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NKs cells are potent killers in ADCC assays with SKOV-3 or Raji cells (A, B)

ADCC assays with SKOV-3 and Raji targets. NK cells were cultured as described in the adjacent figure for 13 days. Prior to setting up the ADCC assay, CD56+CD16+ levels were ascertained to be >30% on NKs using Human Fc gamma RIII (CD16) PerCP-conjugated Antibody (FAB2546C); in our hands, this is the minimum level required for killing via ADCC. Target cells were labeled with Mito Mark Green (SKOV-3 or Raji), and Effectors/NKs were labeled with Janelia Fluor 646 as described (Figure 1). (A) SKOV-3 cells were preincubated with 5 ug/mL Anti-Her2/Trastuzumab (Catalog # MAB9589); (B) Raji cells were preincubated with 10 ug/mL Anti-CD20/Rituximab (MAB9630), both for 30 minutes at room temperature prior to setting up the ADCC assay with the NKs. 0.5:1 E:T ratios were used for the SKOV-3/Trastuzumab model, and NKs and SKOV-3 cells were co-incubated for 2 hours at 37°C. 2:1 E:T ratios were used for the Raji/Rituximab model, and NKs and Raji cells were co-incubated for 2.5 hours at 37°C. In both models, the cells were harvested, stained with antibodies for Human CD3 Alexa Fluor 405 and Human CD56 Alexa Fluor 700, and analyzed by flow cytometry. Importantly, non-NKs (JF 646-CD56- cells) are shown in the Mito Mark dye graphs that depict killing. These results are representative of 3+ experiments.