Zombie aqua fixable viability kit

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 Aqua^TM 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 Aqua^TM 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 LSRFortessa^TM flow cytometer (BD Biosciences) and NovocyteD3000 flow cytometer. Data were further analyzed using FlowJo software.

Tumors were cut into pieces and incubated in RPMI-1640 (Nacalai Tesque, Kyoto, Japan) supplemented with 0.2% collagenase (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and 2 KU/mL DNase I (Sigma-Aldrich, St. Louis, Missouri, USA) for 40 min at 37℃. All material was passed through a 70 µm cell strainer to obtain single cell suspensions. After staining dead cells using the Zombie Aqua Fixable Viability Kit (BioLegend) and blocking Fc receptors with anti-CD16/32 mAb, the cells were stained with mAbs for cell surface antigens.
For intracellular cytokine staining, cells were first stimulated with 10 ng/mL PMA (Sigma-Aldrich) and 1 µM ionomycin (Sigma-Aldrich) in the presence of 10 µg/mL brefeldin A (Sigma-Aldrich) for 4 hours. After staining dead cells using the Zombie Aqua Fixable Viability Kit (BioLegend) and blocking Fcγ receptors with anti-CD16/32 mAb, cells were first stained with mAbs for cell surface antigens. After fixation and permeabilization using Fixation Buffer and Intracellular Staining Perm Wash Buffer (BioLegend) according to the manufacturer’s protocols, cells were then stained with PE-conjugated anti-IL-17A and APC-conjugated anti-IFNγ antibodies. Stained cells were acquired on a CytoFLEX S flow cytometer (Beckman Coulter, Atlanta, Georgia, USA) and analyzed using FlowJo software V.10.6.2 (BD Biosciences).