immediately measured by taking multiple readouts for up to 5 min in a luminometer (Perkin Elmer).