100,000 NK cells were incubated in 96-well tissue culture plates coated with anti-NK1.1 (clone PK136) or isotype-matched control mouse IgG2a as described previously (Nabekura et al., 2015 (link)); incubated with 2.5 ng/ml mouse IL-12 and 2.5 ng/ml mouse IL-18 (R&D Systems); or co-cultured with 105 RMA, RMA expressing m157, or RMA expressing Rae1γ for 5 h at 37°C in the presence of PE-conjugated anti-CD107a mAb and GolgiStop (BD), followed by staining for surface molecules and intracellular IFN-γ as previously described (Nabekura and Lanier, 2014 (link)).
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NK Cell Activation and Degranulation
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Corresponding Organization : Parker Institute for Cancer Immunotherapy
Other organizations : University of Tsukuba
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Variable analysis
independent variables
- Incubation with anti-NK1.1 (clone PK136) or isotype-matched control mouse IgG2a
- Incubation with 2.5 ng/ml mouse IL-12 and 2.5 ng/ml mouse IL-18
- Co-culture with 10^5 RMA, RMA expressing m157, or RMA expressing Rae1γ
- CD107a expression
- IFN-γ production
- Incubation time (5 h at 37°C)
- Presence of PE-conjugated anti-CD107a mAb and GolgiStop
- Positive control: Incubation with anti-NK1.1 (clone PK136)
- Negative control: Incubation with isotype-matched control mouse IgG2a
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