Anti cd56 pe cf594

IFN-γ and TNF-α production by NK cells was evaluated in all samples available for phenotypic analysis after overnight stimulation with or without IL-12 and IL-18 (5 ng/mL) in the presence of 10 µg/mL of brefeldin A (BFA) added during the last 3 h of culture. Surface staining with anti-CD3-PE, anti-CD56-PE-CF594, and anti-CD16-FITC (BD Bioscience, Franklin Lakes, NJ, USA) was performed. Then, cells were fixed with medium A reagent and permeabilized with medium B reagent (Nordic Mubio, Lifespan Biosciences, Seattle, WA, USA) in accordance with manufacturer’s instructions. Cytokine determinations were performed by intracellular cytokine staining (ICS) with anti-IFN-γ-PerCp-Cy5.5 (BioLegend, San Diego, CA, USA) and anti-TNF-α-APC (BioLegend, San Diego, CA, USA) monoclonal antibodies and analyzed by flow cytometry. The CD107a degranulation assay was performed to assess the cytotoxic potential. Cells were stimulated overnight with IL-12 and IL-18 (as above), then incubated with K562 target cells for the last 4 h in the presence of BFA and anti-CD107a-PE-Cy7 (BD Bioscience, Franklin Lakes, NJ, USA).
Data are expressed as the difference between the percentage of cytokine or CD107a-positive⁺ NK cells in the stimulated and unstimulated samples.

IFN-γ and TNF-α release from NK cells was assessed after IL-12 and IL-18 (5 ng/ml) overnight (O/N) stimuli in the presence or absence of specific p38 inhibitors. After 1 h of the stimulus, 10 µg/ml of BFA was supplemented for a further 3 h of culture. Anti-CD3 PE (BD), anti-CD56 PECF594 (BD), and anti-CD16 FITC (BD) were employed to stain the NK cells before fixing them with the medium A reagent and permeabilization with medium B reagent (Nordic Mubio) according to the manufacturer’s instructions. Cytokine production measurements were assessed by ICS after staining with monoclonal antibodies for IFNγ (PerCp-Cy5.5, Biolegend) and TNFα (APC, Biolegend) and subsequently run on FACS cytometry. The cytotoxic activity of NK cells was monitored through CD107a degranulation staining. Specifically, IL-12 and IL-18 were employed to stimulate the inhibitor-treated NK cells overnight (as described above). The cells together with target cells (K562) were then incubated with BFA and CD107 antibody (PECy7, BD) for the last 4 h.
The results are displayed as a ratio (fold change) of cytokine-positive NK cells detectable in inhibitor-treated vs . untreated cells. As control, the percentage of dead cells was evaluated in each inhibitor-treated sample by FACS in order to assess the cell toxicity.