Anti cd16 fitc

This was undertaken according to our previously published methods (33 (link)). The method utilized whole blood analysis with red cell lysis using Easylyse (BD Biosciences) and with anti-CD56 PE, anti-CD16 FITC, anti-CD3 PE Cy5 and anti-CD69 APC (all supplied by BD Pharmingen). Flow cytometric analysis was undertaken on a Facscalibur (BD Biosciences), and using Cellquest Pro software. A minimum number of 10,000 cells were counted.

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.

Samples of EDTA anticoagulated peripheral blood (5 mL) were collected from three groups’ patients. All samples were tested within 6 h of being obtained. Briefly, CD3⁺/CD4⁺/CD8⁺ T-cell, CD19⁺ B-cell, CD3⁻CD16⁺CD56⁺ NK-cell, CD3⁺CD16⁺CD56⁺ NKT-cell, CD4⁺CD25⁺CD127⁻ Treg-cell, CD3⁺HLA-DR⁺ T-cell and CD4⁺HLA-DR⁺ T-cell, CD8⁺ CD28⁺, and CD16⁺CD56⁺CD69⁺ counts (%) were measured by multiple-color flow cytometry with human monoclonal anti-CD3-FITC, anti-CD4-PE, anti-CD8-APC, anti-CD19-PE, anti-CD16-FITC, anti-CD56-APC, anti-CD25-PE, anti-CD127-BV421, anti-HLA-DR-Alexa700, anti-CD28-BV510 and anti-CD69-PE antibodies (BD Multitest) according to the manufacturer’s instructions. The cells were analyzed by flow cytometry system (Becton Dickinson, USA) according to the operating procedure.