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.
Cd56 bv480
Manufactured by BD
Sourced in United States
The CD56 BV480 is a fluorochrome-conjugated monoclonal antibody that binds to the CD56 antigen expressed on natural killer cells, a subset of T cells, and some neural cell types. It is designed for use in flow cytometry applications to identify and enumerate CD56-positive cells within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Aquavivid viability marker, by Thermo Fisher Scientific (2 mentions)
Symphony cytometer, by BD (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Cd20 bv711, by BD (2 mentions)
Igm buv737, by BD (2 mentions)
Igg bv421, by BD (2 mentions)
Cd3 bv480, by BD (2 mentions)
Cd14 bv480, by BD (2 mentions)
Cd16 bv480, by BD (2 mentions)
Cd19 bv650, by BioLegend (2 mentions)
Cd27 apc r700, by BD (1 mentions)
Iga pe, by Miltenyi Biotec (1 mentions)
Fetal bovine serum (fbs), by Avantor (1 mentions)
Cd8 fitc, by BD (1 mentions)
Cd3 apc h7, by BD (1 mentions)
3 protocols using cd56 bv480
1
Detection of SARS-CoV-2-specific B Cells
2
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 .
3
Multiparameter Flow Cytometry Analysis of Immune Cell Subsets
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For analysis of surface markers, fresh PBMCs were incubated for 30 min at room temperature in Stain Buffer (BD Biosciences) with optimal concentrations of the fluorochrome conjugated antibodies. Lymphocyte subsets were determined using the monoclonal antibodies as follows: CD3 APCH7 (Clone-SK-7), CD4 BV480 (Clone -SK-3), CD8 FITC (Clone-RPA-T8), CD62L APC (Clone-Dreg 56), CCR7 PE (Clone-2-L1-A), CD45RA BV421 (Clone-5H9), CD56 BV480 (Clone-NCAM 16.2), and CD16 FITC (Clone-3G8) (BD Biosciences). The cells were further washed with Stain Buffer, fixed with 1% formaldehyde in PBS and acquired to obtain 100,000 gated lymphocyte events on FACS Jazz (BD Biosciences). Gating strategy for identification of different immune cells and subsets was depicted in Supporting Information Material 1. The data was analyzed using FACS Flow Jo software V10.7 (BD Biosciences).
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