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 SP600125 price 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, Belnacasan purchase 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 Rho 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.