Anti cd14 bv510 clone m5e2

Surface antibody stainings were performed in PBS supplemented with 2% heat inactivated FBS for 20 minutes at room temperature. Glut1.- receptor dinding protein (RBD)-eGFP labeling was done in RPMI1640 + 10%FBS + 5% NaN₃ +0.5mM EDTA for 30 minutes at 37°C. The eBioscience Foxp3/Transcription Factor Staining Buffer Set (product protocol B) was used for intracellular staining. The monoclonal mouse anti-human antibodies anti-CD16-BUV395 (clone 3G8, BD Horizon^TM), anti-CD56-BUV737 (clone NCAM16.2, BD Horizon^TM), anti-CD71-BV711 (clone M-A712, BD Horizon^TM), anti-CD57-BV605 (clone NK-1, BD Horizon^TM), anti-CD186 (CXCR6)-PE/Cy7 (clone K041E5, Biolegend), anti-CD127-PE-CF594 (clone HIL-7R-M21, BD Horizon^TM), anti-CD45-BV785 (clone HI30, Biolegend), anti-CD3-AlexaFluor700 (clone UCHT1, Biolegend), anti-CD14-BV510 (clone M5E2, Biolegend), anti-CD19-BV510 (clone HIB19, Biolegend), as well as recombinant human anti-CD98-APC-Vio770 (clone REA387, Milteny Biotec GmbH), Zombie Aqua™(Biolegend) and anti-human GLUT1 (SLC2A1)-eGFP RBD were used for staining. NK cells were defined as CD3 ^neg, CD14 ^neg, CD19 ^neg, CD45+, and were divided into CD56^bright CD16 ^neg NK cells (referred to as CD56^bright NK cells) and CD56^dim CD16+ NK cells (referred to as CD56^dim NK cells).

Cryopreserved samples containing 1-10 × 10⁷ PBMC from DRB1*07:01 (DR7)⁺ convalescent subjects or pre-pandemic negative controls were thawed, washed twice with PBS containing 2% FBS, and resuspended in either complete EHAA or RPMI 1640 with 5% FBS. Staining with a pre-mixed cocktail of DR7:p-streptavidin-PE, DR7:p-streptavidin-PE-Cy7, and DR7:p-streptavidin-APC tetramers at a final concentration of 10 nM for each reagent was for 1 hour at room temperature. Anti-PE and anti-APC magnetic beads (Miltenyi Biotec) were then added, and bead-bound cells were enriched as previously described (22). Cells in the bound and unbound fractions were stained with Zombie Aqua viability dye (BioLegend) and a surface marker antibody panel consisting of anti-CD20^BV510 (clone 2H7, BioLegend), anti-CD14^BV510 (clone M5E2, BioLegend), anti-CD3^AF700 (clone UCHT1, BioLegend), anti-CD4^BV605 (clone OKT4, BioLegend), anti-CD8^BUV395 (clone RPA-T8, BioLegend), anti-CD45RO^APC/Cy7 (clone UCHL1, BioLegend), anti-CXCR3^PE/Dazzle594 (clone G025H7, BioLegend), anti-CXCR5^BV421 (clone J252D4, BioLegend), anti-PD-1^BV785 (clone EH12.2H7, BioLegend), and anti-CCR4^PerCP/Cy5.5 (clone L291H4, BioLegend) for 30 min at room temperature. Data were acquired on an LSR Fortessa X-20 (BD) flow cytometer and analyzed with FlowJo software (BD).