Fix perm cell fixation and permeabilization kit

Antibody-dependent NK cell degranulation was conducted as previously described [83 (link),84 (link)]. Spike or RBD protein was coated on Maxisorp ELISA plate (Thermo Fisher Scientific) and then blocked with 5% BSA. Diluted serum (1:25 dilution) was then added and incubated for 2 hours at 37°C. Human NK cells were isolated from peripheral blood by negative selection using the RosetteSep Human NK cell enrichment cocktail (STEMCELL Technologies - Vancouver, British Columbia, Canada) following the manufacturer’s instructions. Human NK cells were then added to the bound antibody and incubated for 5 hours at 37°C in the presence of RMPI+10% FBS, GolgiStop (BD), Brefeldin A (Sigma - Burlington, Massachusetts, USA), and anti-human CD107a-PE-Cy5 antibody (BD Biosciences). After incubation, cells were washed, stained with CD16-APC-Cy7, CD56-PE-Cy7, and CD3-Pacific Blue (BD Biosciences) and fixed in 4% PFA for 15 minutes. Intracellular staining was performed using the FIX/PERM Cell fixation and permeabilization kit (Thermo Fisher Scientific), and cells were stained for IFNγ-APC and macrophage inflammatory protein-1β-PE (BD Biosciences). Results were reported as percentage of NK cells positive for CD107a, IFNγ, or macrophage inflammatory protein-1β. Flow cytometry was performed with an LSRII (BD), and analysis was performed using FlowJo V10.7.1.

T cells and target cells were co-cultured for 6 h. Two hours after stimulation, 10 ug/ml Brefeldin A (Sigma-Aldrich) was added. Cells were washed and stained after the indicated time. Intracellular cytokine stain for IFN-γ-PE (BD), TNF-α-PacificBlue (Biolegend), and IL-2-APC (BD) was performed using the FIX & PERM cell Fixation and Permeabilization kit (ThermoFisher Scientific) according to the supplier’s information. Intracellular cytokine stains were analyzed using a MACSQuant Analyzer 10 (Miltenyi Biotec).

Antibody-dependent NK cell degranulation was conducted as previously described^{59 (link)}. NVX-CoV2373 Spike protein was coated on Maxisorp ELISA plate (Thermo Fisher), and then blocked with 5% BSA. Antibodies were then added and incubated for 2 hours at 37°C. Human NK cells were isolated from peripheral blood by negative selection using the RosetteSep Human NK cell enrichment cocktail following the manufacturer’s instructions. Human NK cells were then added to the bound antibody and incubated for 5 hours at 37°C in the presence of RMPI + 10%FBS, GolgiStop (BD), Brefeldin A (Sigma), and anti-human CD107a antibody (BD Bioscience). After incubation, cells were washed, stained with CD16, CD56, and CD3 (BD Bioscience), and fixed in 4% PFA for 15 minutes. Intracellular staining was performed using the FIX/PERM Cell fixation and permeabilization kit (Thermo), and cells were stained for interferon-γ and macrophage inflammatory protein-1β (BD bioscience). Flow cytometry was performed with an IQue (Intellicyt) and analysis was performed on IntelliCyt ForeCyt (v8.1).

In brief, plates were coated with AVI-tag gp120, blocked, and patient plasma added before incubating for 2 h at 37 °C. NK cells isolated from a healthy donor's whole blood were added simultaneously with anti-CD107a PE-Cy5 (BD Biosciences), Brefeldin A (Sigma) and Golgi Stop (BD Biosciences) and incubated for 5 h at 37 °C as previously reported [37 ]. NK cells were then stained with anti-CD56 PE-Cy7, anti-CD16 allophycocyanin (APC)-Cy7 and anti-CD3 Alexa Fluoro 700 (BD Biosciences), fixed (FIX&PERM cell fixation and permeabilization kit, Thermo Fisher Scientific, Waltham, Massachusetts, USA), and stained intracellularly with anti-IFNγ-APC and anti-MIP-1β-PE (BD Biosciences). Surface expression of CD107a and intracellular production of IFNγ and MIP1β by NK cells (CD16+/56+CD3−) were then analysed by flow cytometry. For each Fc-assay, a negative control (human purified IgG, Sigma) was included. Mean of signal in the negative controls wells was subtracted as background signal. The gating strategy for the Fc-mediated functions are illustrated in Supplementary Fig. 1.