Mini columns

Extracellular vesicles from milk were isolated by a combination of ultracentrifugation and size exclusion chromatography [24 (link)–26 (link)]. Briefly, 50 μl of fat separated milk was centrifuged at 2,000×g for 10 min at 4°C and then at 10,000×g for 30 min at 4°C. The supernatant was then ultracentrifuged at 100,000×g for 3 hours (TL-100 benchtop ultracentrifuge, Beckman-Coulter). The obtained crude EV pellet was washed in PBS once at 100,000×g for 3 hours. The washed pellet was resuspended in 1 ml of PBS and EVs were purified by mini-size exclusion chromatography (mini-SEC) using 1.5 cm x 12 cm mini-columns (Bio-Rad, Hercules, CA, USA; Econo-Pac columns) packed with 10 mL of Sepharose 2B (Millipore-Sigma, St. Louis, MO). Crude EVs (1.0 ml) obtained from ultracentrifugation were loaded onto the column and five 1 ml fractions corresponding to the void volume peak were collected in PBS. Fraction four was collected and used for subsequent experiments as the ‘EV’ fraction. Given that SEC is a size-dependent assay, we anticipate that the EVs obtained using this approach contain a heterogeneous mixture of exomeres, exosomes, and microvesicles in the size range of 30–200 nm [27 (link)]. The EVs were characterized by nanoparticle tracking analysis (NTA) and Western blotting as per MISEV2018 guidelines [28 (link)]. Antibodies used for Western blot experiments are described in Table 1.

The cells were cultured in three 15 cm culture dishes to ~ 80% confluency for each cell line in triplicates. Then they were harvested by a cell scraper, centrifuged (1000 g, 4 °C, 5 min), washed with PBS, and the cell pellets were frozen at − 80 °C. The frozen cell pellet was resuspended with HNN-lysis buffer (0.5% NP40, 200 mM Na₃VO₄, 1 mM PMSF, 1.2 μM avidin, Complete protease inhibitors without EDTA (Roche, Switzerland)) and subsequently pipetted to dissolve and avoid foaming. The suspension was incubated on ice for 10 min, then transferred to a 2 ml microtube and centrifuged at 14,000 g for 20 min at 4 °C. 250 μl of lysis buffer was added to Bio-Rad Spin Column (Bio-Rad, cat. No 732-6008) to avoid the formation of air bubbles. 100 μl of High Capacity Streptavidin Agarose Resin (Thermo Fisher Scientific, cat. No 20359) were mixed in 750 μl of HNN-lysis buffer, and 200 μl of the prepared beads were added to samples which were then incubated for 15 min at 4 °C on a rotary wheel. The beads were then washed twice with 1 ml of HNN-lysis buffer using Bio-Rad Mini Columns. After washing with lysis buffer, samples were washed three times with 1 ml of HNN buffer (50 mM HEPES, 150 mM NaCl, 50 mM NaF), which contained no detergent and inhibitors. Finally, samples were eluted with 200 μl of 2.5 mM Biotin in HNN buffer three times.

EV-depleted medium was obtained by centrifuging complete medium (supplemented with 10% FBS) at 100,000g for 12 hours and was used for all EV collection experiments. EVs from conditioned media were isolated by SEC (61 (link)–63 (link)). Specifically, 1 ml of J774A.1 conditioned medium was centrifuged at 2000g for 10 min at 4°C and then at 10,000g for 30 min at 4°C. The supernatant was passed through a 0.22-μm pore Millipore filter, and EVs were isolated by mini-SEC using 1.5 cm–by–12 cm mini columns (Bio-Rad, Hercules, CA, USA; Econo-Pac columns) packed with 10 ml of Sepharose 2B (MilliporeSigma, St. Louis, MO) equilibrated with PBS. Conditioned medium (1.0 ml) was loaded onto the column, and five 1-ml fractions corresponding to the void volume peak were collected. Fraction 4 was collected and used for subsequent experiments as the “EV” fraction. Given that SEC is a size-dependent assay, we anticipate that the EVs obtained using this approach contain a heterogeneous mixture, including exomeres, EVs, and microvesicles, in size range of 30 to 200 nm (99 (link)).