Antibody-dependent NK cell degranulation was conducted as previously described59 (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).
Interferon γ and macrophage inflammatory protein 1β
Manufactured by BD
Interferon-γ and macrophage inflammatory protein-1β are lab equipment products. Interferon-γ is a cytokine involved in immune responses, while macrophage inflammatory protein-1β is a chemokine that regulates the migration and activation of various immune cells. These products are used in research settings to study their respective functions, but no further details on intended use can be provided.
Automatically generated - may contain errors
Lab products found in correlation
Forecyt, by Intellicyt (3 mentions)
Maxisorp elisa plate, by Thermo Fisher Scientific (3 mentions)
Anti human cd107a antibody, by BD (3 mentions)
Fix perm cell fixation and permeabilization kit, by Thermo Fisher Scientific (3 mentions)
Brefeldin a, by Merck Group (3 mentions)
Golgistop, by BD (3 mentions)
3 protocols using interferon γ and macrophage inflammatory protein 1β
1
Antibody-Dependent NK Cell Degranulation Assay
2
Antibody-dependent NK Cell Degranulation
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Antibody-dependent NK cell degranulation was conducted as previously described.83 (link) NVX-CoV2373 Spike protein was coated on Maxisorp ELISA plate (Thermo Fisher), and then blocked with 5% BSA. Diluted serum (1:25 dilution) was then added and incubated for 2 h 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 h at 37°C in the presence of RPMI+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 min. 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) (Figure S1 ).
3
Antibody-Mediated NK Cell Cytotoxicity Assay
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Antibody-dependent NK cell degranulation was conducted as previously described59 (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).
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