Immunophenotyping of Neutrophils and Monocytes

The expression of TLR4, NOX2, and CD11b on the surface of neutrophils and monocytes was evaluated by flow cytometry. Whole blood (200 μL) was treated in 100-μL aliquots incubated at 37°C for 1 h untreated (vehicle) or with 10 ng/mL LPS (SIGMA Life Science, Ireland). Fluorochrome-conjugated monoclonal antibodies (mAb) specific for humans CD14-PerCP, CD15-PECy7, NOX2-FITC, CD66b-Pacific Blue, TLR4-APC (BioLegend®, USA), and CD11b-PE (BD Biosciences, UK) were used. The whole blood was then stained with mAb for 15 min. Red blood cells were lysed with BD lysis buffer. Cells were acquired on a FACSCanto II flow cytometer (BD Bioscience) and analyzed using FlowJo version 10 (Tree Star) (2 (link), 14 (link)). Neutrophils were delineated based on SSC-A and CD66b⁺ and monocytes based on SSC-A, CD66b⁻, and CD14⁺ (15 (link), 16 (link)). Monocytes were subdivided into classical, intermediate, and non-classical subtypes. Monocyte subsets were identified as classical: CD14^highCD16^neg/low; intermediate: CD14^highCD16^high; non-classical: CD14^lowCD16^high. A minimum of 10,000 events were collected, and relative expression of antigens was expressed as mean fluorescence intensity (MFI).