Au5811

Circulating leukocyte numbers at different time points after reperfusion were measured by whole-blood analysis using an automated hematological cell-counter (Cell-Dyn Sapphire, Abbott, Santa Clara, CA, USA). Plasma samples were obtained by whole-blood centrifugation at 1850× g and were immediately stored at −80 °C. C-reactive protein (CRP) levels were measured using solid phase DuoSet sandwich ELISA (DY1707, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. Troponin I levels were measured using a clinical chemistry analyzer (AU5811, Beckman Coulter, Woerden, The Netherlands). Histological analysis of collagen, myocardial immune cells, neutrophils, and monocyte infiltration is described in the Appendix A.

For this study, samples were selected from an existing prospective study database of patients (Molecular Diagnosis and Risk Stratification of Sepsis—MARS cohort), who were initially admitted to the ICU for observation following elective surgery, but who developed nosocomial sepsis later during their hospital stay and were consequently re-admitted to the ICU. Median SOFA scores of the patients were 6.5 (range 6–9). Patients’ EDTA plasma was obtained during routine ICU blood collection and 2x 450 µL was stored at −80°C within 4 h after collection. CRP was analyzed in heparin plasma on an AU5811 routine chemistry analyzer (Beckman Coulter, Brea, California). The UMCU institutional review board approved an opt-out method for consent (protocol numbers 10-056C/18-192).
Unless otherwise specified, all chemicals and reagents were obtained from Sigma-Aldrich (Steinheim, Germany). Acetonitrile (ACN) was purchased from Biosolve (Valkenswaard, The Netherlands). Sequencing grade trypsin was obtained from Promega (Madison, WI). The Oasis PRiME HLB plates were purchased from Waters (Etten-Leur, the Netherlands).

We obtained seven saliva samples throughout the experiment using salivettes (Sarstedt, Nümbrecht, Germany) for the quantification of cortisol and alpha-amylase [an indirect marker of adrenergic activity (van Stegeren, Rohleder, Everaerd, & Wolf, 2006 (link))] at the following time points: −10, +5, +20, +30, +65, +90, and +120 min relative to TSST onset. Samples were temporarily stored at 4 °C and subsequently stored at −20 °C. Cortisol and alpha-amylase levels were analyzed as described previously (Vinkers et al., 2013 (link)). In short, cortisol was measured without extraction using an in-house competitive radio-immunoassay, and alpha-amylase was measured using a Beckman-Coulter AU5811 chemistry analyzer. Three out of 497 cortisol samples were not collected (all non-peak values) and these missing values were calculated based on the mean group decline. The area under the curve (AUCi) for cortisol was calculated as previously described (Pruessner, Kirschbaum, Meinlschmid, & Hellhammer, 2003 (link)). The effects of time, stress, group, and their interaction on cortisol and alpha-amylase levels were analyzed using repeated measures ANOVAs using SPSS 23.0.

Serum PRL assay: Peripheral blood was collected using EDTA vacuum blood collection tubes and sent to the clinical laboratory of the hospital for testing on the same day (Equipment: Roche e601; method: electrochemiluminescence). Whole blood was centrifuged and the serum was separated.
Blood biochemical assay: Serum triglycerides, total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL) and fasting blood glucose were measured at baseline and at the end of eight weeks of intervention. 5 mL of whole blood was drawn from the elbow vein at 7:00 a.m. on a fasting basis, and the serum was separated by centrifugation within 2 h after specimen collection. Blood glucose, total cholesterol, HDL and LDL were measured (Equipment: Beckman AU5811).

Serum was analyzed for the function of liver, function of kidney, insulin and blood lipids using automatic hematology analyzer (Beckman AU5811). Biochemistry and hematological analyses were performed in ADICON clinical laboratories (Shanghai, China).