Sera were added to enzyme-linked immunosorbent assay (ELISA) plates coated with RBD (300 ng/well; BEI NR-52309) (37°C 2 hours). CD16a.NK-92 cells (PTA-6967, American Type Culture Collection) with brefeldin A (Biolegend), Golgi Stop (BD Biosciences), and anti-CD107a (clone H4A3, BD Biosciences) were then added (37°C, 5 hours). Cells were stained with anti-CD56 (clone 5.1H11, BD Biosciences) and anti-CD16 (clone 3G8, BD Biosciences) and fixed (4% paraformaldehyde [PFA]). Intracellular cytokine staining to detect interferon-γ (IFN-γ; clone B27, BD Biosciences) and tumor necrosis factor-α (TNF-α; clone Mab11, BD Biosciences) was performed in permeabilization buffer (Biolegend). Markers were measured by flow cytometry as detailed above [8 (link)].
Anti cd56 clone 5.1h11
Manufactured by BD
Anti-CD56 (clone 5.1H11) is a laboratory reagent used for the detection and identification of CD56-positive cells by flow cytometry or other immunoassay techniques. It recognizes the CD56 cell surface antigen, also known as neural cell adhesion molecule (NCAM).
Automatically generated - may contain errors
Lab products found in correlation
Permeabilization buffer, by BioLegend (4 mentions)
Anti cd107a clone h4a3, by BD (4 mentions)
Anti cd16 clone 3g8, by BD (4 mentions)
Brefeldin a, by BioLegend (4 mentions)
Golgistop, by BD (4 mentions)
Clone b27, by BD (3 mentions)
Lsrfortessa, by BD (3 mentions)
Clone mab11, by BD (2 mentions)
Pta 6967, by American Type Culture Collection (2 mentions)
Ifn γ clone b27, by BD (1 mentions)
Nr 52309, by BEI Resources (1 mentions)
4 protocols using anti cd56 clone 5.1h11
1
NK Cell Activation Assay with RBD ELISA
2
NK Cell-Mediated ADNKA Assay for Antigen-Specific Responses
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ADNKA assay was performed as described with modifications (Gunn et al., 2020 (link)). ELISA plates were coated with recombinant RBD antigen (300 ng/well) (Bates et al., 2021c ) (BEI Resources NR-52309). Wells were washed, blocked, and incubated with serial dilutions of sera (1:10, 1:30, 1:90) in duplicate for 2hrs at 37°C prior to adding CD16a.NK-92 cells (PTA-6967, ATCC) (5 × 104 cells/well) for 5hrs with brefeldin A (Biolegend), Golgi Stop (BD Biosciences) and anti-CD107a (clone H4A3, BD Biosciences). Cells were stained with anti-CD56 (clone 5.1H11, BD Biosciences) and anti-CD16 (clone 3G8, BD Biosciences) and fixed with 4% PFA. Intracellular cytokine staining to detect IFNγ (clone B27, BD Biosciences) and TNFα (clone Mab11, BD Biosciences) was performed in permeabilization buffer (Biolegend). Markers were measured using a BD LSRFortessa and analyzed by FlowJo10. CD16 expression was confirmed in all cells. NK cell degranulation and activation were calculated as percent of CD56+NK cells positive for CD107a, or IFNγ or TNFα expression. Representative data from one dilution was chosen by the highest signal to noise ratio for further analyses.
3
ADNKA Assay for SARS-CoV-2 RBD Antibodies
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ADNKA assay was performed as described with modifications (Gunn et al., 2020 (link)). ELISA plates were coated with recombinant RBD antigen (300 ng/well) (Bates et al., 2021c (link)) (BEI Resources NR-52309). Wells were washed, blocked, and incubated with serial dilutions of sera (1:10, 1:30, 1:90) for 2hrs at 37°C prior to adding CD16a.NK-92 cells (PTA-6967, ATCC) (5 × 104 cells/well) for 5hrs with brefeldin A (Biolegend), Golgi Stop (BD Biosciences) and anti-CD107a (clone H4A3, BD Biosciences). Cells were stained with anti-CD56 (clone 5.1H11, BD Biosciences) and anti-CD16 (clone 3G8, BD Biosciences) and fixed with 4% PFA. Intracellular cytokine staining to detect IFNγ (clone B27, BD Biosciences) and TNFα (clone Mab11, BD Biosciences) was performed in permeabilization buffer (Biolegend). Markers were measured using a BD LSRFortessa and analyzed by FlowJo10. CD16 expression was confirmed in all cells. NK cell degranulation and activation were calculated as percent of CD56+NK cells positive for CD107a, or IFNγ or TNFα expression. Representative data from one dilution was chosen by the highest signal to noise ratio for further analyses.
4
Evaluating NK Cell Activation by SARS-CoV-2 RBD
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ADNKA was performed as described (26 (link), 133 (link)). ELISA plates were coated with recombinant RBD (300 ng/well) (BEI Resources NR-52309). Wells were washed, blocked, and incubated with serially diluted samples (1:10, 1:100, 1:1000) in duplicate for 2hours at 37°C prior to adding CD16a.NK-92 cells (PTA-6967, ATCC) (5 × 104 cells/well) for 5hours with brefeldin A (Biolegend), Golgi Stop (BDBiosciences) and anti-CD107a (clone H4A3, BDBiosciences). Cells were stained with anti-CD56 (clone 5.1H11, BDBiosciences) and anti-CD16 (clone 3G8, BDBiosciences) and fixed with 4%PFA. Intracellular cytokine staining to detect IFNγ (clone B27, BDBiosciences) and TNFα (clone Mab11, BDBiosciences) was performed in permeabilization buffer (Biolegend). Markers were measured using a BD LSRFortessa and analyzed by FlowJo10. CD16 expression was confirmed. NK cell degranulation and activation were calculated as %CD56+CD107a+, IFNγ+ or TNFα+. Representative data from one dilution was chosen by the highest signal-to-noise ratio. Experiments were conducted two independent times.
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