Extracellular Vesicles Extracted from Cultured Cells (DELFIA data)

Cells and Cell Culture Human epithelial cells (EPI), human hepatocellular cancer cells (Hep-G2) and human embryotic kidney cells (HEK-293) were purchased from ATCC, Teddington, UK (PCS-300-010, HB-8065 and CRL-1573.3, respectively). The cells were cultured in T175 cell culture treated flasks with filter caps (Thermo Fisher Scientific, Loughborough, UK) in the F-12 Dulbecco's modified eagle medium/nutrient mixture (DMEM-F12, Thermo Fisher Scientific Inc., Santa Clara, CA, USA), supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S), and 0.01% amphotericin B. The cultures were maintained in a humidified incubator at 38.8 °C with 5% CO2 until they reached 70-85% confluence, upon which the medium was replaced with serum-depleted DMEM-F12 to deplete the cell culture media from vesicles present in serum. EV Isolation and Characterisation The conditioned media of the three cell lines (EPI, Hep-G2 and HEK-293) were subjected to differential centrifugation. The samples were centrifuged at 300×g for 10 min to remove the potential presence of whole cells, then the supernatant was centrifuged at 2,000×g for 10 min, followed by a centrifugation step at 20,000×g for 30 min, to eliminate cell debris and apoptotic bodies, respectively. The resulting supernatant was concentrated to a volume of 500 µL using 10 kDa Amicon® ultra centrifugal filter units (Merck Millipore Ltd., Tullagreen, Carrigtwohill, Ireland). EVs were then isolated using size exclusion chromatography (SEC) (qEVoriginal/35 nm, Izon Science Limited, Christchurch, New Zealand). Before EV isolation, the column was pre-washed with 17 mL of filtered Dulbecco’s phosphate-buffered saline (PBS, Sigma Life Science, Dorset, UK) using 0.2 µm Minisart™ syringe (Thermo Fisher Scientific, Loughborough, UK). Subsequently, 500 µL of the concentrated conditioned media sample was introduced through the top of the column. Following sample passage through the column's top filter, PBS was immediately added to the top, and EV fractions 7 to 10, each of 500 µL, were collected as per manufacturer's protocol. EV fractions were then pooled for subsequent characterisation using a ZetaView® nanoparticle tracking analyser (NTA, PMX 120, Particle Metrix GmbH, Inning am Ammersee, Germany), dissociation-enhanced lanthanide fluorescence immunoassay assays (DELFIA; Wallac Oy, Turku, Finland), and scanning electron microscopy (SEM) (Thermo FEI Quanta 200F SEM, Thermo Fisher Scientific, Waltham, MA, USA). DELFIA EV proteins were quantified using DELFIA with slight modifications [10]. Briefly, 20-30 μL of each sample was diluted to 100 μL in PBS, added to high-binding ELISA plates, followed by incubation overnight at 4 °C. The plates were washed three times with DELFIA wash buffer (Revvity, Cambridge, UK). Wells were blocked with 100 μL of 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature. The wells were then washed three times with DELFIA wash buffer. Primary antibodies (CD63 cat. 556019, CD9 cat. 555370 and CD81 cat. 555675, BD Biosciences, USA) were added at 1 μg/mL, and the plates were incubated for 2 h at room temperature. After washing, goat anti-rabbit IgG or goat anti-mouse IgG1 was added (1:2000 in blocking buffer), and the samples were incubated for 1 h at room temperature. The plates were then washed three times with DELFIA wash buffer and incubated for 1 h with 1:1000 streptavidin–europium conjugate in DELFIA assay buffer. After six washes with DELFIA wash buffer, 100 µL of DELFIA enhancement solution was added and the plate was shaken for 2×5 min on the plate reader. Time-resolved fluorimetry was performed using a Pherastar plate reader (BMG Labtech, Ortenberg, Germany), with excitation set at 337 nm, detection set at 620 nm, integration time set at 200 µs, and lag time set at 60 µs.