To study changes in relative quantity of CD184 (CXCR4) and CD62L (L-selectin) on the cell subsets, the median fluorescence intensity (MFI) of the labeled anti-CD184-antibody and anti-CD62L-antibody was analyzed. Samples for measuring hormone concentrations were kept frozen at −70 °C until assay. Cortisol in serum and adrenocorticotropic

hormone (ACTH) in plasma were measured using a commercial assay (Immulite, Siemens Healthcare Diagnostics, Deerfiled, USA). Aldosterone was measured in serum, also using learn more a commercial assay (DPC-Biermann, Bad Nauheim, Germany). Epinephrine and norepinephrine were measured in plasma by standard high-performance liquid chromatography. Sensitivity, intraassay and interassay coefficients of variation were as follows: cortisol 0.2 μg/dL, less than 10%; ACTH 9 pg/mL, less than 9.5%; aldosterone 11 pg/mL, less than 16%; epinephrine 2 pg/mL, less than 6.5%; norepinephrine 5 pg/mL, less than 6%. Sleep stages were determined off-line from polysomnographic recordings following standard criteria (Rechtschaffen and Kales, 1968). For each night, sleep onset (with

reference to lights off at 23:00 h), total sleep time (sum of time spent in sleep stages 1, 2, 3, and 4 and REM sleep), and the time as well as percentage of total sleep time spent in the different sleep stages were calculated. In addition, ICG-001 cost we determined the time between awakening of subjects around 4:00 h for the second administration of spironolactone or placebo and falling asleep again. SWS was defined by the sum of stage 3 and 4 sleep. Analyses of variance (ANOVA) for repeated measurements were calculated on T cell subpopulations (absolute counts, CXCR4 expression, CD62L expression) and hormones. Factors new included were “Condition” (spironolactone versus placebo), “Early/late” (23:00–3:30 h versus 5:00–9:30 h) and ”Time” (reflecting the four time points of measurements during the early and late night, respectively). We included the factor “Early/late” since we expected the

effects of spironolactone only during the early night, when the impact of sleep on T cell migration is evident (see Section 1). Degrees of freedom were corrected according to the Greenhouse-Geisser procedure. In case of ANOVA effects, paired t tests were analyzed at single time points. A p-value <0.05 was considered significant. Data are presented as mean ± SEM. T cells were classified as CD4+ (T-helper) and CD8+ (cytotoxic) T cells, and both of these subpopulations were further divided in naïve, central memory, effector memory and effector T cells. The absolute counts of all subpopulations with the exception of effector CD4+ and effector CD8+ T cells showed a peak during the first night half (23:00–3:30 h) followed by a decline until the last blood sampling at 9:30 h (F(1,10) ⩾ 9.68, p ⩽ 0.01, for main effects of Early/late and F(3,30) ⩾ 8.27, p ⩽ 0.