For PBMC stimulation experiments, IL-6, IL-10, IFN-γ, TNF-α, IL-1β cytokines and CCL-2 chemokine concentrations were measured in supernatants; for both hypoxia and coculture experiments, IL-4, IL-12, IL-10, TNF-α, TGF-β1 concentrations were measured in supernatants using ELISA kits (Human IL-1β, IL-4, IL-6, IL-10, IL-12, IFN-γ, TNF-α Uncoated ELISA and Human/Mouse TGF beta 1 Uncoated ELISA, ThermoFisher Scientific, Waltham, MA, USA) and following manufacturer instructions. An acidification of samples with hydrochloric acid (1N, 40µL) was performed for TGF-β1 concentration measurement.
For hypoxia experiments, results were expressed as ratios of the concentration of TGF-β1 in supernatant (after hypoxia or normoxia) to that of the control condition corresponding to the concentration of TGF-β1 in fresh medium, not used to culture PBMC in normoxia/hypoxia experiments. For coculture experiments, results were expressed as ratios of “PBMC wells” on that of “control wells” containing no PBMC. Medians were obtained for each group.
For hypoxia experiments, results were expressed as ratios of the concentration of TGF-β1 in supernatant (after hypoxia or normoxia) to that of the control condition corresponding to the concentration of TGF-β1 in fresh medium, not used to culture PBMC in normoxia/hypoxia experiments. For coculture experiments, results were expressed as ratios of “PBMC wells” on that of “control wells” containing no PBMC. Medians were obtained for each group.