Human NK cells were enriched from fresh peripheral blood of healthy human volunteer donors’ buffy coats, obtained by Clínica Sanatorio Alemán blood bank at Concepción, Chile.
Briefly, a negative selection kit (Miltenyi) was used to isolate NK cells. 2 µg /ml of SARS-CoV-2 spike recombinant proteins were coated on ELISA High Bind Microplate (Corning) at 4°C overnight, and plates were blocked with 5% bovine serum albumin (BSA) prior to addition of the serum dilutions (1/100) in PBS for 2 hours at 37°C. Unbound antibodies were removed by washing wells 3X with PBS prior to the addition of NK cells. The NK cells were added at 5 x 105 cells/well in the presence of brefeldin A (BioLegend), monensin (BioLegend), and anti-CD107a phycoerythrin (PE) (BioLegend) and incubated for 5 hours at 37°C. NK cells were surface stained with CD56 Alexa Fluor 647 (BioLegend), followed by intracellular staining with IFNγ Alexa Fluor 488 (BioLegend) and MIP1β Brilliant Violet 421 (BioLegend) using the Fix & Perm cell permeabilization kit (Invitrogen) used to detect the production of cytokine/chemokine. Cells were analyzed on a BD LSRFortessa X-20 flow cytometer and data was analyzed using FlowJo software (37 (link)).
Briefly, a negative selection kit (Miltenyi) was used to isolate NK cells. 2 µg /ml of SARS-CoV-2 spike recombinant proteins were coated on ELISA High Bind Microplate (Corning) at 4°C overnight, and plates were blocked with 5% bovine serum albumin (BSA) prior to addition of the serum dilutions (1/100) in PBS for 2 hours at 37°C. Unbound antibodies were removed by washing wells 3X with PBS prior to the addition of NK cells. The NK cells were added at 5 x 105 cells/well in the presence of brefeldin A (BioLegend), monensin (BioLegend), and anti-CD107a phycoerythrin (PE) (BioLegend) and incubated for 5 hours at 37°C. NK cells were surface stained with CD56 Alexa Fluor 647 (BioLegend), followed by intracellular staining with IFNγ Alexa Fluor 488 (BioLegend) and MIP1β Brilliant Violet 421 (BioLegend) using the Fix & Perm cell permeabilization kit (Invitrogen) used to detect the production of cytokine/chemokine. Cells were analyzed on a BD LSRFortessa X-20 flow cytometer and data was analyzed using FlowJo software (37 (link)).
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