FACS-sorted CD3+ cells (2.5×106/ml) were stimulated with CD3/CD28 beads (Life technology, Carlsbad, CA) at a 5:1 ratio. FACS sorted mMDSC were added to the activated CD3 T-cells at 1:1 ratio +/− 750nM Motolimod. Both CD3+ T-cells and mMDSC alone and with Motolimod were used as control. FACS-sorted HLA-DR+CD14+ monocytes were also cultured +/− Motolimod for 20 hours and supernatants harvested. Quantification of secreted cytokines was performed using a cytokine human magnetic kit (Life Technologies) by Luminex (Qiagen) per manufacturer’s instructions and reported as mean of pg/ml of duplicated measurements [22 (link)].
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Quantifying Cytokine Secretion in Immune Cell Interactions
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Corresponding Organization : Institute of Translational Health Sciences
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Variable analysis
independent variables
- Presence of mMDSC cells (added at 1:1 ratio with activated CD3 T-cells)
- Presence of Motolimod (750nM)
- Secreted cytokine levels (measured using a cytokine human magnetic kit and Luminex)
- FACS-sorted CD3+ cells (2.5×10^6/ml)
- CD3/CD28 beads (used to stimulate the CD3+ cells at a 5:1 ratio)
- FACS-sorted mMDSC cells
- FACS-sorted HLA-DR+CD14+ monocytes
- Culture duration of 20 hours
- Positive control: CD3+ T-cells and mMDSC alone
- Negative control: CD3+ T-cells and mMDSC with Motolimod
- HLA-DR+CD14+ monocytes cultured +/- Motolimod
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