One million splenocytes from mixed BM chimeric mice were incubated in 96-well tissue culture plates coated with anti-NKp46 (29A1.4) as described (5 (link)), or with 10 ng/ml mouse IL-12 plus 10 ng/ml mouse IL-18 (R&D Systems), 10 ng/ml PMA plus 1 μg/ml ionomycin (Sigma-Aldrich), or co-cultured with 1 × 105 RMA or m157-transfected RMA cells as described (6 (link)). One million CellTrace Violet-labeled splenocytes were co-cultured with 1 × 105 RMA or m157-transfected RMA cells (fixed in 1% paraformaldehyde) in the absence or presence of 25 ng/ml mouse IL-33 (R&D) and/or 10 ng/ml mouse IL-12 with 50 U/ml human IL-2 (NCI) for 4 days at 37° C.
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Activation and Expansion of NK Cells
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Corresponding Organization : University of California, San Francisco
Other organizations : University of Tsukuba, Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse
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Variable analysis
independent variables
- Anti-NKp46 (29A1.4) coating of tissue culture plates
- Stimulation with 10 ng/ml mouse IL-12 plus 10 ng/ml mouse IL-18
- Stimulation with 10 ng/ml PMA plus 1 μg/ml ionomycin
- Co-culture with 1 × 10^5 RMA or m157-transfected RMA cells
- Presence or absence of 25 ng/ml mouse IL-33 and/or 10 ng/ml mouse IL-12 with 50 U/ml human IL-2
- Splenocyte responses (not explicitly specified)
- One million splenocytes from mixed BM chimeric mice
- Incubation in 96-well tissue culture plates
- CellTrace Violet labeling of splenocytes
- Fixation of RMA or m157-transfected RMA cells in 1% paraformaldehyde
- 4-day co-culture duration at 37°C
- Positive control: Co-culture with RMA or m157-transfected RMA cells
- Negative control: Co-culture with RMA or m157-transfected RMA cells in the absence of any stimulation
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