Cd10 pecf594

Panel 1 included antibodies allowing the gating of PMN-MDSCs in the PBMCs and the analysis of expression of surface markers. It comprised of CD3-BV510, CD56-BV510, CD19-BV510, CD11b-APCH7, CD15-BV711, CD14-BV650, HLA-DR-BV605, CD10-PE-CF594 (Becton Dickinson), CD16-AF700 (BioLegend, Ozyme, Saint-Cyr-l’Ecole, France), CD33-PC7 (eBiosciences, Thermo Fisher), LIVE/DEAD fixable aqua (Life Technologies, Villebon-sur-Yvette, France). LOX-1-PE, CD11b-activated-PE (clone CBRM1/5) (Bio-Legend, Ozyme), CD62L-PE, CD66b-PE, PDL-1-BV421, CXCR1-PE, CD172ab-AF647 (BD Biosciences) were alternately included in Panel 1.
Panel 2 was used for functional analysis. It contained CD15-FITC (eBiosciences, Thermo Fisher), CD3-PC5 (BD Biosciences) and LIVE/DEAD near-IR (Live Technologies,). CD66b-PE, CD10-PE-CF594 and CD16-AF700 were occasionally added. PBMCs were stained 20 min at room temperature with fluorescent reagents pre-mixed in PBS, washed, then fixed with 4% paraformaldehyde and analyzed by FACS within 4 days on LSRII-SORP cytometer (BD Biosciences) equipped with four lasers (405 nm/100 mW, 488 nm/100 mW, 560 nm/50 mW and 630 nm/40 mW). PMT were set using unstained and fully stained samples. Compensations were performed with beads stained with corresponding reagents. Data were exported and analyzed with FlowJo (version 9-2, MacOS X).

Two million of PBMCs were incubated with BD human FC block (BD Biosciences) and then stained with recombinant biotinylated spike S1 + S2 ECD-His (Sino Biological) conjugated with SA-R-Phycoerythrin (PE) and recombinant biotinylated-receptor binding domain  (RBD; BioLegend) conjugated with SA-Allophycocyanin (APC), together with the following fluorescent antibodies (clone; dilution used): CD3-BV650 (clone OKT3; 1:200); CD21-FITC (clone B-LY4; 1:50), CD19-BUV395 (clone SJ25C1; 1:100), CD10-PECF594 (clone HI10A; 1:200), IgM-BV605 (clone G20-127; 1:50), IgD-BV711 (clone IA6-2; 1:100), CD27-BV786 (clone O323; 1:50), CD11c-BB700 (clone 3.9; 1:50), CD20-APCH7 (clone 2H7; 1:100), CD38-BUV737 (clone HB7; 1:400), IgG-PE-Cy7 (clone G18-145; 1:100; all from Becton Dickinson), IgA-Vio blue (clone IS11-8E10; 1:100; Miltenyi Biotec). Following surface staining, cells were washed once with PBS and labeled with Zombie Aqua Fixable Viability Kit (Thermofisher) according to the manufacturer instruction. Cells were fixed in BD fixation solution (BD Biosciences) and acquired with SO LSRFortessa X20 flow cytometer (BD Biosciences). Data analysis was performed using FlowJo v10 (TreeStar, USA).

PBMCs were isolated from whole blood collected from family members and healthy donors by ficoll-histopaque gradient centrifugation. For phenotypic staining, the following monoclonal antibodies were used: CD3-APC-H7, CD4-PerCP-Cy5.5, CD38-PerCP-Cy5.5, CD10-PECF594, CD21-APC, IgG PeCy7, CD14-PerCP, CD123-PE, CD56-PeCy7, CD11c-APC, CD16-APC-H7 (BD Biosciences, San Diego, CA, USA), CD8-APC-EF780, CD27-APC-EF780 (eBioscience, San Diego, CA, USA), CD19-BV650, CD24-BV605 (Biolegend, San Diego, CA, USA) and IgA-PE (Miltenyi Biotech, Bergisch Gladbach, Germany). Naïve B cells were enriched by negative selection using B-cell isolation kit (Stemcell, Vancouver, BC, Canada). Naïve B-cell purity was verified by flow cytometry to 98% purity.