Anti cd45 pe

PBMCs from AML patients were incubated with 10 mg/mL anti-PD-1 (nivolumab, Bristol-Myers-Squibb), anti-TIM-3 (BioLegend) or anti-TIGIT (BioLegend) antibodies for 24 hours in 37°C and 5% CO₂, followed by incubation with 50 ng/mL PMA and 1 mg/mL ionomycin for 1 hour plus 3-hour incubation with brefeldin A (BioLegend). Unstimulated cells were used as control. Cells were then washed in PBS, stained with anti-CD45-PE (EXBIO), anti-CD3-AlexaFluor700 (EXBIO) and anti-CD56-PerCP-Cy5.5 monoclonal antibodies (BioLegend), fixed in fixation/permeabilization buffer (eBioscience), further permeabilized with permeabilization buffer (eBioscience) and intracellularly stained with anti-IFN-γ-PE-Cy7 antibodies (eBioscience).

To assess NK cell functions in AML patients, fresh PBMCs were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA, from Sigma Aldrich) plus 1 μg/mL ionomycin, or with K562 cells at 10:1 PBMC:tumor cell ratio plus overnight incubation with 200 U/mL IL-2 in the presence of anti-CD107a-FITC monoclonal antibody (BioLegend) for 1 h, followed by 3 h incubation with brefeldin A (BioLegend). Cells were then washed in PBS, stained with anti-CD45-PE (EXBIO), anti-CD3-AlexaFluor700 (EXBIO), anti-CD4-ECD (Beckman Coulter), anti-CD8-HV500 (BD Biosciences) and anti-CD56-PerCP-Cy5.5 (BioLegend) antibodies, fixed in fixation/permeabilization buffer (eBioscience), further permeabilized with permeabilization buffer (eBioscience) and intracellularly stained with anti-IFN-γ-PE-Cy7 (eBioscience) and anti-granzyme B-BrilliantViolet421 (BD Biosciences) antibodies.