Magnetic Bead-Based Isolation of Exosomes

Abstract

Exosomes are here defined as extracellular vesicles (EVs) in the approximate size range of 30–100 nm in diameter, and are observed in most body fluids containing typical exosomal markers such as CD9, CD63, and CD81. Potential subpopulations of exosomes can be captured by targeting these markers using magnetic beads. Magnetic beads are versatile tools for exosome isolation and downstream analysis. Here, we describe the workflow of immuno magnetic isolation and analysis of exosomes by flow cytometry, Western immunoblotting, and electron microscopy.

1 Introduction

Extracellular vesicles (EVs) are released from any cells examined so far and found in all body fluids. Exosomes, a subgroup of EVs, ~30–100 nm in diameter, originate by definition from multivesicular bodies (MVBs). However, there is little evidence that all EVs referred to as exosomes actually originate from MVBs. Exosomes contain lipids, carbohydrates, proteins, and nucleic acids and can upon release from the originating cell initiate different functions on the target cells as a way of intercellular communication. Many biological processes are influenced by exosome mediated transfer of information including, but not limited to immune response [1], antigen presentation [2], apoptosis, angiogenesis, inflammation, and coagulation [3] by protein/lipid exchange or initiating downstream signaling pathways in the target cell. Furthermore, exosomes deliver nucleic acid cargo to target cells [4–6] and viral particles utilize the vesicle transport of exosomes for viral spread [5] masking themselves avoiding the host’s immune response.

Interestingly, exosomes are involved in cancer biology where oncogenes have been secreted by tumor cells [7] acting to enhance cancer progression, immune response suppression and causing treatment resistance. Exosomes have therefore been suggested to be used as an early marker for cancer detection to provide optimal treatment and monitor cancer progression.

For exosomes to act as a diagnostic tool, the vesicles need to be enriched and preferentially subfractionated to be characterized. Enrichment of exosomes is generally done by ultracentrifugation (UC), often in combination with sucrose density gradients or sucrose cushions [8]. The scientific community is discussing the best way to utilize UC for exosome pre-enrichment. Exosomes obtained with UC appears heterogenous in terms of size distribution, including 30–100 nm diameter vesicles, but also larger vesicles are present [9]. Larger protein aggregates might also co-aggregate with the target vesicles. Protein aggregates can be removed from the exosome preparation by floating the exosomes on a density gradient which results in a more homogenous size distribution [9]. Other size-based isolation procedure includes ultrafiltration protocols [10]. Finally, precipitation-based protocols using polymers in combination with low-speed centrifugation or filtration have been reported to enrich exosomes [11–13].

Specific capture of exosomes by targeting exosome specific markers increases the purity, removes co-enriched exosomes from media supplements and isolates subpopulations of exosomes if present. Specific capture using a bead-based approach is a versatile method in terms of applications and compatibility with downstream applications such as Western immunoblotting, electron microscopy, qRT-PCR, and flow cytometry. The tetraspanin proteins CD9 and CD81 have been suggested to be common extracellular markers [14] which can be used for specific capture. Latex beads have been used for specific capture. Such a protocol involves numerous centrifugation steps and is challenging in terms of reproducibility. Magnetic separation using nano-sized beads is an alternative to latex beads for exosome capture, isolating a more homogenous population of vesicles in terms of size, morphology, and protein content [9]. These beads however require UC for reduction in sample volume and they are too small to be detected by flow cytometry. Clayton et al. developed a rapid and versatile technique for exosome isolation and analysis using anti-MHC Class II antibodies coated to a larger solid surface, 4.5 μm magnetic beads for downstream applications such as flow analysis [15].

Exosome isolation efficiency will be influenced by factors such as incubation time, temperature, level of surface marker expression, and concentration of target vesicles. The nature and state of the target molecule/structure, characteristics of the antibody–antigen interaction, sample type, concentration and ratio of beads and target molecules will have an influence on the success of the magnetic separation.

Our main focus now is on exosome isolation using magnetic beads (Dynabeads^®) for downstream flow analysis, Western immunoblotting, and electron microscopy. In addition, we address exosome production from cell lines and analysis of pre-enriched exosome preparations prior to magnetic isolation.

2 Materials

2.1 Cell Culture

• 1. SW480 cell line (ATCC).

• 2. Jurkat cell line (ATCC).

• 3. RPMI 1640 Medium (Gibco).

• 4. Heat inactivated fetal calf serum (FCS) (Gibco).

• 5. Integra CELLine CL 1000 Culture System bottles (Integra Biosciences).

• 6. Integra CELLine AD 1000 Culture System bottles (Integra Biosciences).

• 7. Corning cell culture bottles 225 cm² with 0.2 μm ventilation lids (Sigma-Aldrich).

• 8. 100 mM Sodium pyruvate solution (Life Technologies).

2.2 Precipitation

• 1. Total Exosome Isolation Reagent (Life Technologies).

2.3 Immunomagnetic Isolation

• 1. Exosome—Human CD63 Isolation/Detection, (Life Technologies).

• 2. Prototype magnetic beads for isolation and analysis of CD9, CD81, and EpCAM (Life Technologies).

• 3. Isolation buffer: PBS with 0.1 % Bovine serum albumin (BSA), filtered through a 0.2 μm filter.

• 4. 2 mL Sarstedt Tubes.

• 5. Mixer.

• 6. Magnet (DynaMag-2).

2.3.1 Flow Cytometry Analysis

• 1. BD LSR II.

• 2. Anti-CD9 PE (M-L13, BD Biosciences).

• 3. Anti-CD81 PE (JS-81, BD Biosciences).

• 4. Anti-EpCAM PE (EBA-1, BD Biosciences).

2.4 Electrophoresis and Western Immunoblotting

• 1. Lysis buffer: radio immunoprecipitation assay (RIPA) buffer (3.75 mL 4 M NaCl, 1 mL NP40, 0.5 g SDS, 0.61 g Tris Base, deionized H₂O (dH₂O) up to 90 mL, adjust to pH 8 with 5 M HCl, dH₂O up to 100 mL).

• 2. Protease inhibitor: add cOmplete, EDTA-free 25× proteinase inhibitor solution (Roche) to RIPA lysis buffer.

• 3. 2× SDS lysis buffer: 0.58 g SDS, 0.19 g Trizma base, 8.5 mL dH₂O, 2.5 mL 100 % glycerol with or without 1.25 mL β-mercaptoethanol, adjust to pH 6.8 with 10 M HCl. Adjust volume to 12.5 mL with dH₂O.

• 4. Loading Buffer: 4.0 mL dH₂O, 1 mL 0.5 M Tris–HCl pH 6.8, 0.8 mL 100 % glycerol, 1.6 mL 10 % SDS, with or without 0.4 mL β-mercaptoethanol, add bromophenol blue).

• 5. Precast polyacrylamide gels of various polyacrylamide concentrations and gradient gels (e.g., 4–12 %) are commercially available.

• 6. Power supply is required for electrophoresis and Western immunoblotting.

• 7. Electrophoresis running buffer: dissolve 3.03 g Trizma base, 14.4 g glycine, 1 g SDS in 1 L of dH₂O.

• 8. Western transfer buffer: dissolve 15.5 g Trizma base, 72.1 g glycine in 1,000 mL (5×). dilute 200 mL 5× Western transfer buffer in 200 mL methanol and add 600 mL of dH₂O.

• 9. Membrane for protein blotting: PVDF (Bio-Rad).

• 10. Membrane wash solution: TBS-T (2.42 g Tris–HCl, 0.56 g Trizma base, add dH₂O up to 900 mL, adjust pH to 7.60 with 5 M HCl, add dH₂O up to 1,000 mL).

• 11. Blocking solution: dissolve 5 g fat-free dry skimmed milk in 100 ml TBS-T (5 %).

• 12. Antibodies: dilute primary antibody in blocking solution according to manufactures recommendations (see Note 1 ).

• 13. Substrate for antibody signal detection (e.g., SuperSignal West Dura Extended Duration Substrate (Thermo Fisher) (see Note 2 ).

2.5 Transmission Electron Microscopy

Reagents for sample preparation for electron microscopy can be obtained from for example Polysciences, Inc.

• 1. 1 % glutaraldehyde in 200 mM cacodylate buffer (pH 7.4).

• 2. 1.5 % potassium ferricyanide.

• 3. 1 % osmium tetroxide.

• 4. 1.5 % magnesium uranyl acetate.

• 5. 70 % ethanol.

• 6. 90 % ethanol.

• 7. 96 % ethanol.

• 8. Absolute ethanol.

• 9. Epon.

• 10. Ultramicrotome for ultrathin sections.

• 11. Glow discharged copper grids.

• 12. Blocking solution: PBS with 0.5 % BSA.

• 13. Protein A gold.

• 14. 0.3 % uranyl acetate.

• 15. Methyl cellulose.

3 Methods

A protocol for isolation and analysis of exosomes pre-enriched using precipitated exosomes derived from cell culture bottles is described. For some cell lines the release of exosome can be low. An alternative to the cell culture bottles is the Integra CELLine Culture System culture flasks, originally designed for hybridoma cultures, have been used for enhancing the exosome yield. The exosomes released showed identical morphology, phenotype, and immune function [16]. A protocol for isolation and analysis of exosomes pre-enriched using precipitated exosomes derived Integra CELLine Culture System culture flasks is described.

3.1 Exosome Enrichment from Adherent Cells (SW480) Using Cell Culture Bottles

Adherent cells are cultured to 80–90 % confluency in RPMI 1640 with 10 % FCS (see Notes 3 and 4 ), and 1 mM Sodium pyruvate in cell culture bottles (225 cm²) at 37 °C with 5 % CO₂. The medium is removed and 50 mL of fresh medium is added to the culture. After 3 days the cell-conditioned medium is removed and two centrifugation steps are performed on the pre conditioned medium prior to pre-enrichment (300 × g for 10 min at 2–8 °C and 2,000 × g for 30 min at 2–8 °C).

3.2 Exosome Enrichment from Cells in Suspension (Jurkat) Using Cell Culture Bottles

Pre-cultured Jurkat cells are seeded at a density of 0.4 × 10⁶ cells per mL in fresh RPMI medium and grown for 3 days at 37 °C with 5 % CO₂. The cell suspension is subjected to two centrifugation steps (300 × g for 10 min at 2–8 °C and 2,000 × g for 30 min at 2–8 °C).

3.3 Exosome Enrichment from Adherent Cells (SW480) Using Integra CELLine AD 1000

Adherent cells are cultured to confluency in RPMI 1640 with 10 % FCS and 1 mM Sodium pyruvate in Integra CELLine Culture System bottles [16] at 37 °C with 5 % CO₂. After culturing for 7 days the cell-conditioned medium is harvested. Two centrifugation steps are performed on the medium before pre-enrichment (400 × g for 10 min at 2–8 °C and 2,000 × g for 30 min at 2–8 °C).

3.4 Exosome Enrichment from Cells in Suspension (Jurkat) Using Integra CELLine CL 1000

3 × 10⁷ cells in 15 mL medium are cultured in RPMI with 10 % FCS in Integra CELLine Culture System bottles [16]. The cell-conditioned medium is harvested after 7 days. The medium is centrifuged twice before pre-enrichment (400 × g for 10 min at 2–8 °C and 2,000 × g for 30 min at 2–8 °C).

3.5 Exosome Pre-enrichment Using Precipitation

• 1. Harvest cell culture media.

• 2. Centrifuge the cell media at 350 × g for 10 min at 2–8 °C.

• 3. Transfer the supernatant to a new tube.

• 4. Centrifuge the supernatant at 2,000 × g for 30 min at 2–8 °C.

• 5. Supernatant can be frozen at this stage.

• 6. Transfer the required volume of cell-free culture media to a new tube and add 0.5 volumes of the Total Exosome Isolation (from cell culture media) reagent.

• 7. Mix the culture media–reagent mixture well by vortexing, or pipetting up and down until there is a homogenous solution.

• 8. Incubate samples at 2–8 °C ON.

• 9. Centrifuge the samples at 2–8 °C at 10,000 × g for 1 h (fixed angle rotor).

• 10. Aspirate and discard the supernatant. Exosomes are contained in the pellet at the bottom of the tube for swing-out rotors or at the tube-wall near the bottom for fixed angle rotor (see Note 4 ).

• 11. To remove all the liquid leave the tube upside down on a filter paper for 3–4 min.

• 12. Resuspend the pellet in a convenient volume of PBS (see Note 5 ).

• 13. Once the pellet is resuspended, the exosomes are ready for downstream analysis or further purification through affinity methods or aliquotation and freezing in −80 °C (see Notes 5 and 6 ).

3.6 Isolation of Exosomes Using Magnetic Beads for Downstream Flow Analysis

Analysis of exosomes by flow cytometry is limited by the resolution provided by the instrument. The analysis of individual exosomes similar to cell analysis by flow cannot be done, while “swarm analysis,” looking at many exosomes as one larger object is feasible. Such analysis will provide information about the average expression level of the marker in question for the whole population. Magnetic beads (micrometer sized) used as solid support for flow analysis of exosomes can serve several purposes: (1) the beads are detected by the flow instrument and trigger data collection, (2) enable easy washing/handling of the exosomes without the need for UC, and (3) add an additional level of resolution to the analysis if used.

3.7 Validation of the Method

The protocol is based on isolation using 200,000 magnetic beads. The protocol can be scaled up by increasing volumes and reagents proportionally.

3.7.1 Preparation of Pre-enriched Exosome Sample

• 1. Titrate the volume of the pre-enriched exosome solution. Start with approximately 25 μg of total protein (see Note 7 ).

• 2. Add Isolation Buffer to 100 μL final volume per 20 μL magnetic beads (see Note 8 ).

Day 1.

• 1. Resuspend the magnetic beads by mixing for >10 min or vortexing for 30 s.

• 2. Transfer 20 μL magnetic beads into an appropriate tube (see Note 8 ).

• 3. Wash the magnetic beads by adding 200 μL of Isolation Buffer (see Subheading 2) and mix well.

• 4. Place the tube on the magnet for 1 min and discard the supernatant.

• 5. Remove the tube from the magnet and add pre-enriched exosome solution titrated with Isolation Buffer (100 μL final volume) to the magnetic beads. Mix well (see Note 9 ).

• 6. Incubate the tube ON (18–22 h) at 2–8 °C with mixing (see Note 10 ).

Day 2.

• 7. Centrifuge the tube for 3–5 s to collect the sample at the bottom of the tube (see Note 11 ).

• 8. Wash the bead-bound exosomes by adding 300 μL of Isolation Buffer. Mix gently by pipetting (do not vortex).

• 9. Place the tube on the magnet for 1 min and discard the supernatant.

• 10. Remove the tube from the magnet and add 400 μL of Isolation Buffer. Mix gently by pipetting.

• 11. Place the tube on the magnet for 1 min and discard the supernatant.

• 12. Resuspend the bead bound exosomes in 300 μL Isolation Buffer.

The bead bound exosomes are now ready to be stained for flow cytometry.

3.7.2 Staining for Flow Cytometry

The staining antibody should be titrated for optimal signal to noise ratio, starting with the manufacturer’s recommended concentration for staining 1 × 10⁶ cells (Fig. 1).

Fig. 1

Flow cytometry analysis of exosome after specific isolation with magnetic beads. (a) Showing forward/side scatter plot of bead-bound exosomes. (b–d) Illustrating staining with anti-human EpCAM, -CD81 and -CD9 after specific isolation using magnetic beads coated with anti-human EpCAM, -CD81 and -CD9 respectively. (e) Demonstrating good correlation of microgram exosomal protein input and flow signal output (signal to noise). (f) Showing the effect of increasing amount of PE-conjugated staining antibody used for flow analysis

• 1. Transfer desired staining antibodies to a flow tube (see Note 12 ).

• 2. Add 100 μL bead-bound exosomes to the tube. Mix gently by pipetting (see Note 13 ).

• 3. Incubate for 45–60 min at RT protected from light on a sample shaker (~1,000 rpm) (see Note 14 ).

• 4. Wash the bead-bound exosomes by adding 300 μL of Isolation Buffer. Mix gently by pipetting (do not vortex).

• 5. Place the tube on the magnet for 1 min and discard the supernatant.

• 6. Repeat the washing steps once and resuspend in the desired volume of Isolation Buffer for flow cytometry analysis.

3.7.3 Western Immunoblotting Analysis of Exosomes Isolated with Magnetic Beads

Western immunoblotting analysis of pre-enriched exosomes prior to magnetic isolation provides an overview of the total content of proteins in the pre-enriched sample. Magnetic separation of exosomes offers advantages such as increased resolution by removing potential protein aggregates or other non-exosomal vesicles and isolation of potential exosome subpopulations for comparison analysis. Analysis of exosomes by Western immunoblotting is a challenge in terms of sensitivity. Titration of the amount of exosomes used and the amount of magnetic beads added to the exosomes are important in addition to careful selection of high sensitivity substrate (Fig. 2).

Fig. 2

Western immunoblotting analysis of exosomes isolated with magnetic beads coated with anti-CD9 (A, B ) or -CD81 (C ) and processed for electrophoresis and Western immunoblotting. The blots were labeled for CD9 (A), CD63 (B ), or CD81 (C) using antibody clones TS9, TS63, or M38, respectively. Lane 1 shows pre-enriched exosomes prior to magnetic isolation. Lane 2–5 show exosomes isolated with increasing amounts of magnetic beads

• 1. Titrate the volume of the pre-enriched exosome solution (see Note 15 ).

• 2. Add Isolation Buffer (see Subheading 2) to 100 μL final volume per 100 μL magnetic beads.

• 3. Resuspend the magnetic beads by mixing for >10 min or vortexing for 30 s.

• 4. Transfer 100 μL magnetic beads into an appropriate tube (see Note 16 ).

• 5. Wash the magnetic beads by adding 500 μL of Isolation Buffer and mix well.

• 6. Place the tube on the magnet for 1 min and discard the supernatant.

• 7. Remove the tube from the magnet, and add pre-enriched exosome solution titrated with Isolation Buffer (100 μL final volume) to the magnetic beads. Mix well.

• 8. Incubate the tube ON (18–22 h) at 2–8 °C with mixing.

• 9. Centrifuge the tube for 3–5 s to collect the sample in the bottom of the tube.

• 10. Wash the bead-bound exosomes by adding 300 μL of Isolation Buffer. Mix gently by pipetting (do not vortex).

• 11. Place the tube on the magnet for 1 min and discard the supernatant.

• 12. Remove the tube from the magnet, and add 400 μL of Isolation Buffer. Mix gently by pipetting (do not vortex).

• 13. Place the tube on the magnet for 1 min and discard the supernatant.

• 14. Add 10 μL of lysis buffer (e.g., 1× RIPA buffer with proteinase inhibitor) to bead bound exosomes, mix well, and incubate at 2–8 °C for 15 min to lyse the exosomes.

• 15. Add 10 μL 2× SDS lysis buffer to the sample and mix well.

• 16. Add 1.5 μL Loading Buffer and mix well.

• 17. Incubate at 95 °C for 5 min (see Note 17 ).

• 18. Place the tube in the magnet (see Note 18 ) and load the supernatant on the gel (depending on well capacity) (see Note 19 ).

• 19. Transfer samples to PVDF membranes by wet transfer (100 V for 1 h).

• 20. Block the membranes in 5 % nonfat dry milk or 5 % BSA in TBS-T (see Notes 20 ).

• 21. Incubate ON at 2–8 °C with primary antibody diluted in 5 % nonfat dry milk.

• 22. Wash extensively in TBS-T.

• 23. Incubate with HRP-conjugated secondary antibody for 60 min at RT (see Note 21 ).

• 24. Detect signal by using for example SuperSignal West Dura or Femto Substrate (Thermo Scientific) (see Note 2 ).

3.7.4 Ultrastructural Analysis of Exosomes Isolated with Magnetic Beads

Exosomes isolated with magnetic beads can be visualized at the ultrastructural level by electron microscopy. The exosomes on the surface of the magnetic beads can also be subjected to stereological analysis for estimation of the vesicle density on the bead surface. Exosome isolation for downstream electron microscopy resembles the magnetic isolation for downstream flow in terms of maximizing the amount of exosomes per magnetic bead (Fig. 3).

Fig. 3

Ultrastructural analysis of exosomes isolated with magnetic beads from pre-enriched exosomes. Exosomes were isolated with magnetic beads coated with anti-CD81 and processed for electron microscopy. The micrographs show fractions of the magnetic bead surface and demonstrate magnetic isolation of exosomes pre-enriched from cell culture using ultracentrifugation (a), cell culture using precipitation (b), CELLine using ultracentrifugation (c), or CELLine using precipitation (d). Arrows indicate exosomes on the surface of magnetic beads. Bar, 200 nm

• 1. Mix 50 μL of magnetic beads coated with primary Ab with 250 μL of pre-enriched exosomes (see Note 16 ).

• 2. Incubate ON at 2–8 °C.

• 3. Centrifuge the sample shortly on a table centrifuge in order to collect all material.

• 4. Wash the magnetic beads to remove unbound targets with 500 μL PBS with 0.1 % BSA followed by mixing.

• 5. Apply the tube to a magnetic field and remove the supernatant.

• 6. Add 1 % glutaraldehyde in 200 mM cacodylate buffer (pH 7.4) to immunomagnetic beads covered with exosomes for 1 h at RT (see Note 22 ).

• 7. Wash repeatedly in dH₂O.

• 8. Incubated in cacodylate buffer containing 1.5 % Potassium ferricyanide and 1 % Osmium tetroxide for 1 h.

• 9. Incubate in 1 % tannic acid for 30 min at RT.

• 10. Incubate in 1.5 % magnesium uranyl acetate for 30 min at RT.

• 11. Dehydrated the sample in 70 % ethanol for 10 min, 90 % ethanol for 10 min, 96 % ethanol for 10 min and Absolute ethanol for 4 × 15 min.

• 12. Embed the samples in Epon.

• 13. Ultrathin sections are prepared using an ultramicrotome and stained with lead citrate (see Note 23 ).

3.7.5 Western Immunoblotting Analysis of Pre-enriched Exosomes from Cell Culture Prior to Magnetic Isolation

• 1. 7.5 μL pre-enriched Exosomes (see Note 24 ).

• 2. 1.9 μL 5× RIPA.

• 3. 10 s of sonication.

• 4. Incubate for 15 min at 2–8 °C.

• 5. 9.4 μL 2× SDS lysis buffer with or without β-mercaptoethanol to sample (see Note 25 ).

• 6. 1 μL Loading buffer with or without β-mercaptoethanol.

• 7. Incubate for 5 min at 95 °C (see Note 17 ).

• 8. Proceed with electrophoresis, Western immunoblotting and labeling as described in Subheading 3.7.6.

3.7.6 Western Blotting Analysis of Pre-enriched Exosomes from CELLine CL 1000/AD 1000 Prior to Magnetic Isolation

• 1. 1 μL pre-enriched Exosomes (see Note 24 ).

• 2. 1 μL 2× RIPA.

• 3. 10 s of sonication.

• 4. Incubate for 15 min at 2–8 °C.

• 5. Add 2 μL 2× SDS lysis buffer with or without β-mercaptoethanol to sample (see Note 25 ).

• 6. 0.5 μL Loading buffer with or without β-mercaptoethanol.

• 7. Incubate for 5 min at 95 °C (see Note 17 ).

• 8. Proceed with electrophoresis, Western blotting and labeling as described in Subheading 3.7.6.

3.7.7 Ultrastructural Analysis of Pre-enriched Exosomes by Electron Microscopy

Pre-enriched exosomes can be subjected to immunolabeling and ultrastructural analysis using electron microscopy. The amount of exosomes present, morphology, and the presence of surface markers can be investigated. The protocol is performed at RT on a clean surface (Fig. 4) (see Note 26 ).

Fig. 4

Size comparison of exosomes pre-enriched using ultracentrifugation (a), cell culture precipitation (b), and Integra CELLine CL 1000 precipitation (c). The size distribution is measured by the Nanosight^® LM10 instrument. The profiles are essentially very finely segmented histograms, indicating the number of particles per milliliter (in millions) for each size in bins of 1 nm increment from 0 to 1,000 nm. (d) Pre-enriched exosomes were transferred to glow discharged hexagonal copper grids and labeled with mouse anti-human CD81 or mouse anti-human CD63 (inset) followed by rabbit anti-mouse antibody and colloidal gold coated with protein A (10 nm). Samples were stained with uranyl acetate and protected with methyl cellulose. Bar, 100 nm

• 1. Adsorb exosomes by placing a carbon-coated copper grid to a drop of pre-enriched exosomes (see Note 27 ) and incubate for 15 min.

• 2. Wash the grids briefly in PBS.

• 3. Move the grid to the blocking solution and incubate for 10 min.

• 4. Move the grid to a drop of primary antibody and incubate for 15 min.

• 5. Wash the grid on 4 drops of PBS for a total of 10 min.

• 6. If primary antibody is mouse origin move the grid to a drop of rabbit anti-mouse secondary antibody for 15 min.

• 7. Wash grids on 4 drops of PBS for a total of 10 min.

• 8. Move the grid to a drop of protein A gold and incubate for 15 min (see Note 28 ).

• 9. Wash grids on four drops of PBS for a total of 15 min.

• 10. Wash grids on four drops of dH₂O.

• 11. Move the grids to a drop of methylcellulose with 0.3 % uranyl acetate and incubate for 15 min on ice (see Note 29 ).

• 12. Remove excess methyl cellulose and uranyl acetate (see Note 30 ).

4 Notes

• 1. To find optimal signal titration dilution series of primary antibody is recommended. For targets with the similar size (kDa) as heavy or light chain antibody TrueBlot^® Ultra Ig HRP is recommended (Rockland antibodies & assays).

• 2. It is recommended to use the most sensitive system available for detection of small amounts of target proteins. Thermo Scientific™ SuperSignal™ West Dura Chemiluminescent Substrate is optimized for high sensitivity (mid-femtogram) and long signal duration.

• 3. To ensure that the isolated exosomes originate from cells of interest, including controls of medium with FCS alone is essential. FCS may contain exosomes that could contaminate the cell derived exosomes. If contamination is a problem, exosome depleted FCS should be used. An alternative is to grow the cell line in the absence of FCS. Some cell lines can handle up to 12 h in media without FCS.

• 4. It is essential to have high cell viability to reduce the release of apoptotic bodies.

• 5. Exosomes are contained in the pellet at the bottom of the tube but not always visible. When using fixed angle rotor, it’s important to mark the tube’s position to retrieve the exosomes at the “outer” part of the tube wall. For large scale pre-enrichment 24 mL of cell culture medium is mixed with 12 mL of Total Exosome Isolation reagent. After incubation and centrifugation the pellet is dissolved in 500 μL PBS and transferred to a new tube. In addition, 500 μL of PBS are added to the original tube to collect any remaining exosomes and transferred to the new tube. The pre-enriched exosomes are now in 1 mL PBS.

• 6. For short-term storage, exosomes can be left at 4 °C for up to 1 week. For long-term storage we recommend −80 °C. We recommend to aliquot the exosomes into multiple tubes to avoid freeze/thaw cycles, which may cause damage to the exosomes and reduce their numbers. Morphologically the exosomes do not change upon storage at 2–8 °C. However, changes in biological function might occur.

• 7. Exosomes, at 30–100 nm, are too small to be seen using a regular microscope. There is a few options to determine the concentration of exosomes in samples, e.g., the NanoSight and Izon instruments. The Nanosight instrument enables counting and sizing of nanoparticles (10–1,000 nm size) using light scattering and browning motion, while the Izon instrument accomplishes the same thing using nanopore analysis. Although very different in methodology, both technologies allow one to study particles at 10–1,000 nm in size.

• 8. Maximum of 50 μL per 20 μL magnetic beads if sample is prepared using the precipitation technique and maximum 100 μL per 20 μL magnetic beads if sample is prepared by UC.

• 9. The Dynabeads show linear capture efficiency up to 120 μg of exosome input for exosomes pre-enriched with precipitation measured by flow cytometry.

• 10. It is important to use tubes that allow proper mixing. V-Shaped tubes are not recommended. The amount of Dynabeads used during isolation depends on downstream application. For flow cytometry few beads are used in order to ensure maximum amounts of exosomes to dock onto the bead surface (same apply for downstream electron microscopy). A maximum total surface area for exosome docking is required for downstream western blotting.

• 11. Good mixing is critical to successful exosome isolation.

• 12. For optimal binding of exosomes to the bead surface it is recommended to incubate up to 21 h (reason for incubation at 2–8 °C). Shorter incubation times can be applied if the amount of exosomes pulled out of the solution is not critical.

• 13. To collect the sample after isolation, a short centrifugation step is recommended using a bench top centrifuge.

• 14. In terms of volume of staining antibody it is recommended to use the supplier recommendation for 1 × 10⁶ target cells.

• 15. Start with approximately 25 μg of total protein. Maximum of 50 μL pre-enriched exosomes per 100 μL magnetic beads if sample is prepared using precipitation technique and maximum 100 μL pre-enriched exosomes per 100 μL magnetic beads if sample is prepared by ultracentrifugation. Estimation of amount of protein can be performed by Micro BCA™ Protein Assay Kit (Thermo Scientific), qBit (Life Technologies), or Bio-Rad protein assay.

• 16. More magnetic beads can also be added if complete exosome depletion is required. The protocol can be scaled up by increasing volumes and reagents proportionally. To achieve greater depletion of exosomes, increase the number of magnetic beads by 2–25 times per 100 mL (final volume) of sample. In contrast to flow cytometry that uses relatively few magnetic beads in order to allow as many exosomes to dock onto each bead as possible, the total bead surface provided is critical. A large total bead surface will ensure that a sufficient number of exosomes are docked onto the bead surface to provide a strong signal for Western immunoblotting. This translates into the use of more beads during immuno magnetic separation for downstream Western immunoblotting compared to downstream flow cytometry.

• 17. Lower temperature might be tested if aggregation occurs.

• 18. Exosome lysate with magnetic beads can also be loaded on the gel. Magnetic beads will remain in the upper part of the gel during electrophoresis. Some of the target can then be observed at the top of the blot.

• 19. Such as 10 % or 12 % TGX SDS–polyacrylamide gels (Bio-Rad) or NuPage Bis-Tris minigel (Life Technologies).

• 20. Other blocking solutions might be tested such as 5 % BSA in TBS-T.

• 21. Presence of azide might inhibit the HRP reaction.

• 22. Magnetic beads with exosomes that have been exposed to fixative (glutaraldehyde) need longer incubation time on the magnet in order to collect the beads.

• 23. When contrasting Dynabeads coated with exosomes, lead citrate might be excluded if the contrast is too strong. Iron oxide grains can be seen as black spots evenly distributed throughout the magnetic bead.

• 24. It is recommended to titrate the amount of exosome input in order to find the optimal exosome input for the detection system.

• 25. Many of the primary antibodies for labeling of tetraspanins (e.g., CD9, CD63, and CD81) require non-reducing conditions.

• 26. A clean surface can be obtained by using for example Parafilm.

• 27. Glow discharge carbon coated copper grids to improve adsorption by making the surface hydrophilic. The drop of pre-enriched exosomes can be as small as 5–10 μL. Cover the grids during incubation with a lid to avoid any disturbance of the grid.

• 28. For double labeling different sizes of protein A gold are used (e.g., 5–20 nm).

• 29. In order to obtain the best possible contrast and resolution several different solutions should be tested. (1) Aqueous uranyl acetate: A 1–3 % solution of uranyl acetate dissolved in dH₂O can be used to negatively stain many samples. The stain has a low pH so this solution is not recommended for particles that are unstable in acid conditions. (2) Neutral phosphotungstic acid: A 1–3 % solution of phosphotungstic acid is made up in dH₂O and the pH is adjusted to 7 using sodium hydroxide. This is a useful stain for many samples but is especially good for viruses that dissociate at low pH. The stain produces less contrast than the uranyl acetate. (3) Ammonium molybdate: Make up a 1 % solution of ammonium molybdate in dH₂O. This solution has also been used to negatively stain thawed, thin cryosections of fixed cells.

• 30. Remove excess methyl cellulose and uranyl acetate by using a filter paper and place the grid perpendicular to the filter paper. Barely touch the grid to the filter paper and move the grid until no methyl cellulose is removed. There should only be a thin layer of methyl cellulose covering the exosomes on the grid.