There are various methods for isolation of exosomes, including ultracentrifugation, precipitation-based methods, and immunobeads. Here, we describe a method (Total exosome isolation reagent from cell culture media, ThermoFisher) that only requires general laboratory equipment and hence is broadly applicable to most laboratories.
Total exosome isolation reagent from cell culture media
Manufactured by Thermo Fisher Scientific
The Total exosome isolation reagent from Thermo Fisher Scientific is a product designed to isolate extracellular vesicles, including exosomes, from cell culture media. It is a reagent-based method for the separation and concentration of these vesicles.
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Lab products found in correlation
Exosome spin column, by Thermo Fisher Scientific (2 mentions)
Exosome depleted fbs, by System Biosciences (2 mentions)
Syto rnaselect green fluorescent cell stain, by Thermo Fisher Scientific (1 mentions)
Fv3000, by Olympus (1 mentions)
Exosome depleted fcs, by Thermo Fisher Scientific (1 mentions)
Total exosome isolation reagent from serum, by Thermo Fisher Scientific (1 mentions)
Acriflavine, by Merck Group (1 mentions)
Nanoparticle tracking analysis, by Malvern Panalytical (1 mentions)
Pcdna3 myc3 nrf2, by Addgene (1 mentions)
Mission lightswitch luciferase assay reagent, by Merck Group (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Rpmi 1640 medium, by Sartorius (1 mentions)
9 protocols using total exosome isolation reagent from cell culture media
1
Exosome Isolation Using Reagent
2
Exosome Isolation and Characterization
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Total exosome isolation reagent from cell culture media (Thermo Fisher Scientific) was used to isolate exosomes following the manufacture's protocol. After that, larger vesicles and cell debris were removed again by passing the samples through the 220 nm filter.
Membrane permeable SYTO™ RNASelect™ green fluorescent cell stain (Thermofisher) was used to fluorescently label the RNA present inside the exosomes, as described by the manufacturer. Briefly, isolated exosomes were incubated at 37 °C for 20min with the stain at a final concentration of 40 μM, and then the remaining free dye was removed by exosome spin columns (MWCO 3 000, Invitrogen). As controls, culture medium without cells was subject to the same isolation and staining procedures. Stained exosomes were visualized by confocal laser scanning microscope equipped with a 20x lens (FV3000, Olympus), irradiated by 488 nm.
To verify the presence of exosomes, TEM imaging was performed with a JEOL 1200EX, 80 kV microscope. A 10 μL drop was incubated on a TEM grid for 1 min, wicked dry, rinsed with water, and stained with uranyl acetate prior to TEM imaging.
Membrane permeable SYTO™ RNASelect™ green fluorescent cell stain (Thermofisher) was used to fluorescently label the RNA present inside the exosomes, as described by the manufacturer. Briefly, isolated exosomes were incubated at 37 °C for 20min with the stain at a final concentration of 40 μM, and then the remaining free dye was removed by exosome spin columns (MWCO 3 000, Invitrogen). As controls, culture medium without cells was subject to the same isolation and staining procedures. Stained exosomes were visualized by confocal laser scanning microscope equipped with a 20x lens (FV3000, Olympus), irradiated by 488 nm.
To verify the presence of exosomes, TEM imaging was performed with a JEOL 1200EX, 80 kV microscope. A 10 μL drop was incubated on a TEM grid for 1 min, wicked dry, rinsed with water, and stained with uranyl acetate prior to TEM imaging.
3
Isolation of Extracellular Vesicles from BMDM
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For isolation of extracellular vesicles, the Total Exosome Isolation Reagent from cell culture media (Thermo Scientific) was used. After differentiation, BMDMs were seeded in a density of 1 × 107 cells/10 ml dish. FCS supplement in BMDM media was replaced by exosome‐depleted FCS (Thermo Scientific). In order to avoid carryover of LPS after the LPS priming step, BMDMs were rinsed twice with pre‐warmed PBS before stimulation with ATP. After ATP stimulation, cell culture media was harvested and centrifuged at 2,000 ×g for 30 min at 4°C to remove cells and debris. The supernatant was transferred into a new tube and mixed with the reagent mixture well by vortexing. Samples were incubated overnight at 4°C. After incubation, samples were centrifuged at 10,000 ×g for 1 h at 4°C. The supernatant was carefully discarded. Extracellular vesicles were resuspended in PBS. To remove ATP and possible contaminants, Exosome Spin Columns (MW3000, Thermo Scientific) were used according to the manufacturer's protocol. The protein content of the EVs was determined using BCA protein assay (Pierce), and subsequent stimulations and injections were carried out using equal amounts of EV protein.
4
Exosome Isolation from Cell Culture and Serum
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Cells were plated 200,000 cells per well in a 12-well plate, and medium was conditioned for 48 h, followed by harvesting for exosome isolation. Medium was collected from each well and used for separate exosome preparations. Exosomes were isolated using the Total Exosome Isolation Reagent from Cell Culture Media (Thermo Fisher Scientific) according to the manufacturer's instructions. Exosome pellets were resuspended in 100 µl of 0.2 µM filtered PBS and stored in small aliquots to avoid freeze-thaw cycles. In select assays, cells were treated with 10 µM acriflavine (Sigma-Aldrich) and the medium was conditioned for 2 h, followed by harvest of the medium and exosome isolation. Exosomes were harvested from mdx mouse serum using the Total Exosome Isolation Reagent from Serum (Thermo Fisher Scientific) according to the manufacturer's instructions.
5
Fibroblast-Derived Exosome Isolation
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Cells were plated 120,000 cells per well in a 12-well plate, conditioned media was harvested after 48 hours and exosomes were isolated with Total Exosome Isolation Reagent from Cell Culture Media (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. The exosome pellet was resuspended in 100 μL of 0.2 μM filtered PBS. Exosomes isolated from a normal human adult dermal fibroblast cell line (Lonza, Basel, Switzerland) were used as an inert control.
6
Exosome Isolation and Characterization
7
Nrf2 Activation Assay Protocol
8
Exosomal Characterization in Ovarian Cancer
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A2780, TOV-21, and SKOV-3 cells were grown in 75 cm3 flasks. Based on the cytotoxicity results obtained, cells were treated with α-mangostin and/or cisplatin at a concentration equal to the IC25 values for a given condition. Cells were treated for 24 h. The control was untreated ovarian cancer line cells. In addition, fibroblast cells, NHDF, were cultured. Exosomes were isolated from each culture after treatment and non-treatment control. Isolation of exosomes was performed using Total Exosome Isolation (from cell culture media) reagent (Invitrogen) from 10 mL samples of the medium. The isolation was carried out according to the producer’s instructions. Before treatment, cells were cultured for at least 24 h in an exosome-depleted FBS medium. Treatment and isolation were performed in triplicates.
9
Extracellular Vesicle Isolation from Neutrophils and Trophozoites
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For EV isolation, 7.5 × 106 neutrophils or 7.5 × 105 trophozoites were centrifuged at 1400 rpm for 5 min and washed 3 times with PBS pH 7.4. After last washing, neutrophils or trophozoites were resuspended in 3 ml of free-serum RPMI-1640 medium (Biological Industries) and culture in 24 well-plates for 1 h at 37°C. After incubation, the culture supernatant was collected in conical tubes and centrifuged at 1400 rpm for 5 min. Free cell supernatant was obtained collecting only 2.5 ml of culture media after centrifugation and filtered through 0.22 μm. The EVs were purified using Total Exosome Isolation (from cell culture media) reagent (Invitrogen, cat 4478359) according to manufacturer instructions. EVs were resuspended in filtered PBS pH 7.4 and protein concentration was determined with Nanodrop 2000 equipment (Thermo Fisher). For EVs derived from trophozoites-neutrophil coculture, 2.5 ml of free-serum RPMI-1640 containing 7.5 ×106 neutrophils were placed in 24 well-plate and immediately were added 0.5 ml of the same medium containing 7.5 × 105 trophozoites (ratio 10:1; final volume of 3 ml). Coculture was incubated for 1 h at 37°C and the methodology was the same as described above.
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