In brief, nTreg were isolated, using the MACS® protocol

(see above), from peripheral blood samples taken at 08:30 hr, which were then incubated at 37° in 5% CO2 for 30 min with 1 μg/ml of Simulect® (Novartis, Basel, Switzerland), a CD25-neutralizing antibody. nTreg were then washed twice with phosphate-buffered saline (PBS) and used for functional assays as described above. To analyze whether hormone levels at the time of T-cell isolation influenced Tres and nTreg activities, we measured cortisol, melatonin, prolactin, growth hormone and noradrenalin levels in serum or plasma using commercially available assays. For cortisol and growth hormone analysis the Immulite® system was used (Immulite; DPC-Biermann GmbH, Bad Nauheim, Germany). Prolactin was measured using an immunoradiometric assay (Prolactin IRMA; DPC-Biermann GmbH) EGFR inhibitor and melatonin was measured using a radioimmunoassay (Bühlmann Laboratories

AG, Schönenbuch, Switzerland). Noradrenalin was analysed using standard high-performance liquid chromatography with subsequent electrochemical detection (Chromsystems, Munich, Germany).34 In order to investigate whether the correlational data obtained regarding the influence Selumetinib concentration of hormones on Tres cytokine secretion can be proven in an in vitro system, we isolated Tres, using the MACS protocol (see above), from peripheral blood collected at 08:30 hr. These purified Metalloexopeptidase Tres were then incubated (37°, 5% CO2) for 2 hr with physiological serum levels of cortisol (12 μg/dl; Sigma-Aldrich, Munich, Germany), melatonin (50 pg/ml; Sigma-Aldrich), or prolactin (20 ng/ml, R&D, Munich, Germany) in X-VIVO 15. After incubation, cells were washed twice, cultured as described above and the supernatants collected for analysis of cytokine concentrations. To ensure that the subjects slept well in the sleep condition, sleep quality was monitored using polysomnographic electroencephalogram (EEG) recordings.

EEG measurements were analyzed according to previously published standards.32 The mean time for sleep onset was 22·6 ± 5·6 min. Sleep time was 451 ± 6·2 min: time in stage 1 sleep was 26·3 ± 4·1 min; time in stage 2 sleep was 236 ± 23·1 min; time in slow wave sleep (SWS) was 77·8 ± 10·5 min; and time in rapid eye movement (REM) sleep was 76·8 ± 9·8 min. Latencies (with reference to sleep onset) were 19·3 ± 5·2 min for SWS and 172·1 ± 36·8 min for REM sleep. In all six subjects, SWS predominated during the first half of the night (49·3 ± 5·5 min versus 28·5 ± 9·6 min for the first half of the night and the second half of the night, respectively), while REM sleep dominated during the second half of the night (7·9 ± 2·6 min versus 70·3 ± 8·5 min for the first half of the night and the second half of the night, respectively). Hence, all subjects slept normally during the night of the experiment.