Cell culture and stimulation.  PBMCs were cultured in complete RPMI-1640 culture medium supplemented with 7.5% heat-inactivated foetal calf serum (Sigma-Aldrich, St. Louis, MO, USA) and plated on 24-well plates. For stimulation, cells were incubated with

anti-CD3/anti-CD28-coated beads (Invitrogen Dynal AS, Oslo, Norway) at a bead:cell ratio of 1.0. Proliferation assay.  For cell proliferation assay, PBMCs were labelled with carboxyfluorescein diacetate (CFSE) (Molecular Probes, Inc., Eugene, OR, USA) according to the manufacturers recommendations. At the end of the culture period, the CFSE labelled cells were stained with anti-CD4-APC, anti-CD8-PerCP and anti-CD25-APC-Cy7 monoclonal antibodies (mAbs) (BD Pharmingen, San Diego, CA, USA), Navitoclax concentration washed and then run immediately on the flow cytometer. The BD FACS Aria (Becton-Dickinson, Franklin Lakes,

NJ, USA) was used for all measurements of our study. Cell death assay.  Cells Idelalisib order were stained with anti-CD4-PE-Cy7, anti-CD25-FITC and anti-CD8 APC-Cy7 mAbs (BD Pharmingen). After 10 min of incubation in dark, propidium iodide was added and samples were incubated for 10 more min. The samples were then analysed immediately on flow cytometer. Intracellular FoxP3 assay for the identification of Tregs.  For analysis of Foxp3, cells were first stained for the expression of CD4 and CD25 surface molecules with anti-CD4 APC and anti-CD25 FITC mAbs (both BD PharMingen). Cells were fixed and permeabilized based on the manufacturer’s recommendations (Fixation/Permeabilization solution,

Permeabilization solution, eBioscience, San Diego, CA, USA). Anti-Foxp3 PE mAb (eBioscience) was then used for intracellular staining (40 min at 4 °C), and corresponding isotype control was also included. Cells were washed once and analysed immediately on flow cytometer. Surface markers of T lymphocytes.  To determine check the activation, maturation markers and Th1/Th2 polarization of CD4+ and CD8+ lymphocytes, the following mAbs were used in combinations: anti-CD4 PE-Cy7, anti-CD8 APC-Cy7, anti-CXCR3 APC (Th1), anti-CCR4 PE (Th2), anti-CD62L PE-Cy5, anti-CD25 FITC, anti-CD69 APC, anti-CD45RO PE, anti-HLA-DR PerCP, anti-CD45RA FITC (all purchased from BD Pharmingen). The cells were incubated with mAbs for 20 min in dark, washed once and analysed on flow cytometer. Statistical analysis.  Median [range] of the variables is reported. Hettmansperger–Norton trend test was applied to investigate the trend of the changes with increasing hyperoxia time [18]. P values <0.05, two tailed, were considered significant. We did not correct for multiplicity. Mann–Whitney U test was used for comparison of two groups. Data are summarized in Table 1 for cell cultures without T cell stimulation and in Table 2 for experiments with anti-CD3/CD28 bead stimulation.