Parafilm

The isolated products from each experiment were processed in the same way. Paraformaldehyde was first added to the sample with a final concentration of 4% (w/v) and then incubated for 20 min at room temperature. A 100-μl droplet of isolated exosome sample was then placed on a sheet of Parafilm (VWR, USA). A 300-mesh copper grid support film (Electron Microscopy Sciences, USA) was placed on the droplet with membrane side down to allow the sample absorption for 20 min. Then, this grid was transferred to a 100-μl droplet of distilled water for 2 min. This process was repeated three times. This grid was then transferred to a 100-μl droplet of uranyl-acetate solution for negative staining for 8 min. Last, the grid was rewashed using distilled water and left to dry at room temperature. The sample was finally observed under TEM (FEI Tecnai G2 Twin, FEI Company, USA).

The NSS and SS from the 10 volunteers (five women and five men) between 25 and 67 years of age were collected in the morning between 9 and 11 a.m. and for 5 min (Figure 1). The NSS was naturally collected in 15 ml centrifuge tubes (VWR, PA, USA), while for the SS, the volunteers chewed a piece of Parafilm™ during the collection (30 (link)). After this, the saliva samples were centrifuged at 15,000 g and 4°C during 15 min (31 (link)). The supernatants from the NSS and SS were pooled into two different tubes. The pH was determined in both the saliva mixtures SS (pH = 7.7) and NSS (pH = 7.6). Then, aliquots of 1.5 ml of saliva were prepared in Eppendorf tubes (VWR, PA, USA) and were frozen (−80°C) until the experiments. This storage technique has been previously proven not to affect the saliva EA (32 (link)). Figure 1 shows a schematic summary of the experimental procedure.
The volunteers did not report oral diseases. The donors were not allowed to eat or drink anything 1 h before the saliva collection. The participants were informed about the purpose of the study, and they gave their written consent to participate. These experiments were authorized by the Bioethical Committee of the Spanish National Council of Research (CSIC), Spain.

Chicken (Gallus gallus domesticus) eggs (Winter Egg Farm, Cambridge, UK) were incubated in a digital cabinet incubator (OVA Easy 380, Brinsea) until Hamburger Hamilton developmental stage 24 (HH24). A small window was made and 500-1000 epicardial cells were administered into the extra-embryonic vessels. HESCs were fully differentiated to epicardial cells before administration into the chicken embryos. H9 (GFP)-derived epicardial cells are referred to as GFP⁺ and FRSC-derived epicardial cells as mStrawb⁺. The window was covered with parafilm (VWR) and eggs were placed horizontally in the incubator until HH34.

The chemical stability studies of amitriptyline in neat solvents and in the Franz cell receptor medium were conducted for 96 h. A known quantity of amitriptyline was dissolved in the receptor medium or neat solvent. These solutions were placed in Eppendorf® tubes and sealed with Parafilm® before being placed in a shaker (VWR, UK) at 32 ± 1 °C. Samples were taken at 0, 24, 48, 72, and 96 h. The samples were diluted with methanol where necessary and analysed using HPLC or LC-MS.