Anti cd3 bv421

Animals were sacrificed at the end of the experiment and their blood was collected. Red blood cells were lysed with lysis buffer (Sigma Aldrich, R7757) and white blood cells (WBCs) were stained with anti-CD11b-PE (BioLegend, cat#: 101207, clone: M1/70), anti-Gr1-biotin (Invitrogen, cat#: 13-5931-81, clone: RB6-8C5) and streptavidin-PerCP eFluor™710 (Invitrogen, cat#: 46-4317-82), anti-CD19-APC (Invitrogen, cat#: 17-0193-80, clone: eBio1D3) and anti-CD3-BV421 (BioLegend, cat#:100227, clone: 17A2) antibodies to quantify immune cell populations. Flow cytometry was performed using an LSRFortessa (BD Biosciences) and results were analysed using FlowJo software (Tree Star Inc.). The student’s t-test was used for statistical analysis, using Prism (GraphPad software).

PBMCs, purified monocytes, or PBMCs depleted in monocytes from different donors were treated with ORF8 produced in 293T cells for 16 h at 37 °C and 5% CO₂. As control, PBMCs were treated with an identical volume of conditioned media collected from 293T cells transfected with a control plasmid (pc DNA 3.1). Alternatively, PBMCs were treated with a commercially available ORF8 (SARS-CoV-2 ORF8 (aa16-121), His Tag (RP-87666), Thermo Fisher Scientific, Waltham, MA, USA) at different concentrations (from 3.2 pg/10⁶ PBMCs to 250 ng/10⁶ PBMCs). Following treatment, cells were stained with anti-CD3 BV-421 (BioLegend, San Diego, CA, USA, Cat. 300434), anti-CD19 BV-421 (BioLegend, San Diego, CA, USA, Cat. 302233), anti-CD14 PerCP-Cy5.5 (BD, Franklin Lakes, NJ, USA, Cat. 550787), anti-CD56 PE (BD, Franklin Lakes, NJ, USA, Cat. 340363), anti-CD16 FITC (BD, Franklin Lakes, NJ, USA, Cat. 555406) and LIVE/DEAD Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific, Waltham, MA, USA, Cat. L34966) for 25 min at 4 °C to identify the monocytes and NK cells among the PBMC population and to measure their surface level of CD16. After staining, the cells were fixed with 2% PFA and stored at 4 °C until the samples were acquired on a LSRII cytometer (BD Biosciences, Mississauga, ON, Canada). Data analysis was performed using FlowJo v10.5.3 (Tree Star, Ashland, OR, USA).

Peripheral blood mononuclear cells (PBMCs) were isolated via centrifugation on a Ficoll–Paque PLUS gradient (GE Healthcare, Uppsala, Sweden). Neutrophils were further isolated after dextran sedimentation and hypotonic lysis as previously described (20 (link)).
Fresh EDTA anti-coagulated peripheral blood (100 μl) was incubated with antibodies for 30 min in the dark. The following antibodies were used: Anti-CD66b-PE-cy7, anti-TLR2-FITC, anti-TLR4-APC, anti-CXCR1-APC, anti-CXCR2-FITC, anti-C5aR-APC, anti-CD64-PE, anti-CD177-APC, anti-CD62L-FITC, anti-CD11b-PE, anti-CD49d- FITC, anti-C5L2-PE, anti-CD3-BV421 and anti-PD-1-PE were obtained from Biolegend (San Diego, California, USA); anti-CD4-BV421, anti-CD8-APC-cy7, anti-CD38-FITC, and anti-HLA-DR-PerCP were purchased from BD Biosciences (Franklin Lakes, New Jersey, USA); anti-PD-L1-PE were bought from eBioscience (San Diego, California, USA). Isotype-matching antibodies were used as negative controls. After lysing the red blood cells, the remaining cells were washed and fixed for flow cytometry analysis.