Qev size exclusion chromatography column

Exosomes were fluorescently labeled using Vybrant^® DiD (Life Technologies) according to the manufacturer’s instructions with modifications (11 (link)). Briefly, exosomes were incubated for 10 min with DiD (1:1,000 dilution in PBS) at room temperature and re-purified using qEV size exclusion chromatography columns (Izon Science Ltd.).

The culture supernatants of EO771 cells at approximately 60–70% confluence were harvested after 16 h conditioning in serum-free media (11 (link)). Exosomes were isolated as previously described (6 (link)). Briefly, cells and debris were cleared from the supernatant by centrifugation (500 g, 10 min), followed by filtration using 0.22 μm filters (Merck Millipore). Cell-free supernatants were concentrated by ultrafiltration through Centricon Plus-70 Centrifugal Filter (100 kDa; Merck Millipore), spun at 3,500 g at 4°C. Exosomes were subsequently purified by overlaying concentrated samples on qEV size exclusion chromatography columns (Izon Science Ltd.) followed by elution with PBS. Finally, the elute from qEV columns were concentrated using Amicon Ultra-4 10-kDa nominal molecular weight centrifugal filter units (Merck Millipore) to a final volume of approximately 200 µL.

Leishmania late-log phase promastigotes were washed three times with sterile PBS and resuspended in M199 medium without FBS. Approximately 10⁸ promastigotes were placed in each microtube and incubated for 4 h at 37° or 26°C for L. major^GFP+ or L. tarentolae^GFP+, respectively, to release EVs in the culture medium. Parasite viability was measured by Resazurin before and after incubation for 4 h. At the end of 4 h of incubation, the samples were centrifuged at 1,800 g, and the supernatants were filtered through 0.45-µm sterile syringe filters and subjected to serial centrifugation at 4°C as follows: 500 g for 10 min, 1,500 g for 10 min, and 10,000 g for 10 min. Then, the supernatant of the last step of serial centrifugation was concentrated using Amicon Ultra-15-3K and passed through a 0.5-ml qEV size-exclusion chromatography column (Izon Science, Christchurch, New Zealand) according to the manufacturer’s protocol. The final products (purified EVs) derived from L. major^GFP+ and L. tarentolae^GFP+ are herein referred to as mEV and tEV, respectively (Supplementary Figure S1).

EVs were isolated from stimulated whole saliva using size-exclusion chromatography, as described previously [44 ]. In brief, the saliva samples were centrifuged at 300 rpm for 10 minutes to remove debris, and then diluted 1:2 with 0.1 μm filtered PBS. A qEV size-exclusion chromatography column (iZON Science, Oxford, UK) was equilibrated by washing the column with 15 ml of 0.1 μm filtered PBS; 1 ml of the diluted saliva was then applied to the column and 16 fractions, each 500 μl in volume, were collected by continuously adding 0.1 μm filtered PBS to the column. To standardise the procedure, elution time frames were recorded when reaching fractions 7, 12 and 15, and the number of eluted drops in fraction 10 was also recorded. A new column was used for each saliva sample. The eluted fractions 8 − 10 (containing the majority of microvesicles and exosomes present in the samples) were concentrated for 80 minutes at 30 °C in a MiVac centrifugal vacuum concentrator (SP Scientific, Suffolk, UK) from a volume of 500 μl to approximately 250 μl. Fractions 8–10 were collected into a joint fraction and the protein concentration was determined using Qubit Fluorometric Quantitation (ThermoFisher Scientific, Oslo, Norway). A volume of the diluted stimulated whole saliva (100 μl) and the joint fractions from each participant were then sent for proteomic analysis while preserved on dry ice.