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.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.