Data acquisition was performed on a Navios Flow Cytometer (Beckman Coulter, Brea, US). Our instrument was daily calibrated with Flow Check (Beckman Coulter, Brea, US) and Flow Set (Beckman Coulter, Brea, US) calibration beads to control the optical and fluidic stability of the device and for a performance validation throughout the study. To minimize autofluorescence and the improper analysis of cell doublets, cells were first put through a forward scatter area and forward scatter height gate to identify single cells. Leukocytes were then gated out from dead cells and debris on the basis of labeling with CD45. For the monocyte panel, among the CD45+ cell population, monocytes were identified on a CD14/SS dot-plot. Intracellular tumor necrosis factor-α (TNFα) and mHLA-DR results were expressed as mean fluorescence intensity (MFI) of the entire monocyte subpopulation. For the lymphocyte panel, two complementary gating strategies were used. First, in order to phenotype IL-10 producing lymphocytes in stimulated tubes, on a IL-10 (x-axis) and SS (y-axis) dot-plot gated on in CD45+ leukocytes, we selected IL-10 producing lymphocytes (IL-10+SSClow cells). CD3, CD19, and CD4 expressions were then characterized on these cells based on CD4 (y-axis) and CD3 or CD19 (x-axis) dot-plots. Second, so as to evaluate the impact of sepsis on IL-10 production capacity on beforehand identified lymphocyte subpopulations, B cells were identified on a CD19/SS dot-plot and T cells on a CD3/SS dot-plot among the CD45+ cell population. Finally, CD4- and CD4+ T cells were gated among CD3+ cells on a CD3/CD4 dot-plot. The percentages of IL-10 expressing cells among these three lymphocyte subpopulations were then evaluated. Positivity threshold was defined based on isotype values set up at 1%. A minimum of 5,000 target cells (monocytes or lymphocytes) were systematically acquired to ensure robustness of results. Of note, both in patients and donors, the majority of monocytes were able to produce TNF-α; which was not the case for IL-10. Thus TNF- α results expressed as MFI possessed a better dynamic range compared with percentages which saturated at 100%. In addition, TNF-α results expressed as percentages and MFI were strongly correlated (Data not shown).

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