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.