Salivette

Cortisol samples were collected using Sarstedt Salivettes (Sarstedt AG & Co, Nümbrecht, Germany). Samples were stored in a refrigerator shortly after collection and were centrifuged following the visit completion and stored at −80 F prior to processing by the Oregon Clinical Translational Research Institute Core Laboratory. Saliva samples from each subject at baseline, after stress battery, and during the post stress assessments were run in the same assay batch.
Values for cortisol levels were obtained using a commercially available ELISA kit (Salimetrics, State College, PA 16803).
Subjective stress was rated with a VAS ranging from 0 (no stress) to 100 (extremely stressed).
The positive and negative affect schedule (PANAS) [35 (link)] was used to assess positive and negative affect at the time of the assessment. Item for alertness was used as a proxy measure of alertness.

A sample of 70 ml of venous blood was collected in heparinized tubes (BD, Germany). To control for circadian rhythms and other potential confounds, samples were obtained between 8.00 a.m. and 9:30 a.m. after 12 h overnight fasting. Peripheral blood mononuclear cells (PBMCs) were immediately isolated and cryopreserved until assayed (see below for details).
Serum was collected in serum separator tubes (BD, Germany) and allowed to clot for 30 min at room temperature in the dark. Next, samples were centrifuged for 5 min, after which serum was aliquoted and stored at −20°C until analysis. Saliva samples were collected on 2 consecutive days at 8:00 a.m. and 10.00 p.m. using Sarstedt salivettes (Sarstedt, Germany) at home by the participants (within a week of their clinical visit) and shipped back to the lab in pre-stamped envelopes provided by the study team. All samples arrived within 7 days and were immediately processed and stored until assayed (see below for details).

Saliva samples were collected with Sarstedt salivettes (Sarstedt, Germany) in order to measure cortisol and alpha-amylase. Saliva was separated from the cotton swab by centrifugation and stored at -80°C until analyzed. Cortisol was determined with a competitive electrochemiluminescence immunoassay ECLIA using a Modular Analytics E602 immunoassay analyzer on the Roche Cobas 8000 from Roche Diagnostics (Mannheim, Germany). Determination of salivary alpha-amylase was performed using a colorimetric method with Ethylidene Protected Substrate (EPS) on the Roche Cobas 8000 C702 and C502 (Mannheim, Germany).

Saliva was collected using Salivettes^® (Sarstedt, Nümbrecht, Germany) or by direct placement in a plastic tube. Volunteers were required to not eat for at least half an hour prior to sampling and to rinse their mouths with water 10 min before taking the sample. Salivettes^®, after being placed in the oral cavity, were chewed for 2 min, then centrifuged at 8000 rpm for 5 min. The obtained samples were frozen and stored at −20 °C until analysis.

On each measurement day, eight saliva samples were collected using cotton rolls (Salivettes, Sarstedt, Sevelen, Switzerland). The subjects were asked to refrain from heavy physical exercise and drinking alcohol 48 h beforehand, as well as to avoid physical exertion and caffeine (e.g., coffee, coke, energy drinks, black tea) on the day of the study examination. Participants were also asked not to eat or drink, to chew gum and to use a toothbrush 2 h prior to study participation. Subjects were instructed to chew the cotton rolls for 1 min immediately after waking up, 30 and 45 min later, and at 8:00 am, 10:00 am, 12:00 pm, 4:00 pm, and 8:00 pm. The first sample was taken while still lying in bed. After the collection, the samples were stored in the participants’ own refrigerators prior to storage in the study refrigerator at −20°C. After completion of the data collection, biochemical analyses were conducted at the biochemical laboratory of the department of Clinical Psychology and Psychotherapy at the University of Zurich. Salivary cortisol levels were analyzed using an immunoassay with time-resolved fluorescence detection (Dressendörfer et al., 1992 (link)), while the activity of sAA was determined using a kinetic colorimetric test (see Boesch et al., 2014 (link)).