Oxidative Burst Assay

The oxidative burst of neutrophils was determined according to the manufacturer’s instructions. Briefly, 100 μl of heparinized whole blood was pretreated by incubating in an ice bath for 10 min. LPS (Sigma Aldrich, St. Louis, MO, USA) at a final concentration of 100 ng/ml or the same volume of PBS was added. The burst assay samples were incubated for 10 min at 37.0°C in a water bath. Dihydrorhodamine (DHR) 123 (AAT Bioquest, Sunnyvale, CA, USA) was added and the sample was mixed for another 10 min. After staining, erythrocytes were lysed, and the remaining cells were fixed and then collected for flow cytometry analysis.