Anti tlr4 apc

Neutrophil surface receptor expression was assessed in whole blood. Briefly, 100 μL of heparin treated blood was dispensed into 5 mL tubes and stored 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-CD11b-APC (BD Bioscience, clone ICRF44), anti-CD18-PE (BD Bioscience, clone 6.7), anti-TLR2-Alexa-647 (BD Bioscience, clone 11G7) or anti-TLR4-APC (ThermoFisher, clone HTA-125) or their relevant concentration-matched isotype control for 30 min on ice in the dark. Following incubation, cells were washed twice in cold PBS and erythrocytes lysed and leukocytes fixed using 1% fix/lyse solution (ThermoFisher Scientific). Following fixation, cells were washed twice and resuspended in 300 μL PBS/1%BSA for analysis by flow cytometry. All flow cytometry analyses were conducted on a BD FACSCanto II (BD Bioscience, United States) flow cytometer equipped with 3-lasers using the Duke Cancer Institute Core Facility, which maintained daily quality controls of the machine. Analyses were completed on 10,000 neutrophils and data analyzed using FCS Express 6 (FCS Express, United States).

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.