Saliva samples were collected using Sarstedt Cortisol Salivette® cotton swab collection tubes. Participants were asked to chew on the swabs for 2 minutes to stimulate saliva flow. Samples were stored at −20°C until analysis. The free salivary cortisol was measured using a commercially available enzyme-linked immunosorbent assay kit (Demeditec Diagnostics, Kiel, Germany, DES6611). The inter- and intra-assay coefficients of variation were below 10 and 7%, respectively.
Salivette cortisol
Manufactured by Sarstedt
Sourced in Germany
The Salivette Cortisol is a device used for the collection of saliva samples for the measurement of cortisol levels. It provides a standardized and hygienic method for collecting saliva specimens.
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29 protocols using salivette cortisol
1
Salivary Cortisol Measurement Protocol
2
Salivary Cortisol Sampling and Analysis
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Saliva samples were collected with swabs (Cortisol Salivette®; Sarstedt, Nürmbrecht, Germany). Participants chew on the swabs for 30–60 s to stimulate saliva flow. Saliva samples were stored at −20 °C until the samples were thawed and centrifuged at 3000 rpm for 3 min. Salivary cortisol was analyzed using a commercial chemiluminescence immunoassay (CLIA; IBL, Hamburg, Germany) with intra-assay precision of 3.0% and inter-assay precision of 4.2%.
3
Salivary Cortisol Dynamics During Stress
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Saliva samples were taken at seven timepoints during the testing session (see Fig. 1 ) using Cortisol Salivettes® (Sarstedt, Nürnbrecht, Germany). We obtained a first baseline sample ca. 10 min after arrival and a second baseline sample after another 20-min adaptation phase, then, one directly before the stress or control protocol, in the short break in the middle of the stress /control protocol and directly after. Another saliva sample was taken after the post run of the cognitive task (about 15 min after cessation of stress/control protocol) and the last one 45 min after the stress/control protocol ended to capture the regeneration phase adequately. To collect saliva, participants put a synthetic fiber swab in the mouth for 1 min, allowing to absorb secreted saliva. Participants were instructed not to chew on the swab to avoid tactile stimulation which in turn is known to increase saliva flow rate. All samples were stored at − 20 °C until they were sent to the laboratory for analysis. For cortisol analysis, salivettes were thawed and centrifuged for 2 min at 1000 rpm to collect saliva. Further analysis was performed using an enzyme immunoassay (IBL International, Hamburg, Germany, Cortisol ELISA, REF RE52611) according to the manufacturer’s instructions. Average cortisol levels were taken from duplicate runs if intra-assay variation was below 10%.
4
Salivary Cortisol Measurement Protocol
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To evaluate stress objectively, we measured cortisol levels in participants' saliva. As cortisol levels fluctuate during the day, saliva samples were collected between 12:00 a.m. and 1:00 p.m. Saliva samples were collected using Cortisol Salivettes (SARSTEDT AG & CO.; Nümbrecht, Germany). Participants were asked to rinse their mouths with water for 10 minutes before taking the saliva sample. After rinsing, participants chewed a cotton roll for 45 to 60 seconds and placed it in the Salivette. The Salivettes were then hermetically sealed and sent on the same day to the test laboratory (Daacro GmbH & Co. KG; Trier, Germany) for evaluation. Cortisol levels were measured in nanomole/liter.
5
Salivary Cortisol Measurement Protocol
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Saliva was collected by swab (Salivette® Cortisol, Sarstedt AG & Co, Nümbrecht, Germany) at 1500 h on days 1 and 2 of the environmental challenge. The swabs then were centrifuged (1000× g at room temperature) for 2 min to obtain saliva, which was stored at −80 °C until analysis. Cortisol concentrations in saliva samples were measured by enzyme-linked immunosorbent assay (ELISA) using commercial kits as per the manufacturer’s instructions (Cat no. 1-3002-5, Salimetrics LLC, State College, PA, USA).
6
Humor, Stress, and Visual Perception
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Participants in the humor group were shown movie clips depicting either harmless but funny mishaps of humans and animals while participants in the control group watched neutral interactions of humans and animals. The clips were displayed on a 17.30-in. screen (HP ProBook 470 G5, Hewlett-Packard Development Company, Palo Alto, CA, USA) with a resolution of 1600 × 900 Px. Saliva samples were collected via Salivette Cortisol (SARSTEDT AG & Co., Nümbrecht, Germany) and used to determine individual cortisol levels as a marker for physiological stress. Pain tolerance and subsequently stress levels were induced using a Digitimer DS5 Isolated Bipolar Constant Current Simulator (https://www.digitimer.com ). The visual stimuli of the visual search task were presented with Matlab 2018b (https://www.Mathworks.com ) using Psychtoolbox 3 (http://psychtoolbox.org/ ). Visual stimuli appeared in purple on a grey background and were displayed on a 27-inch CRT monitor (resolution: 1920 × 1080 Px, refresh rate: 120 Hz).
7
Salivary Cortisol Measurement for Stress Assessment
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Participants provided saliva samples using synthetic sticks in plastic tubes (Salivette® Cortisol, Sarstedt, Nümbrecht, Germany), labeled to indicate day of study and time of assessment. Samples were stored during the study week in participants’ home freezer, afterwards at −31°C at Humboldt University Berlin, and subsequently shipped to Dresden LabService GmbH (Prof. Clemens Kirschbaum) for cortisol assaying; extremely low and high values were double-checked. The data were screened for compliance with the collection protocol (Hoppmann et al., 2018 (link)).
As an indicator of physiological stress, we calculated for each study day the area under the curve with respect to ground (AUCg), derived from the trapezoid formula using the discrete cortisol measurements and the time between measurements (Pruessner et al., 2003 (link)). We calculated AUCg for days on which the two first cortisol measurements (upon waking and 30 min later) and in total, at least 3 cortisol measurements per day were available. Higher AUCg scores can be interpreted as reflecting higher overall physiological stress levels (Hoppmann et al., 2018 (link)).
As an indicator of physiological stress, we calculated for each study day the area under the curve with respect to ground (AUCg), derived from the trapezoid formula using the discrete cortisol measurements and the time between measurements (Pruessner et al., 2003 (link)). We calculated AUCg for days on which the two first cortisol measurements (upon waking and 30 min later) and in total, at least 3 cortisol measurements per day were available. Higher AUCg scores can be interpreted as reflecting higher overall physiological stress levels (Hoppmann et al., 2018 (link)).
8
Salivary Cortisol and Blood Biomarkers during Gaming
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A saliva sample was taken 15 min before and 15 min after the gaming session to determine the cortisol level (Cort). For this purpose, a cotton roll was soaked with saliva in the mouth for 1–2 min (Salivette® Cortisol, SARSTEDT AG & Co., Nümbrecht, Germany). The saliva was then isolated by centrifugation and analyzed using an enzyme-linked immunosorbent assay. Pre-gaming, 5, 10, 20, and 30 min into the gaming phase, as well as 10 min post-gaming capillary blood samples were taken from an hyperemized earlobe. These were used to determine lactate and blood glucose concentrations (BIOSEN S-Line Lab+, EKF-diagnostic GmbH, Barleben, Germany).
9
Saliva Cortisol Measurement Protocol for Surgical Anxiety
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Saliva samples were obtained with commercially available collection devices (Salivette Cortisol, Sarstedt, Nümbrecht, Germany). Saliva was recovered by centrifugation (1000×g, 2 min, 4 °C) and was stored at − 20 °C until assayed. Salivary cortisol concentrations were measured using an enzyme-linked immunosorbent assay (Cortisol ELISA, IBL International, Hamburg, Germany) according to the manufacturer’s instructions. Cross-reactivity of the anti-cortisol antibody with other relevant steroids was 7.0% (11-deoxycortisol), 4.2% (cortisone), 1.4% (corticosterone), 0.35% (progesterone), and < 0.01% (testosterone, estrone, estradiol, estriol). Intra- and interassay variances were 4.8% and 5.9%, respectively. For comparison of cortisol data from pre- and postoperative days with the day of surgery, salivary cortisol levels of the two collection times (11 am and 1 pm) on preoperative day 3 (T1), on preoperative day 2 (T2), and on postoperative day 2 (T6) were averaged. By measuring the cortisol levels 2 days after surgery, we intended to circumvent that elevated levels of cortisol induced by immediate postoperative pain might have confounded evaluation of anxiety.
10
Salivary Cortisol Measurement Protocol
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The saliva samples of each participant were collected using a salivette cortisol (Sarstedt, Nümbrecht, Germany). All saliva samples were taken at the same hour of the day to avoid any variations due to circadian rhythm. Participants were instructed to not eat, drink, or brush their teeth for 30 min before saliva collection. The cotton sliver of the salivette was taken out and put in the sublingual for 1/2 min, and then put back into the salivette. The samples were centrifuged at 2000 rpm for 2 min. Saliva flow was collected from the salivette’s bottom and stored at −20 °C for further laboratory analysis.
Salivary cortisol was detected using a specific commercial enzyme-linked immunosorbent assay kit (item no. 500360 Cayman chem, Ann Arbor, MI, USA). Saliva samples (50 µL) were used, and the assay was performed according to manufacturer-recommended procedures. The sensitivity provided by the manufacturer is approximately 35 pg/mL, with a detection range from 6.6–4000 pg/mL. The samples were analyzed in duplicate [25 ].
Salivary cortisol was detected using a specific commercial enzyme-linked immunosorbent assay kit (item no. 500360 Cayman chem, Ann Arbor, MI, USA). Saliva samples (50 µL) were used, and the assay was performed according to manufacturer-recommended procedures. The sensitivity provided by the manufacturer is approximately 35 pg/mL, with a detection range from 6.6–4000 pg/mL. The samples were analyzed in duplicate [25 ].
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