Qevoriginal 70 nm

CCM was thawed and concentrated in preparation for EV isolation by size-exclusion chromatography followed by additional concentration of the EV sample. All three steps were performed according to the manufacturer’s instructions. Briefly, Centricon® Plus-70 centrifugal filter units (10,000 kDa cutoff, MilliporeSigma) were primed with 50 mL of 0.1 N sodium hydroxide, followed by 65 mL of 0.22 µm-filtered PBS. The retentates of the priming buffers were discarded. Then, 50 mL portions of CCM were concentrated to 500 µL, which was loaded into a SEC column (qEVoriginal, 70 nm, IZON Science) that had been primed with 13 mL of 0.22 µm-filtered PBS. SEC-output fractions of 500 µL each were collected. The fractions with highest abundance of EVs but with the least protein contaminants are fractions 7 to 10 according to the product manual and our previous findings [27 ]. For each replicate, the EV fractions (i.e., 7 to 10) of five SEC column isolations were combined and concentrated from 10 mL to 200 µL. Of this final EV-product for each replicate, 2 µL was used for counting and TEM. The remainder was used for RNA extraction (Fig. S1b).

EVs were isolated from 500 µL of the plasma using qEV Original/70 nm (Izon, Christchurch, New Zeland) size-exclusion chromatography (SEC) columns, according to the manufacturer’s instructions. The size and concentration of collected EVs were analyzed by Nanoparticle Tracking Analysis (NTA) (NanoSight LM10, Malvern Instruments Ltd., Malvern, UK). The EVs were checked by transmission electron microscopy (TEM), as previously described [20 (link)]. EV protein expression was analyzed by western blot, as previously described [20 (link)], using an anti-flotillin-1 (1:10,000 dilution, ab41927, Abcam), anti-PD-L1 (1:1000 dilution, 13684, Cell Signaling, Danvers, MA, USA) and anti-B7-H3 antibody (1:1250 dilution, MAB1027-100, R&D Systems Biotechne, Minneapolis, MN, USA). Anti-rabbit (ECL-antirabbit IgG, NA934V, Amersham, dilution: 1:2500) or anti-mouse (ECL-anti mouse IgG, NA931V, Amersham, 1:2000 dilution) secondary antibodies were used.

Bacterial cultures (500 ml) were centrifuged at room temperature (6,000 × g, 15 min) and the supernatants filtered through 0.22-µm top filters (Nalgene, Thermo Scientific). The supernatants were then concentrated 1,000-fold using 100-kDa ultrafiltration units (Amicon, Merck Millipore) by successive centrifugations (2,500 × g). The concentrated suspension of EVs was recovered in TBS buffer (Tris-buffered saline; 150 mM NaCl, 50 mM Tris-Cl, pH 7.5) and further purified by size exclusion chromatography (qEV original, 70 nm; iZON) as recommended by the manufacturer (30 (link), 77 (link)). The resulting fractions containing EVs in TBS buffer were pooled (1.5 ml) and further concentrated to achieve approximately 50 µl using 10-kDa centrifugal filter units (Amicon, Merck Millipore). The aliquots were stored at −20°C or used immediately.