Cellstar tube

Biofilms were removed from the inner surface of bath toys using an electric toothbrush (Oral-B^®, Advanced Power) as follows: one half of each bath toy was put into a sterile beaker and submerged in 100–150 mL ultrapure water. The biofilm was then removed by brushing the bath toys’ surface for approximately 2 min and this suspension was collected in 50 mL tubes (CellStar^® Tube, Greiner Bio-One). The whole procedure was repeated once with fresh ultrapure water to make sure all biofilm was removed. Biofilm clumps and clusters were subsequently dispersed with a sonication needle (Sonopuls HD 2200, Bandelin Sonorex, Rangendinen, Germany) for 30 s at 50% power and 40% intensity. Thereafter, the biofilm suspensions of one bath toy were combined in a sterile SCHOTT^® bottle and the volume was filled up with ultrapure water to a total volume of 500 mL. The toothbrush heads were replaced for each sample to avoid cross contamination.

Before quantification of cortisol in hairs, the samples had to be processed extensively first. In short, approximately 250 mg of hair was placed in a 50 mL Cellstar tube (Greiner Bio-one, Kremsmünster, Austria) covered with aluminium foil for protection against UV-light. After washing with 10 mL 2-propanol (1.09h634, Emsure®, Merck, Kenilworth, NY, USA) the hairs were dried at 37 °C for four days. Next, approximately 35 mg of clean hair was weighed off and placed in a microtube (clickcap, 2.0 mL Trefflab, Nolato Treff AG, Degersheim, Switserland). Three beads (3.2 mm, Cat. No 11079132 BioSpec Products, Bartlesville, OK, USA) were added to each tube. The hairs were now ground with a Tissuelyser II (Cat. No 85300, Qiagen, Hilden, Germany). For cortisol extraction [43 (link)] the hair samples were incubated with slow rotation for 24 h with 1 mL of methanol (1.06009.2500, Emsure). After centrifuging of the samples, 600 μL of the supernatant were transferred to a new tube. The extracts were dried in a Speedvac (Automatic Environmental Speedvac, AES1000, Savant Instruments, Woonsocket, RI, USA) with medium temperature for 2.5 to 3 h. The precipitate was then dissolved in 100 μL of phosphate buffer (assay diluent provided in the assay kit) [36 (link)] for 24 h on a shaker set at 300 rpm (KS250, Janke & Kunkel, Breisgau, Germany).

Oral fluid samples were obtained from healthy volunteers and patients with OSCC with their informed consent. Ethical approval was obtained from the Tokyo Dental College of Ethics Committee (approval number: 1-16-46R, 702) and the Japanese Foundation for Cancer Research (approval number: JFCR 2013-1112). This study was conducted according to the principles of the Declaration of Helsinki, and the informed consent was obtained from all participants. Donors were negative for a history of HIV, autoimmune disorders, hepatitis, and malignancy. The donors were asked to avoid eating, drinking, smoking, or oral hygiene procedures for at least 1 h before sampling. At 9 AM, the donors were directed to expectorate 5 mL of unstimulated OF into a 50 mL Cellstar tube (227270, Greiner Bio-One, Kremsmünster, Austria). The sample was placed on ice upon collection and then immediately stored at − 80 °C until analysis in subsequent experiments. Basic information, including age, sex, and tumor location, for all patients and the healthy controls is summarized in Table S1.