2.4 Isolation of human peripheral blood mononuclear cells (PBMCs)
Freshly taken EDTA-blood from healthy subjects (HS) was diluted with PBS (1:4), stratified over 10 mL of Ficoll-Paque and then centrifuged at 1660 rpm for 30’ at 20 °C. The mononuclear cell layer was aspired and transferred into a 50 mL conical tube and washed two times with PBS by centrifugation at 1000 g for 10’. The cell pellet was suspended in 1 ml of PBS and was pre-incubated (20 minutes at 37°C) with scalar concentration (150-300-600 ng/ml) of methylprednisolone (Sigma Aldrich, USA) or betamethasone (Sigma Aldrich, USA), which corresponded to the levels detected in serum of patients upon glucocorticoid administration.18 After incubation, samples were stimulated with LPS (40 pg/ml; Sigma Aldrich, USA) for 10’ at 37°C. Cells were centrifuged for 3’ at 3000 rpm. Supernatants and pellet were stored at −80 °C for analysis of sNox2dp, H2O2, p47phox and pAKT.