Cd14 percp

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).

Cells from healthy donors were pelleted by centrifugation after 24 or 72 h exposure to SARS-CoV-2, as described above, before staining for cytoflorometry analysis. PBMC isolated from COVID-19 patients and healthy donors were stained immediately after isolation. Cells were incubated with FcR Blocking Reagent according to manufacturer’s protocol (Miltenyi Biotec, 130-059-901). Cells from healthy donors, used for the exposure to virus in vitro, were fixed using Cytofix/Cytoperm kit (BD Biosciences, 51-2090KZ) according to manufacturer’s instructions, before staining. Staining was performed with CD3-PE (BD Biosciences, 552127), CD14-PerCP (BioLegend, 301848), CD19-APC-H7 (BD Biosciences, 560252) and 10 μg/mL GN_mAb_Env01-biotin (GeNeuro, murine antibody) in Perm/Wash buffer (BD Biosciences, 512091KZ). HERV-W ENV expression was revealed using Streptavidin FITC conjugate (eBioscience, 11-4317-87). Background noise was assessed using mouse isotype control (mIgG1-FITC, Miltenyi Biotec 130-113-199) and Streptavidin FITC conjugate (eBioscience, 11-4317-87). Stained cells were acquired on a BD LSR Fortessa (BD Biosciences). Fluorochrome emissions from the pool of antibodies were compensated using Ultra-Comp eBeads plus Compensation beads (Invitrogen, 01-3333-42). Data were analyzed with FlowJo software (v.10).