Heraeus megafuge 16r

Colitis was induced in rats as described earlier and the rats were euthanized on day 8. Blood samples were collected in clot activator tubes (Add Surgifield Medicals, Middlessex, England) and centrifuged (Heraeus Megafuge 16R, Thermo Scientific) at 3000 rpm for 30 min at 4°C to obtain the serum. The serum was assayed quantitatively for TNF-α and IL-6 by using their respective Picokine ELISA kits as instructed by the manufacturer.

Saliva collection was performed by using sterile kits containing SalivaBio oral swabs and sampling tubes (Salimetrics, Carlsbad, CA, USA). After collection, saliva samples were immediately processed by centrifugation at 4 °C (pre-chilled centrifuge) and 1500× g for 15 min (Heraeus Megafuge 16R, Thermofisher Scientific, Waltham, MA, USA). From the total sample volume, one aliquot of 300 µL was used for the analysis of oxytocin, and two additional aliquots of 200 μL for cortisol and DHEA. All aliquots were handled on ice and immediately stored at −80 °C until further analyses. Oxytocin and cortisol measurements were prioritized when insufficient amounts of saliva were available (which was the case for n = 3 participants). The final sample size with valid DHEA measurement was n = 25 for t₀ and n = 23 for t₁. To avoid an artificial reduction in the available dataset for statistical analyses, all available biological measures were included in the analyses.

Plasma isolated from EDTA blood (25 mL) was centrifuged (Thermoscientific Heraeus Megafuge 16R) with 4800× g for 30 min at 4 °C to remove cell debris. Then plasma was diluted 1:1 in ice-cold PBS (Life Technologies) and filtered through a 0.22 µm sterile filter (TPP) and ultracentrifuged (Sorvall discovery M120) at 150,000× g for 8 h. After one washing step in PBS and another ultracentrifugation for 4 h the pellet containing exosomes were resuspended in PBS and protein content was evaluated by the BCA-based protein assay. Exosomes were isolated from cell culture supernatants of 60–70% confluent tumor cell lines, as described above. The amount of Hsp70 derived from exosomes of the FBS in fresh medium was subtracted. Size and uniformity of the exosomes were characterized by dynamic light scattering on a Zetasizer NanoS instrument (Malvern Instruments, Malvern, UK) and by their protein content was determined by Western blot analysis using antibodies directed against β-actin (A228; Sigma-Aldrich), Hsp70 and Grp75 (SPS-825; StressMarq Biosciences Inc.).

Optical density was measured for cell growth determination at 600 nm by a photometer (UV1700 Pharmaspec Shimadzu UV-VIS Spectrophotometer, Shimadzu Europa GmbH, Duisburg, Germany). Furthermore, cell dry weight (CDW) was determined by drying of double washed cell pellets obtained from 1 mL culture broth. Samples were centrifuged at 4500 rpm for 6 min by a centrifuge (Thermo Scientific Heraeus Megafuge 16R, Thermo Fisher Scientific Inc., Waltham, MA, USA), and dried at 60 °C until reaching constant weight.