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 .
Econo pac columns
Manufactured by Merck Group
Sourced in United States
Econo-Pac columns are chromatography columns designed for efficient and convenient separation and purification of biomolecules. They provide a simple and reliable solution for routine preparative and analytical scale chromatography.
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6 protocols using econo pac columns
1
Isolation and Characterization of Milk Extracellular Vesicles
2
Isolation of Extracellular Vesicles by SEC
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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)).
3
Plasma Exosome Isolation by Mini-SEC
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Frozen plasma specimens were thawed and centrifuged at 2,000×g for 10 min at 4°C and then at 10,000–14,000×g for 30 min at 4°C. Clarified plasma was passed through 0.22 µm-pore Millipore filter and used for exosome isolation by SEC performed using 1.5 cm×12 cm mini-columns (Bio-Rad, Hercules, CA, USA; Econo-Pac columns) packed with Sepharose 2B (Sigma-Aldrich, St. Louis, MO, USA). The column bed volume is 10 mL. Prior to applying clarified plasma, the column is washed with 20 mL of phosphate-buffered saline (PBS), and a porous frit is placed at the top of the gel to prevent its disturbance during subsequent elution with PBS. Clarified plasma (0.5–1.0 mL) was loaded onto the column and five 1 mL fractions corresponding to the void volume peak were collected. Fractions # 3, #4 and #5 were tested for protein content, morphology by transmission electron microscopy (TEM) and in functional assays. In preparation for western blots, the fractions were concentrated using 300,000 MWCO VivaSpin 500 Centrifugal Concentrators (Sartorius Corp, New York, NY, USA) by centrifugation at 5,000×g for 2–15 min, depending on the content. Supplementary Fig. 1 shows the schema for isolation of plasma exosomes by the mini-SEC method.
4
Exosome Isolation from AML Plasma
5
Exosome Isolation by Size-Exclusion Chromatography
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After centrifuged at 500g for 5 min of blood, serum placed in − 80 °C. Serum specimens were thawed and centrifuged at 2000g for 10 min at 4 °C and then at 12,000g for 30 min at 4 °C.
Clarified serum was passed through 0.22 μm-pore Millipore filter and used for exosome isolation by SEC performed using 1.5 ~ 12 cm mini columns (Bio-Rad, Hercules, CA, USA; Econo-Pac columns) packed with Sepharose 2B (Sigma-Aldrich, St. Louis, MO, USA). The column bed volume is 10 ml. Prior to applying clarified serum, the column was washed with phosphate-buffered saline (PBS) 20 ml, and a porous frit was placed at the top of the gel to prevent its disturbance during subsequent elution with PBS. Clarified serum 1.0 ml was loaded onto the column and five 1 ml fractions corresponding to the void volume peak were collected. Fractions No. 3, 4 and 5 were tested for protein measurements, western blot, proteomic analysis, morphology by transmission electron microscopy (TEM) and proteomic analysis [12 (link)].
Clarified serum was passed through 0.22 μm-pore Millipore filter and used for exosome isolation by SEC performed using 1.5 ~ 12 cm mini columns (Bio-Rad, Hercules, CA, USA; Econo-Pac columns) packed with Sepharose 2B (Sigma-Aldrich, St. Louis, MO, USA). The column bed volume is 10 ml. Prior to applying clarified serum, the column was washed with phosphate-buffered saline (PBS) 20 ml, and a porous frit was placed at the top of the gel to prevent its disturbance during subsequent elution with PBS. Clarified serum 1.0 ml was loaded onto the column and five 1 ml fractions corresponding to the void volume peak were collected. Fractions No. 3, 4 and 5 were tested for protein measurements, western blot, proteomic analysis, morphology by transmission electron microscopy (TEM) and proteomic analysis [12 (link)].
6
Exosome Isolation from Plasma
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Plasma samples were pre-clarified by centrifugation first at 2000 g for 10 min at 4º C and then at 10,000g for 30 min. Filtration through 0.22 µm Millipore filter was then performed, and a 1 mL aliquot of clarified plasma was used for exosome isolation by mini-Size-Exclusion Chromatography (SEC) as previously described [10 ]. Briefly, 1.5 cm×12 cm mini-columns (Bio-Rad, Hercules, CA, USA; Econo-Pac columns) were packed with Sepharose 2B (Sigma-Aldrich, St. Louis, MO, USA). A porous frit was placed on top of the gel to minimize disturbance during exosome isolation. Each column was washed with 20 mL of PBS and 1.0 mL of clarified plasma was loaded onto the column and eluted with PBS. One mL fractions were collected, with the bulk of exosomes eluting in the fraction #4. The EM morphology, the vesicle size range, numbers of nanoparticles in the fraction #4 and their molecular cargos were evaluated as previously described [10 ].
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