Fitc conjugated anti human cd8

Freshly thawed PBMC were used for surface and intracellular staining and analysis. The following monoclonal antibodies were used: fluorescein isothiocyanate (FITC)-conjugated anti-human CD3 (BD Pharmingen, USA), FITC-conjugated anti-human CD4 (BD Pharmingen, USA), FITC-conjugated anti-human CD8 (BD Pharmingen, USA), FITC-conjugated anti-human CD107a (BD Pharmingen, USA), phycoerythrin (PE)-conjugated anti-human Galectin-9 (Biolegend, USA), PE-conjugated anti-human TIM-3 (R and D Systems, USA), allophycocyanin (APC)-conjugated anti-human CD56 (BD Pharmingen, USA), APC-conjugated anti-human CD8 (BD Pharmingen, USA), APC-conjugated anti-human TIM-3 (R and D Systems, USA) APC-conjugated anti-human FoxP3 (eBioscience, USA). Control antibodies included isotype-matched FITC-conjugated, PE-conjugated and APC-conjugated mouse antibodies (all from BD-Pharmingen, USA).

Blood samples were mixed with 2 mL of ACK lysis buffer and incubated for 10 min to deplete red blood cells; after that, cells were incubated with human Fc receptor blocking buffer (eBioscience) at 4°C for 10 min followed by the incubation with specific antibodies or isotype-matched controls at 4°C for 30 min at the manufacturers’ recommended concentrations. PECY7-conjugated anti-human CD3, FITC-conjugated anti-human CD8, PECY5-conjugated anti-human CD56, PE-conjugated mouse IgM isotype control and PE-conjugated anti-human CD160 (Clone BY55; all from BD Biosciences) were used for flow cytometry analysis. Samples were washed with FACS buffer (2% BSA in PBS, 0.09% sodium azide). Pellets were resuspended in 300 mL of FACS buffer. Samples were acquired on a Cytomics FC 500 MPL (Beckmam Coulter) and analyzed by FlowJo software (TreeStar, Inc.) as reported previously [24 (link)].

PBMCs were washed once with 1 ml ice-cold FACS buffer (2% FCS in PBS) and the cell density was adjusted to 1 × 10⁶ cells/100 μl. APC-conjugated anti-human CD3 monoclonal antibody (BD, USA), FITC-conjugated anti-human CD8 (BD, USA), PE-conjugated anti-human CD279 monoclonal antibody (BD, USA) and FVS620 (BD, USA) were added and incubated in the dark for 15 min at room temperature. For FoxP3 staining, PBMCs were first stained with FITC-conjugated anti-Human CD4 monoclonal antibody (BD, USA), APC-conjugated anti-Human CD25 monoclonal antibody (BD, USA) and FVS620 (BD, USA) for 15 min in the dark at room temperature. After fixation and permeabilization, the cells were stained with PE-conjugated anti-human FoxP3 monoclonal antibody (BD, USA) overnight at 4°C. The cells were then washed three times with 1 ml of FACS buffer, resuspended in 0.5 ml of FACS fixing buffer (BD, USA) and acquired using CytoFLEX S flow cytometer (Beckman, USA). The data was analyzed by FlowJo software version VX (ThreeStar, San Carlos, CA, USA).