Anti cd57 fitc

PBMCs were pre-incubated with an anti-mouse CD16/32 Fc blocker (BD Pharmingen, USA), followed by anti-FVD-APC-Cy7 (all supplied by eBioscience, San Diego, CA, USA) to exclude dead cells. After washing with FACS staining buffer, cells were treated with fluorochrome-conjugated monoclonal antibodies for 40 min at 4°C. The monoclonal antibodies used in this study were as follows: anti-CD3-PerCP-Cy5.5, anti-CD3-PE-Cy7, anti-CD4-AF700, anti-CD8-PE, anti-CD8-APC, anti-CD28-APC, anti-CD45RA-FITC, anti-CD45RO-PE-Cy7, anti-CD57-FITC, anti-TCR gamma/delta-FITC, fixable viability dye-APC-Cy7, anti-interferon (IFN)-γ-PE-Cy7, and anti-tumor necrosis factor (TNF)-α-APC (all supplied by eBioscience, San Diego, CA, USA). For intracellular staining, surface-stained cells were stimulated with phorbol-myristate acetate/ionomycin/brefeldin A/monensin for 5 h, and then fixed and permeabilized using a Fixation/Permeabilization Buffer kit (eBioscience, San Diego, CA, USA). The permeabilized cells were washed and resuspended in 1% formaldehyde and stained with anti-IFN-γ-PE-Cy7 and anti-TNF-α-APC. Multicolor flow cytometry was performed using a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA), and the data were analyzed by FlowJo V10 software (FlowJo, LLC, Ashland, OR, USA).

PBMCs were incubated with directly fluorochrome-conjugated monoclonal antibodies for 40 min at 4 °C. The antibodies used in this study were anti-CD3-PerCP-Cy5.5, anti-CD3-PE-Cy7, anti-CD4-AF700, anti-CD8-PE, anti-CD8-APC, anti-CD28-APC, anti-CD57-FITC, fixable viability dye-APC-Cy7, anti-IFN)-γ-PE-Cy7, anti-TNF-α-APC, anti-IL-17A-APC, anti-perforin-PerCP-Cy5.5, anti-granzyme B-PE, and anti-FVD-APC-Cy7 (all supplied by eBioscience, San Diego, CA, USA). PBMCs were stimulated with phorbol-myristate acetate/ionomycin/brefeldin A/monensin for 5 h. The cells were fixed and permeabilized using a Fixation/Permeabilization Buffer kit (eBioscience, San Diego, CA, USA). The permeabilized cells were washed and resuspended in 1% formaldehyde and further stained for intracellular cytokines and cytotoxic molecules with anti-IFN-γ-PE-Cy7, anti-TNF-α-APC, anti-IL-17A-APC, anti-perforin-PerCP-Cy5.5, and anti-granzyme B-PE. Multicolor flow cytometry was performed using a BD FACSCanto II Flow Cytometer (BD Biosciences, San Jose, CA, USA), and the data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

The cell sorting of NK cell subpopulations was performed on the FACSVantage DiVa (BD Biosciences, San Jose, CA, USA). The following antibodies were used: anti-CD3-PC7, anti-CD56-APC (both from Beckman Coulter, USA), anti-CD57-FITC (eBioscience, USA), and anti-HLA-DR-PE (Sony Biotechnology, Japan).

Cryopreserved human PBMCs and hepatic mononuclear cells were thawed and then incubated with fluorochrome-conjugated monoclonal antibodies for 30 min at 4 °C. The cells were then preincubated with anti-CD16/32 Fc blocker (BD Pharmingen, Franklin Lakes, NJ, USA), which was followed by staining with the Live/Dead marker anti-FVD-APC-Cy7 (all supplied by eBioscience, San Diego, CA, USA). The fluorochrome-conjugated antibodies used were anti-CD3-PerCP-Cy5.5, anti-CD3-PE-Cy7, anti-CD4-AF700, anti-CD8-PE, anti-CD8-APC, anti-CD28-APC, anti-CD57-FITC, anti-IFN-γ-PE-Cy7, anti-TNF-α-APC, anti-IL-17A-APC, anti-perforin-PerCP-Cy5.5, and anti-granzyme B-PE (all supplied by eBioscience). Following this, the PBMCs and hepatic mononuclear cells were stimulated with phorbol-myristate acetate/ionomycin/brefeldin A/monensin for 5 h ex vivo, after which they were fixed and permeabilized using a Fixation/Permeabilization Buffer kit (eBioscience). The permeabilized cells were washed with FACS buffer; resuspended in 1% formaldehyde; and immunostained for intracellular cytokines using anti-IFN-γ-PE-Cy7, anti-TNF-α-APC, anti-IL-17A-APC, anti-perforin-PerCP-Cy5.5, and anti-granzyme B-PE fluorochrome-conjugated antibodies. The stained cells were analyzed using a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA), and the data were analyzed using FlowJo software (Treestar, Ashland, OR, USA).