NK cells were stimulated at indicated time points with HNSCC targets for 6 hours in presence of 1 ng/mL rhIL15. Effector to target (E:T) ratio was 5:1, unless otherwise indicated, with anti-CD107a antibody (Biolegend; RRID:AB_1227509) for 6 hours, with Golgi Plug/Stop present in the last 5 hours. When indicated, NK cells were pre-incubated, IgG1 Isotype control (5μg/mL; BioLegend; RRID:AB_2801451), anti-NKG2D (5μg /mL; BioLegend; RRID:AB_2810480), anti-CD2 (5 μg/mL; BD-Biosciences; RRID:AB_395731) or anti-CD226 (5μg/mL; BioLegend; RRID:AB_1279155) blocking antibodies 30 minutes before incubation with tumor targets. In Cetuximab experiments, tumor cells were pre-incubated with anti-EGFR antibody cetuximab (10μg/mL; Lily) prior to incubation with NK cells. Cells were stained for flow cytometry analysis as described previously.19 Degranulation (CD107a), TNF and IFN-γ were assessed by flow cytometry.16 (link),20 (link),26 (link) Data were acquired on a Gallios flow cytometer (Beckman Coulter) and analyzed using FlowJo software (Tree Star v10.6; RRID:SCR_008520).
Protocol detail
NK Cell-Mediated Cytotoxicity Assay
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Corresponding Organization : Washington University in St. Louis
Other organizations : University of Pittsburgh Medical Center
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Variable analysis
independent variables
- Presence of HNSCC targets
- Presence of rhIL15 (1 ng/mL)
- Effector to target (E:T) ratio (5:1, unless otherwise indicated)
- Presence of anti-CD107a antibody
- Incubation with IgG1 Isotype control (5μg/mL), anti-NKG2D (5μg /mL), anti-CD2 (5 μg/mL) or anti-CD226 (5μg/mL) blocking antibodies
- Presence of cetuximab (10μg/mL) pre-incubated with tumor cells
- Degranulation (CD107a)
- TNF production
- IFN-γ production
- Incubation time (6 hours)
- Presence of Golgi Plug/Stop in the last 5 hours
- Not explicitly mentioned
- IgG1 Isotype control (5μg/mL)
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