cNK and ML NK cells were incubated with tumor targets at 5:1 ET ratio as previously described.13 (link),14 (link) When indicated, cNK and ML NK cells were pre-incubated with anti-NKG2D, anti-NKp46, anti-NKp30, and anti-DNAM1 (5 µg/mL; all Biolegend) or a combination of anti-NKG2D, anti-NKp46 and anti-DNAM for 30 minutes before co-incubation with tumor targets. Cells incubated with Isotype control were used as control. At the end, the cells were stained with anti-CD56 (clone N901), anti-CD3 (clone UCHT1), anti-NKG2A (clone Z199), and anti-CD45 (clone J33) followed by intracellular staining with anti-IFN-γ (clone B27). Data was acquired in a Gallios flow cytometer (Beckman Coulter) and analyzed using FlowJo software (Tree Star v10.8).
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Cytotoxicity Assay of NK Cells
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Corresponding Organization : Washington University in St. Louis
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Variable analysis
independent variables
- Type of NK cells (cNK and ML NK cells)
- Pre-incubation with antibodies (anti-NKG2D, anti-NKp46, anti-NKp30, anti-DNAM1, or a combination of anti-NKG2D, anti-NKp46 and anti-DNAM)
- NK cell-mediated cytotoxicity against tumor targets
- Effector to target (E:T) ratio (5:1)
- Incubation time with tumor targets
- Positive control: Cells incubated with Isotype control
- Negative control: Not explicitly mentioned
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