Qev original 70 nm column

A total of 60 ml of culture‐conditioned medium (CCM) from each cell line was centrifuged at 1000 × g for 5 min at 4°C to remove cells and cellular debris. 3 kDa molecular weight cut off (MWCO) Centricon Plus‐70 centrifugal filters (Millipore Sigma) were used to concentrate the initial volume to 1.5 ml. Size exclusion chromatography (SEC) was done with qEV Automated Fraction Collectors (AFC; Izon Science, Cambridge, MA) and qEV original 70 nm columns (Izon Science, Cambridge, MA). Columns were left at room temperature for 30 min and washed with phosphate‐buffered saline (PBS). 0.5 ml of concentrated CCM was loaded onto each of three columns, and 0.5 ml fractions were collected by adding additional PBS to the column. EV‐enriched fractions (SEC; fractions 7–9) were pooled altogether from the three columns used for each sample and further concentrated using 3 kDa MWCO Amicon Ultra‐15 Centrifugal Filters to a final volume of 1 ml. 50‐μl aliquots were stored at –20°C for downstream assays.

The medium of both M0- (non-induced control) and M1-like macrophages was replaced with fresh D˗MEM, supplemented with 10% v/v exosome-depleted FBS for 48 h, before culture-conditioned medium (CCM) collection. The collected CCM was previously centrifuged twice at 700 × g for 5 min and 2000 × g for 10 min at 4 °C and then filtered through a 0.22 μm filter unit, in order to remove debris and detached cells [26 ]. Later, the starting volume was concentrated by using Centricon Plus-70 centrifugal filters (Millipore Sigma) up to a final volume of 1 mL. The samples’ purification was performed through size exclusion chromatography (SEC) with qEV original 70 nm columns (Izon Science, Cambridge, MA). All the columns were rinsed with PBS, and then 1 mL of each sample was loaded onto each column, followed by fractions’ extraction. Finally, the recovered volume was concentrated by using 50 kDa MWCO Amicon Ultra-15 centrifugal filters up to a final volume of 1 mL [27 (link)]. When required, fluorescent EVs were labeled after isolation by 1 h of incubation at 37 °C with carboxyfluorescin diacetate succinimidyl ester (CFDA-SE). This latest, after passing the membranes, undergoes hydrolysis of the diacetic groups, thus coverting into the fluorescent state CFSE [28 (link), 29 (link)]. The fluorophore excess was removed by the use of centrifugal filters.

Either 500 ml supernatant from WJMSC culture or 1 ml blood plasma sample from aGvHD patients was spun down at 400 x g for 10 min to remove cell debris. Then, the supernatant was spun down at 2000 x g for 30 min to remove apoptotic bodies. This was followed by ultracentrifugation spins at 10,000 x g for 1.5 h and 100,000 x g centrifugation for 1.5 h, both at 4°C. The pellet was washed with PBS and spun 100,000 x g centrifugation for 1.5 h at 4°C. Finally, the pellet was resuspended in PBS. To enrich for small EVs, extracellular vesicle pellets were further passed through qEVoriginal/70 nm columns (Izon Science, USA) according to the protocol provided by the manufacturer. The size of the sEVs was measured by NanoSight LM10 system and data was analysed using the NTA software v2.3 (NanoSight Ltd; United Kingdom).

Blood plasma from the healthy controls was thawed at 4 °C and 500 µl platelet-poor plasma was transferred to a qEV original 70 nm column (Izon Science, Oxford, UK) for EV isolation by size exclusion chromatography (SEC). To ensure similar processing as the control samples, RA samples were thawed and centrifuged at 15,000g for 15 min at 4 °C prior to SEC. For all samples, the EVs were eluted in 500 µl filtered PBS per fraction and EV enriched fractions 7–9 were pooled, as according to the manufacturer’s recommendation. Freshly isolated EV aliquots were used for transmission electron microscopy (TEM), while EV aliquots used for ExoView analysis were stored at − 80 °C.

EVs were isolated by a size-exclusion chromatography (SEC) technique using a qEVoriginal-70 nm column (Izon Science, New Zealand). The procedure was performed according to the protocol provided by the company with some modification. In brief, sera were thawed at room temperature, then centrifuged at 10,000× g for 10 min. Before loading the samples, columns were flushed with at least 15 mL of 0.22 μm-filtered PBS. Centrifuged sera (500 μL each) were loaded onto the loading frit. Once all of the sera entered the column, filtered PBS (10 mL) was used to flush the columns. The flow-through fluid was collected from fractions 1 (F1) to 11 (F11), 500 μL per each fraction. Fractions F1 to F4 (2 mL) were voided, while F5 to F11 was stored at −80 °C before use in the following experiments as shown in Figure 2A. All of the isolation steps were performed in a cell culture hood.