Swab storage tube

To collect saliva samples, a Salimetrics Children’s Swab (Salimetrics State College, PA) was cut in half to create two ~ 2″ long swabs. Swabs were held on the inside bottom of the cheek pocket for one minute, one swab on the left side and one on the right side. A treat was held in front of the dog to encourage salivation. The swabs from each dog were then placed in a 2 ml Swab Storage Tube (Salimetrics, State College, PA) and placed on ice until all saliva samples were collected. Saliva was released from the swabs by centrifugation for 20 min at 3000 rpm and 10 °C. Saliva was pipetted from each sample into a 2 ml low retention tube (Sigma-Aldrich Corp., St. Louis, MO). The entire saliva sample was diluted 1:4 with sterile ddiH₂O. These were further diluted 1:100 and 1:250 with sterile ddiH₂O and refrigerated at 4 °C until the next day for analysis.
Total protein in saliva samples was measured using the Bio-Rad protein assay (Bio-Rad, Hercules, CA) according to the manufacturer’s directions. Samples were assayed in triplicate. The Canis familiaris allergen, Can f 1, was determined using an ELISA kit (Indoor Biotechnologies, Charlottesville, VA), according to the manufacturer’s directions, and 100 μl of the saliva sample (either diluted 1:100 or 1:250, depending on the concentration of total protein in the saliva sample). These samples were assayed in duplicates.

During one month, individual saliva samples were collected using an infant swab (Salimetrics, USA) immediately before each experimental session. Based on the literature47 (link)48 (link), we trained the monkeys to chew the swab for about 80 s and then spit it out into a sterile kidney dish. Immediately after collection, the swab was transferred into a swab storage tube (Salimetrics, USA) and centrifuged for 15 min at 3500 rpm. The extracted saliva was then stored at −80 °C until assayed. Samples were analyzed in duplicate using an enzyme immuno-assay for salivary cortisol following manufacturer’s instructions (Salimetrics, USA). The cortisol data were standardized [X _standardized = (X-mean)/standard deviation] in order to pool data from both animals.

No biological samples were from patients or subjects other than the involved authors in this study, and saliva samples were obtained from the two male co-authors. There were no ethical concerns involved in this study. The biological samples were collected in three days consecutively (6 samples/person). They were required to rinse their mouths with water 30 min before each harvest. The SalivaBio Oral Swab (Salimetrics, LLC., CA, USA), a synthetic swab specifically designed to improve volume collection, was then placed under the tongue, and held for 1 min 30 s, followed by preservation in a swab storage tube (Salimetrics, LLC., CA, USA) at −20°C. Each tube collected around 2 mL saliva samples. The tools are shown in Figure S8. Alcohol consumption was prohibited for the duration of the saliva collection. These saliva samples were used only for ELISA and practicability of our microfluidic system, where biological information is kept confidential. Therefore, the study also did not involve any research on sex and gender, ancestry, race or ethnicity.