Anti cd107a pe cy7

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.

PBMCs stimulated with the same peptides were collected together, washed twice, and stained with FV450 (BD) for 15 min at room temperature (RT) and anti CD107a PE‐Cy7, anti CD3 FITC, anti CD45 APC‐Cy7, anti CD4 PE, anti CD8 APC, and anti CD137 PE‐Cy5 (BD) for 30 min at 4 ℃. Cells were washed twice in FACS buffer, fixed and permed using Fixation/Permeabilization kit (PeproTech), and stained for intracellular IFN‐γ (anti IFN‐γ BV510, Biolegend) for 15 min at 4 ℃. Cells were washed twice with FACS buffer before analyzed with Canto II (BD) or CytoFLEX (Beckman) flow cytometer.