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.
Cd19 bv650
Manufactured by BioLegend
Sourced in United States
CD19-BV650 is a fluorescently-conjugated monoclonal antibody that binds to the CD19 cell surface antigen. CD19 is a glycoprotein expressed on the surface of B cells and is commonly used as a marker to identify and quantify B cells in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Iga pe, by Miltenyi Biotec (2 mentions)
Cd3 percp cy5, by BioLegend (2 mentions)
Hla dr fitc, by BioLegend (2 mentions)
Cd3 bv510, by BioLegend (2 mentions)
Super bright complete staining buffer, by Thermo Fisher Scientific (2 mentions)
Cd14 bv421, by BioLegend (2 mentions)
Cd64 bv785, by BioLegend (2 mentions)
Siglec 1 pe, by BioLegend (2 mentions)
Cd66b percp cy5, by BioLegend (2 mentions)
Northern lights 3000, by Cytek (2 mentions)
Cd38 apc, by BioLegend (2 mentions)
Cd16 apc cy7, by BioLegend (2 mentions)
Trustain fcx, by BioLegend (2 mentions)
Cd86 pe cy7, by BioLegend (2 mentions)
Cd20 bv711, by BD (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Cd16 bv480, by BD (2 mentions)
Symphony cytometer, by BD (2 mentions)
Aquavivid viability marker, by Thermo Fisher Scientific (2 mentions)
11 protocols using cd19 bv650
1
Isolation and Phenotypic Analysis of Naïve B Cells
2
Comprehensive Immune Cell Phenotyping
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Directly conjugated antibodies were purchased from BD Biosciences (San Jose, CA, USA): CCR7-PECy7 (3D12), CD3-PerCPCy5.5 and CD3-V500 (UCHT1), Foxp3-AF488 (259D/C7), CD25-AF700 (M-A251), CD4-APCH7 (RPA-T4), CXCR5-AF647 (RF8.B2), CD45RA-PECF594 (HI100), CD19-APCCy7 (SJ25C1), IgM-FITC (G20-127), IgD-PECF594 (IA6-2), CD38-V450 (HB7), CD138-PerCPCy5.5 (MI15), CD21-APC (B-LY4), CD27-PE (M-T271), IgG-BV605 (G18-145) or BioLegend (San Diego, CA, USA): PD1-PE (EH12.2H7), CD19-BV650 (HIB19), CD57-PB (HCD57), ICOS-FITC and ICOS-PerCPCy5.5 (C398.4A), OX40-PECy7 (BER-ACT35), CD40L-BV605 (24–31).
3
Multiparametric Analysis of IFNβ-Stimulated Leukocytes
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One milliliter of blood collected in sodium heparin tubes was stimulated with 10 ng IFNβ or unstimulated for 24 h. Proteomic Stabilizer PROT1 (Smart Tube, Inc.) was added and incubated for 10 min at room temperature prior to freezing at −80°C. Samples were thawed, and single‐cell suspensions of blood leukocytes were obtained using Thaw‐Lyse buffer (Smart Tube, Inc) according to manufacturer's instructions. Following incubation with human Fc receptor blocker, TruStain FcX (Biolegend Inc.), and Super Bright Complete Staining Buffer (Thermo Fisher), cells were stained with fluorescently conjugated antibodies: CD14 BV421, CD64 BV785, HLA‐DR FITC, Siglec‐1 PE, CD38 APC, CD19 BV650, CD66b PerCP/Cy5.5, CD16 APC/Cy7, CD86 PE/Cy7, and CD3 BV510 from Biolegend, acquired with a Cytek Northern Lights 3000 (Cytek Biosciences) and analyzed using FlowJo.
4
Detection of SARS-CoV-2-specific B Cells
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To detect SARS-CoV-2-specific B cells, we conjugated recombinant RBD proteins with Alexa Fluor 488 or Alexa Fluor 594 (Thermo Fisher Scientific) according to the manufacturer’s protocol. Approximately 10 × 106 frozen PBMC from 13 convalescent donors were prepared in Falcon® 5ml-round bottom polystyrene tubes at a final concentration of 14 × 106 cells/mL in RPMI 1640 medium (GIBCO) supplemented with 10% of fetal bovine serum (Seradigm), Penicillin- Streptomycin (GIBCO) and HEPES (GIBCO). After a rest of 2h at 37°C and 5% CO2, cells were stained using Aquavivid viability marker (GIBCO) in DPBS (GIBCO) at 4°C for 20min. The detection of SARS-CoV-2-antigen specific B cells was done by adding the RBD probes to the following antibody cocktail: IgM BUV737, CD24 BUV805, IgG BV421, CD3 BV480, CD56 BV480, CD14 BV480, CD16 BV480, CD20 BV711, CD21 BV786, HLA DR BB700, CD27 APC R700 all from BD Biosciences; CD19 BV650 from Biolegend and IgA PE from Miltenyi. Staining was performed at 4°C for 30min and cells were fixed using 2% paraformaldehyde at 4°C for 15min. Stained PBMC samples were acquired on Symphony cytometer (BD Biosciences) and analyzed using FlowJo v10.7.1 (TreeStar). In each experiment, PBMC from unexposed donors (total of n = 9) were included to ensure consistent specificity of the assay.
5
Multiparameter Flow Cytometry Panel
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PD-1 FITC (1:300; J43; eBioscience), NK1.1 PE (1:300; PK136; eBioscience), CD3e PE-Dazzle594 (1:300; 145-2C11; BioLegend), CD3e PE (1:200; 145-2C11; eBioscience), FoxP3 PerCP-Cy5.5 (1:200; FJK-16s; eBioscience), CD8a PE-Cy7 (1:2000; 53-6.7; eBioscience), CD8a APC (1:600; 53-6.7; eBioscience), CD8a PerCP-Cy5.5 (1:400; 53-6.7; eBioscience), TCRb AF700 (1:100; H57-597; BioLegend), CD45 BV785 (1:1000; 30-F11; BioLegend), CD4 BV711 (1:400; GK1.5; BioLegend), CD19 BV650 (1:400; 6D5; BioLegend), CD19 PE (1:500; 1D3; eBioscience), CD11b BV605 (1:1000; M1/70; BioLegend), CD11b BV510 (1:400; M1/70; BioLegend), CD11c BV605 (1:400; N418; BioLegend), Siglec-F PE (1:300; E50-2440; BD Biosciences), F4/80 APC (1:200; BM8; BioLegend), Ly-6G AF 700 (1:300; 1A8; BioLegend), MHC II BV650 (1:4000; M5/114.15.2; BioLegend), CD64 BV421 (1:200; X54-5/7.1; BioLegend), TNF FITC (1:300; MP6-XT22; BioLegend), IFNg PE-Cy7 (1: 1000; XMG1.2; BD Biosciences), Eomes PE (1:200; W17001A; BioLegend), Ki-67 BV 650 (1:200, 11F6; Biolegend), GzmB FITC (1:100; GB11; BioLegend). Gating strategy is depicted in Supplementary Fig. 6d .
6
Detecting SARS-CoV-2-Specific B Cells in PBMCs
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To detect SARS-CoV-2-specific B cells, we conjugated recombinant RBD proteins with Alexa Fluor 488 or Alexa Fluor 594 (Thermo Fisher Scientific) according to the manufacturer’s protocol. Approximately 107 frozen PBMCs from 12 convalescent donors were prepared at a final concentration of 14 × 106 cells/mL in RPMI 1640 medium (GIBCO) supplemented with 10% of fetal bovine serum (VWR, Radnor, PA, USA), Penicillin-Streptomycin (GIBCO) and HEPES (GIBCO). After a rest of 2 h at 37 °C and 5% CO2, cells were stained using Aquavivid viability marker (GIBCO) in DPBS (GIBCO) at 4 °C for 20 min. The detection of SARS-CoV-2-antigen specific B cells was accomplished by adding the RBD probes to the following antibody cocktail: IgM BUV737, IgG BV421, CD3 BV480, CD56 BV480, CD14 BV480, CD16 BV480, and CD20 BV711, all from BD Biosciences, Franklin Lakes, NJ, USA and CD19 BV650 from Biolegend, San Diego, CA, USA. Staining was performed at 4 °C for 30 min and cells were fixed using 2% paraformaldehyde at 4 °C for 15 min. Stained PBMC samples were acquired with a Symphony cytometer (BD Biosciences) and analyzed using FlowJo v10.7.1 (TreeStar, Woodburn, OR, USA). The gating strategy is shown in Figure S2 .
7
Nasal B and MAIT Cell Immunophenotyping
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Immunophenotyping of nasal B and MAIT cells obtained by curettes was performed as described previously (29 (link)). In brief, cells were dislodged from curettes and stained with LIVE/DEAD Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific) and an antibody cocktail containing, among others, Epcam-PE, HLADR-PE/Cy7, CD66b-FITC, CD19-BV650 (all BioLegend), CD3-APC/Cy7, CD14-Percp/Cy5.5 (BD Biosciences), and CD45-PACOrange (Thermo Fisher Scientific) for B cells, while the cocktail for MAIT cells also included CD8–BV785 and TCRva7.2-BV711 or TCRva7.2–PE/Texas Red and CD45-BV510 (BioLegend). Samples were acquired on a BD LSR II flow cytometer and analyzed using FlowJo X. Fluorescence-minus-one controls for each of the included antibodies were used to validate results during setup of all of the panels used. Samples with less than 500 immune cells or 250 epithelial cells (11.9% of all nasal samples) were excluded from further analysis. A full list of all antibodies used for flow cytometry is provided in Supplemental Table 2 ).
8
Multiparametric Immune Cell Analysis
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Cell suspensions obtained from skin samples were fluorescently labelled with Fixable Viability Dye eFluor® 780 (eBioscience). Samples were then stained with antibodies against the following cell surface markers: CD45-APC, CD45-AF700, CD3-PerCP/Cy5.5, CD4-FITC, CD8-PE/Cy7, GR1-FITC, GR1-PE/Cy7, F4/80-PE, F4/80-BV786, CD11b-Percp/Cy5.5 (Biolegend), and CD19-BV650 (Biolegend). Data were acquired with a LSRFORTESSA X-20 (BD) or Accuri (BD) with subsequent analysis using the FlowJo software.
9
Immunophenotyping of Tumor-Infiltrating Cells
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Cells were dislodged from the curette by repeated pipetting with PBS+ as described previously (19 (link), 27 (link)). Cells were spun down at 440 × g for 5 min and resuspended in PBS++ containing LIVE/DEAD fixable aqua dead cell stain (Thermo Fisher). After 15 min incubation on ice, an antibody cocktail, which included EpCam-PE, HLADR-PECy7, CD66b-FITC, CD19-BV650 (all BioLegend), CD3-APCCy7, CD14-PercpCy5.5 (BD Biosciences), and CD45-PACOrange (Thermo Fisher), was added to the cells. Following a further 15-min incubation on ice, cells were filtered over a 70-μm filter (Thermo Fisher). Cells were spun down (440 × g for 5 min), resuspended in PBS containing 0.5% heat-inactivated fetal bovine serum and 5 mM EDTA (Invitrogen), and acquired on a flow cytometer (LSR II; BD). All cells per tube were acquired, and samples with less than 500 immune cells or 250 epithelial cells were excluded from further analysis (9/40 samples; 22.5%). The numbers of acquired cells per population were used as counts. Flow cytometry data were analyzed using FlowJo version 10 (Tree Star Inc., San Carlos, CA, USA).
10
Flow cytometry analysis of PBMCs
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Peripheral blood mononuclear cells (PBMCs) isolated from buffy coats (Red Cross) using standard gradient density centrifugation were spinoculated with vectors at 2671 g for 2 hours, RT, MOI5. After 20 hours, PBMCs were harvested using 0.05% Trypsin-EDTA, seeded in 96-well plates, washed using FACS buffer (PBS (Phosphate Buffered Saline), 2.5% FBS, 10 mM EDTA) with 0.01% sodium azide, stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFisher) and incubated for 1 hour in CD56-BV650, CD19-BV650, HLA-DR-BV421, CD14-AF700, CD11c-PE-Dazzle594, CD303-PerCP-Cy5.5, CD1c-APC-Cy7, CD141-PE-Cy7, CD80-BV711 and CD40-BV605 (BioLegend), CD86-BUV737, CD16-BV786 (BD Biosciences). After washing, cells were fixed with Cytofix (BD Biosciences), resuspended in FACS buffer, and analyzed using a BD Fortessa flow cytometer and FlowJo v10.8 Software (BD Life Sciences).
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