Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy human donors by density gradient centrifugation (Ficoll-Paque™ Plus, GE Healthcare). ADCC was measured by the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega Corporation). In brief, 1×104 SK-BR-3 cells or Ramos cells (target cells) were incubated with ten-fold gradient diluted antibodies (for ADCC curve) or 1 μg/mL of trastuzumab (for three donors ADCC assay) in complete RPMI 1640 medium (without phenol red, Thermo Fisher Scientific) at 37 °C for 30 min, and then 1.5×105 PBMCs (effector cells) were added with an effector/target ratio of 15:1. After 4 h of incubation, lactate dehydrogenase (LDH) activity in the supernatants was measured. Each measurement was performed with three repeats. The percentage of ADCC was calculated as follows: % Cytotoxicity = (experimental lysis - spontaneous effector lysis - spontaneous target lysis) / (maximum target lysis - spontaneous target lysis) × 100.

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