Cotton swab

HPA-axis functioning of each patient was assessed by analyzing cortisol levels in saliva throughout the day at measurement moments 0, 16 (at the end of the intervention), and 52 weeks after randomization. Saliva was collected at home, 5 days' post-planned sessions; upon awakening timepoint 1 (T1), 30 (T2), 45 (T3), and 60 (T4) min after awakening in saliva tubes with cotton swabs (Sarstedt, Germany). A fifth sample was collected at 2,200 h (T5). Moreover, 0.5 mg dexamethasone was ingested before sleeping, and a sixth sample of saliva was collected the next morning upon awakening (T6). A DST was done to assess the negative feedback mechanisms of the HPA axis. The participants were instructed not to smoke, eat, drink, or brush teeth within 15 min before saliva collection. Moreover, participants were instructed to report other potential CAR-interfering factors (e.g., sleeplessness before the sampling day, having the flue or flu-like symptoms) on the provided forms. Samples with reported potential interfering factors were excluded for further analyses. Samples were stored at refrigerators, and participants were instructed to return the collected samples by mail upon collection of T6. Upon arrival, the collected saliva was stored at −20°C.

To examine sAA and salivary cortisol stress responses, saliva samples were collected using cotton swabs (Sarstedt Ltd., Germany). Participants were instructed to gently chew on the swab for 1∼2 min. We ensured that baseline cortisol and sAA levels were stable by collecting the first salivary sample 10 min after the participants arrived at the laboratory. Salivary samples were taken three times during the course of the experiment: at baseline (i.e., 10 min after the participant’s arrival), 10 min after stress or control manipulation (when cortisol was expected to rise in the stress condition), and after anger regulation (approximately 20 min after stress or control manipulation). All samples were immediately stored in a sterile tube and kept at -20°C until analysis. Salivary cortisol and sAA were assayed using ELISA kits (Cortisol Parameter Assay Kit, R&D Systems, Inc., United States and Canada; Human Amylase ELISA Kit, Assaypro LLC., St. Charles, MO, United States).

The participants were trained to collect sufficiently large (0.5 mL) and clean saliva samples during the training session. They received written and verbal instructions according to the guidelines published by Pandi-Perumal et al. (2007) (link). During the experimental sessions, samples were collected 45 min and immediately before the manipulation (nap vs. video), immediately after and 45 min after (corresponding to approximately to 2:30, 3:15, 4:45, and 5:30 p.m.). All saliva samples were collected under supervision using cotton swabs (Salivette, Sarstedt, Nümbrecht, Germany), labeled with a code, and then stored at -20°C until the end of data collection. Saliva samples were analyzed using a commercially available competitive, enzyme-linked immunosorbent assay kit (Melatonin direct Saliva ELISA) by the laboratory of the manufacturer (IBL International, Hamburg, Germany). The process has an analytic sensitivity of 0.3 pg/mL, a functional sensitivity of 1 pg/mL, an intra-assay coefficient of variation of 6.1%, and an interassay coefficient of variation of 7.6% in the range of the expected values.