Whole blood samples were collected from patients after sepsis was diagnosed. Cells were stained with anti-CD45-Brilliant Violet 570™ (BioLegend), anti-CD3-FITC (BioLegend), and anti-CD56-PE (BioLegend). For MX1 and intracellular cytokine staining, 250uL blood from each sample was resuspended and incubated in RPMI 1640 culture medium containing 3% FBS (Mediatech). The cells were then stimulated with a cell activation cocktail (with Brefeldin A) (eBioscience) for 5 h at 37°C. Cells were stained with Zombie AquaTM fixable viability kit (BioLegend), anti-CD45-Alexa Fluor®700 (BioLegend), anti-CD3-Percp-cy5.5 (BioLegend), and anti-CD56-FITC (BioLegend). After surface staining, fixation, and permeabilization, we used anti-MX1-AF647 (Abcam), and anti-IFN-ɑ (Invitrogen). Brilliant Violet 421-conjugated donkey anti-rabbit IgG was used as the secondary antibody (BioLegend).
For mice NK cells, cells were stained with Zombie AquaTM fixable viability kit (BioLegend), anti-NK 1.1-FITC (BioLegend) and anti-MX1 (Proteintech). Alexa Fluor 647-conjugated donkey anti-rabbit IgG was used as the secondary antibody (BioLegend). Data were collected on the LSRFortessaTM flow cytometer (BD Biosciences) and NovocyteD3000 flow cytometer. Data were further analyzed using FlowJo software.
For mice NK cells, cells were stained with Zombie AquaTM fixable viability kit (BioLegend), anti-NK 1.1-FITC (BioLegend) and anti-MX1 (Proteintech). Alexa Fluor 647-conjugated donkey anti-rabbit IgG was used as the secondary antibody (BioLegend). Data were collected on the LSRFortessaTM flow cytometer (BD Biosciences) and NovocyteD3000 flow cytometer. Data were further analyzed using FlowJo software.