Neutrophil Surface Receptor Profiling in Whole Blood

Neutrophil surface receptor expression was assessed in heparin-treated fresh whole blood similar to previously described (18 (link)). Briefly, 100µL of heparin-treated blood was dispensed into 5mL tubes at 4°C in the dark. Cells were stained with anti-CXCR2-PE (ThermoFisher, clone 5E8-C7-F10), anti-CD16-FITC (BD Bioscience, clone 3G8), anti-TLR2-Alexa-647 (BD Bioscience, clone 11G7), and anti-TLR4-APC (ThermoFisher, clone HTA-125) or their relevant concentration-matched isotype control for 30 minutes at 4°C in the dark. Following incubation, cells were washed twice in at 4°C PBS and erythrocytes lysed, and leukocytes fixed using 1% fix/lyse solution (ThermoFisher Scientific). Following fixation, cells were washed twice and resuspended in 300µL PBS/1%BSA for analysis by flow cytometry.