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.
Protocol detail
Evaluating NK Cell Activation by SARS-CoV-2 RBD
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NR-52309
PTA-6967,
brefeldin A
Golgi Stop
anti-CD107a (clone H4A3,
anti-CD56 (clone 5.1H11,
anti-CD16 (clone 3G8,
clone B27,
permeabilization buffer
BD LSRFortessa
Brefeldin a
Buffer
Cell degranulation
Cells
Clone
Cytokine
Dilution
Elisa
Golgi
Ifnγ
Intracellular
Nk cells
Tnfα
Corresponding Organization : Parkland Health & Hospital System
Other organizations : Oregon Health & Science University
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Variable analysis
independent variables
- Dilution of samples (1:10, 1:100, 1:1000)
- NK cell degranulation (%CD56+CD107a+)
- NK cell activation (IFNγ+, TNFα+)
- Recombinant RBD (300 ng/well) used to coat ELISA plates
- CD16a.NK-92 cells (PTA-6967, ATCC) used as effector cells
- Incubation time of 2 hours at 37°C
- Incubation of CD16a.NK-92 cells for 5 hours with brefeldin A, Golgi Stop, and anti-CD107a
- Staining with anti-CD56, anti-CD16, anti-IFNγ, and anti-TNFα
- Measurement using a BD LSRFortessa and analysis with FlowJo10
- Positive controls: Not explicitly mentioned
- Negative controls: Not explicitly mentioned
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