方法文章

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

DOI：

10.3791/65574

⸱

2023年9月1日

 ,  ,  ,  ,  ,  ,  ,  ,  , 
1Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University

* These authors contributed equally

本文内容

摘要

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This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.

摘要

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Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.

This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.

Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

引言

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The gut is widely recognized as the organ harboring the most abundant microbial communities in the human body, with over 90% of bacteria involved in colonization and multiplication1,2. Extensive evidence has demonstrated that the gut microbiota modulates the gut microenvironment and simultaneously interacts with dysfunction in distant organs, primarily through an impaired intestinal barrier3,4. Mounting evidence indicates a correlation between the imbalance of gut microbiota and the progression of inflammatory bowel disease (IBD)5,6, as well as cognitive disorders through the gut-brain axis5,6,7,8. Bacterial extracellular vesicles (BEVs) produced by bacteria play significant roles in these pathological processes.

BEVs are nanoscale particles encapsulating bacterial derivatives, with diameters ranging from 20 to 400 nm. They have been demonstrated to facilitate interactions between bacteria and their host organisms9,10. Despite their invisibility, these particles have garnered increasing attention from researchers due to their prospective broad applications as diagnostic biomarkers, therapeutic targets, and drug delivery vehicles11. Human feces, often used as biospecimens for studying BEVs, predominantly sourced from gut bacteria, contain a complex mixture of water, bacteria, lipids, proteins, undigested food residue, and exfoliated epithelial cells among others. The intricate fecal composition poses challenges to the isolation and purity of BEVs, thereby impeding a comprehensive, objective, and realistic analysis of BEVs. Hence, effective strategies to minimize interference from contaminating components and enhance the yield of BEVs have emerged as critical issues warranting immediate attention.

Existing isolation strategies largely rely on techniques such as ultra-high-speed centrifugation (UC), density gradient centrifugation (DGC), and size exclusion chromatography (SEC)12,13,14,15,16,17. Currently, DGC is one of the most widely applied methods in the field of BEV separation, encompassing two sedimentation-floating modes, "Top-down" and "Bottom-up", which are determined by the initial loading position of the sample. These methodologies differentiate extracellular vesicles (EVs) from other components based on size and density disparities, yielding variable purity and recovery rates. Prior research has indicated that single-approach strategies are insufficient for adequately separating EVs from soluble proteins in body fluid samples, such as lipoprotein in blood18 and Tamm-Horsfall protein in urine19. Additionally, the size distribution of eukaryotic extracellular vesicles (EEVs) often overlaps with that of BEVs, thereby necessitating further methodological enhancements to optimize BEV yield. Consequently, advancing the study of BEVs hinges on the development of effective separation and purification methodologies. Notably, Tulkens et al15 employed an orthogonal biophysical strategy to separate fecal BEVs from EEVs, in which the centrifugation time of a Bottom-up DGC mode was up to 18 h. In contrast, this study reduced it to 7 h, greatly saving the gradient-ultracentrifugation time and simplifying the process.

In the present study, we isolated and purified fecal BEVs employing two DGC modes under optimized buffer conditions, after enriching BEVs with a range of differential centrifugation speeds, from low to extremely high velocity. Evaluations based on morphology, particle size, and concentration indicated a commendable performance by this enhanced method. This study could serve as the foundation for future research, extending its applications to a broader domain, and offering insights into the heterogeneity of BEVs within the human body. It also contributes to the standardization of BEV separation and analysis techniques.

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方案

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The Ethics Committee of Nanfang Hospital, Southern Medical University, sanctioned this study, which was conducted with the informed consent of the participants. All methods employed herein adhered to the standard operating guidelines furnished by the International Human Microbiome Standards (IHMS: http://www.microbiome-standards.org/). All subsequent liquid handling procedures were mandated to be carried out within a biosafety cabinet or an ultra-clean bench.

1. Collection and aliquoting of fecal samples

  1. Distribute a stool sampler, a sealed bag, and an iced box, and provide comprehensive instructions to the participants on how to procure and preserve the samples.
  2. Instruct each participant to collect their fecal sample using the provided sampler, and to transport it to the lab at a temperature of 4 °C within a 24-h window.
  3. Upon receipt at the lab, use a sterile spoon to aliquot less than 3.5 g of feces into a pre-weighed 50 mL centrifuge tube. Notate the fecal sample's weight on the tube for subsequent pre-treatment procedures.
    PAUSE POINT: If immediate processing of the samples is not feasible, allocate the sample for long-term storage at -80 °C after appropriate notation.

2. Feces sample preparation

  1. Chill 500 mL of phosphate-buffered saline (PBS) at 4 °C. Filter the pre-chilled PBS through a 0.22 µm Polyethersulfone (PES) filter using a 50 mL syringe into ten 50 mL centrifugation tubes.
  2. Position two 50 mL tubes on ice, each containing 3.5 g of fecal samples. Add 35 mL of PBS to each tube.
    NOTE: Calculate the required amount of PBS for feces dissolution, ensuring a maximum sample concentration of 10% (w/v). If processing additional samples, employ more tubes as needed.
  3. Shake the samples at 300 rpm for 2 h at 4 °C, or place them for 8 h at least at 4 °C until the feces are completely suspended visibly.

3. Differential speed centrifugation

  1. Chill the high-speed refrigerated centrifuge to 4 °C.
  2. Adjust the weight of the two tubes containing the samples prepared in Step 2.3 with PBS to reach a total weight of 0.1 g.
  3. Centrifuge the samples at 3, 000 × g for 20 min at 4 °C.
  4. Carefully pipette the supernatant into two clean 50 mL centrifuge tubes, leaving approximately 1 mL above the pellet. Use a disposable plastic Pasteur pipette for this process.
  5. Adjust the weight of the two tubes with PBS to reach a total weight within ± 0.1 g.
  6. Centrifuge the transferred supernatant at 12, 000 × g for 30 min at 4 °C.
  7. Aspirate the supernatant using a 20 mL syringe, remove the syringe needle, and filter it through 0.22 µm filters into 50 mL tubes. At this point, the supernatant volume should be approximately 30 mL.
    ​NOTE: If a significant amount of impurity is still visible after the 12,000 × g centrifugation, it is necessary to repeat the centrifugation step at 12,000 × g for 30 min at 4 °C. Failure to do so may result in a significant loss of BEVs due to pore blockage during filtration.

4. Ultra-high-speed centrifugation

  1. Clean the rotor and bucket by wiping them with 75% (v/v) alcohol to eliminate any residual contamination.
  2. Position a 38.5 mL ultracentrifuge tube in the tube holder.
    NOTE: Select the appropriate ultracentrifuge tube based on the sample volume. Avoid ultraviolet radiation and unsuitable chemical reagents for sterilizing centrifugal tubes as indicated in the product manual.
  3. Transfer approximately 30 mL of the filtered fecal supernatant from Step 3.7 into the ultracentrifuge tube.
  4. Fill the ultracentrifuge tube with approximately 8 mL of PBS, leaving a 3 mm gap from the tube's opening.
  5. Place the two ultracentrifuge tubes with the samples in opposing ultracentrifugation buckets, for example, bucket 1 corresponds to 4 (1-4), 2-5, 3-6.
  6. Adjust the weight of the two buckets with PBS to achieve a total weight within ± 0.005 g.
  7. Install all buckets onto the rotor, regardless of whether the tubes are loaded.
  8. Initiate the vacuum and centrifuge the samples at 160,000 × g for 70 min at 4 °C.
  9. Release the chamber vacuum, and open the door once Ready is displayed on the Home page of the instrument. 
  10. Remove the rotor from the ultracentrifuge.
  11. Move the buckets from the rotor to the rack, and retrieve the tubes using a nipper.
  12. Discard the supernatant, which will contain visible brown pellets at the bottom.
  13. Resuspend the pellets by repeatedly pipetting them up and down using a 1,000 µL pipette with 1 mL of prechilled (4 °C) PBS until completely suspended. Fill the tube with approximately 37 mL of PBS, leaving a 3 mm gap from the opening.
  14. Perform another ultracentrifugation following Steps 4.5-4.11 at 160,000 × g for 70 min at 4 °C to remove partially attached contaminating components from the tube walls.
  15. Discard the supernatant, invert the two ultracentrifuge tubes for 5 min, and clear any remaining solution on the inner wall with wipers.
    NOTE: Cleaning the inner wall minimizes interference from residual contamination adhering to the ultracentrifuge tube wall. Choose wipers that will not influence the BEV analysis, particularly regarding particle size.
  16. Resuspend the pellets in each tube by pipetting them up and down with 1.2 mL of pre-chilled (4 °C) PBS using a 1,000 µL pipette.
  17. Transfer 1.2 mL of the PBS/BEV solution from one ultracentrifuge tube to a clean 1.5 mL microtube.
    PAUSE POINT: The PBS/BEV solution collected from one ultracentrifuge tube can proceed to Step 6. Store the solution from the other tube at -80 °C.

5. Solution preparation for density gradient centrifugation

  1. Preparation of 0.02 M HEPES buffer
    1. Combine 0.477 g of HEPES powder and 0.8 g of NaCl with 90 mL of autoclaved deionized water.
    2. Adjust the pH to 7.2 by adding 1 M sodium hydroxide (NaOH). Bring the volume to 100 mL with deionized water and filter the solution through 0.22 µm PES membranes.
      CAUTION: Sodium hydroxide is a strong caustic alkali. Operators must handle and prepare it carefully in a fume cupboard.
  2. Preparation of density gradient buffer
    1. Calculate the required volume of each density gradient buffer based on the number of ultracentrifuge tubes for density gradient centrifugation (Step 6) (In this protocol, the volumes for 60%, 50%, 40%, 20%, and 10% gradient solutions were 2.5 mL, 3 mL, 6 mL, 6 mL, and 6 mL, respectively).
    2. For the preparation of a 50% (w/v) iodixanol working solution, mix the 0.02 M HEPES buffer and 60% (w/v) iodixanol stock solution in a volume ratio of 1:5 (0.5 mL:2.5 mL).
      NOTE: Use a disposable 20 mL syringe to withdraw the iodixanol stock solution and avoid introducing air.
    3. To prepare iodixanol buffers with different concentrations, combine the 50% iodixanol working solution from Step 5.2.2 with the HEPES buffer obtained in Step 5.1 using a 1,000 µL pipette according to the proportions shown in Table 1.
      ​NOTE: Perform Step 5 on an ultra-clean bench with the lights off. Store the opened iodixanol stock solution in the refrigerator at 4 °C to prevent bacterial growth. Keep the iodixanol solution and HEPES buffer protected from light.

6. Establishment of a density gradient centrifugation system

  1. Top-down DGC mode
    1. Combine 500 µL of PBS/BEV solution isolated from Step 4 with 3 mL of PBS. Gently mix the solutions using a 1,000 µL pipette to obtain 3.5 mL of PBS/BEV solution.
    2. Place a 31 mL ultracentrifuge tube on a foam plate with holes and label it as "↓".
    3. Vertically add 3 mL of the 50% iodixanol solution to the bottom of the tube using a 1,000 µL pipette.
    4. Tilt the tube to a 70° angle and position a tube holder or other support slightly above the level of the foam plate, below the opening of the tube.
    5. Add 3 mL of 40% iodixanol solution on top of the 50% iodixanol solution using a 1,000 µL pipette.
    6. Add 3 mL of 20% iodixanol solution on top of the 40% iodixanol solution using a 1,000 µL pipette.
    7. Add 3 mL of 10% iodixanol solution on top of the 20% iodixanol solution using a 1,000 µL pipette.
    8. Add 3.5 mL of PBS/BEV solution from Step 6.1.1 on top of the 10% iodixanol solution using a 1,000 µL pipette.
    9. Gently return the tubes to an upright position.
      NOTE: After pipetting, the stratification should be visible.
  2. Bottom-up DGC mode
    1. Place a 31 mL ultracentrifuge tube on a foam plate with holes and label it as "↑".
    2. Vertically add 2.5 mL of the 60% iodixanol stock solution to the bottom of the tube. Mix it gently with 500 µL of PBS/BEV solution isolated from Step 4 using a 1,000 µL pipette to obtain 3 mL of 50% iodixanol/BEV solution.
    3. Tilt the tube to a 70° angle and position a tube holder or other support slightly above the level of the foam plate, below the opening of the tube.
    4. Add 3 mL of the 40% iodixanol solution on top of the 50% iodixanol/BEV solution using a 1,000 µL pipette.
    5. Add 3 mL of the 20% iodixanol solution on top of the 40% iodixanol solution using a 1000 µL pipette.
    6. Add 3 mL of the 10% iodixanol solution on top of the 20% iodixanol solution using a 1000 µL pipette.
    7. Add 3.5 mL of PBS on top of the 10% iodixanol solution using a 1,000 µL pipette.
    8. Gently return the tubes to an upright position.
    9. At this point, 200 µL of the suspension obtained in Step 4.17 remained, which can be analyzed subsequently as a group named "UC".
      ​NOTE: When transferring solutions in Step 6, always keep the pipette tip against the ultracentrifuge tube wall perpendicular to the axis of the tube.

7. Density gradient centrifugation and fraction collection

  1. Adjust the weight of the two tubes with PBS to achieve a total weight within ± 0.005 g.
  2. Place the tubes in the buckets according to Step 4.5 and subject them to centrifugation at 160,000 × g for 7 h at 4 °C.
  3. Collect the fractions using a pipettor (1000 µL) from top to bottom against the side wall in the following order: 3 mL, 2 mL, 1 mL, 1 mL, 1 mL, 1 mL, 1 mL, 1 mL, 1.5 mL, and 3 mL into a 38.5 mL ultracentrifuge tube.
    ​NOTE: Maintain the line of sight at the same level as the liquid surface.
  4. Conduct ultracentrifugation for each fraction obtained in Step 7.3 according to Step 4 at 160,000 × g for 70 min at 4 °C.
  5. Remove the supernatant and invert the ultracentrifuge tubes for 5 min.
  6. Resuspend the pellets in 200 µL of pre-chilled (4 °C) PBS using a 200 µL pipette.
  7. Transfer the PBS/BEV solution into a clean 1.5 mL microtube.
  8. Label the fractions from F1 to F10 and analyze them based on Step 8.
    PAUSE POINT: If the samples obtained in Step 7.8 cannot be processed immediately, store them at -80 °C.

8. Characterization and quantitative analysis of the collected fractions

  1. Determine the absorbance values (OD 340 nm) of each fraction using a microplate detector with a blank control to calculate their corresponding density.
    NOTE: Replace PBS/BEV solution (Step 6.1.1 and Step 6.2.2) with PBS to establish a blank control.
    1. Prepare 100 µL each of 0%, 5%, 10%, 12.5%, and 20% iodixanol solution diluted by 0.02 M HEPES based on 50% stock solution (Step 5.2.2) as the standards, corresponding to densities of 1.0058 g/mL, 1.0318 g/mL, 1.058 g/mL, 1.0708 g/mL, and 1.111 g/mL, respectively.
    2. Add 50 µL of each fraction from the blank control (prepared in Step 7.3) and 50 µL of each standard solution (prepared in Step 8.1.1) to duplicate wells of a 96-well plate.
    3. Set the wavelength to 340 nm, measure the endpoint optical density, and calculate the density of each fraction.
    4. Identify F4-F9 as the range of BEV fractions based on their density.
  2. Determine the protein concentration of the BEV fractions using the bicinchoninic acid assay (BCA).
    1. Dilute the substance tenfold in PBS provided by the manufacturer to prepare a 0.5 µg/µL standard protein solution.
    2. Add the standard protein solution (prepared in Step 8.2.1) with varying volumes according to the reagent instructions, and supplement each well of a 96-well plate with PBS to a total volume of 20 µL.
    3. Add 20 µL of the samples prepared in Step 7.8 and the samples left after UC defined in Step 6.2.9 to each well.
    4. Mix reagents A and B in a ratio of 50:1, and transfer 200 µL to each well.
    5. Incubate the plate in a 37 °C water bath for 30 min.
    6. Measure the absorbance value using a microplate reader, calculate the protein concentration (µg/µL), and determine the protein content (µg) of the samples based on the standard curve (x: optical density; y: protein concentration) generated from the values of the standard solutions diluted in Step 8.2.2.
  3. Characterize the BEV fractions defined in Step 8.1.4 using transmission electron microscopy (TEM) to verify the presence of BEVs. All procedures below should be performed on ice.
    1. Apply 10 µL of each isolated F4-F9 fraction from Step 7.8 and the samples left after UC defined in Step 6.2.9 onto Formvar/Carbon supported copper grids for 20 min, and blot with filter paper.
    2. Rinse the samples with 100 µL of PBS three times for 1 min each, and blot with filter paper.
    3. Fix the samples with 100 µL of 1% (w/v) glutaraldehyde for 5 min and blot with filter paper.
    4. Wash the grids with 100 µL of PBS ten times for 2 min each, and blot with filter paper.
    5. Stain the grids with 50 µL of 1.5% (w/v) uranyl acetate for 10 min, and blot with filter paper.
      CAUTION: Uranyl acetate is radioactive and extremely toxic when in contact with the skin or inhaled. Handle solutions with uranyl acetate in a fume cupboard and follow appropriate safety measures.
    6. Transfer the grids onto a 1% (w/v) methylcellulose drop for 5 min and blot with filter paper.
    7. Store the air-dried grids in a dark, dust-free environment until observation.
    8. Capture images and analyze them using a TEM.
  4. Characterize the BEV fractions defined in Step 8.1.4 using nanoparticle tracking analysis (NTA) to count the number of particles.
    1. Calibrate the system using 110 nm polystyrene particles.
    2. Wash the sample pool using PBS.
    3. Dilute the samples from different fractions acquired in Step 7.8 and the samples left after UC defined in Step 6.2.9 to a concentration in the range of 105-109 particles/mL, with an optimized concentration of 107 particles/mL.
    4. Record and analyze at 11 positions with three replications, maintaining the temperature around 23-30 °C.
  5. Analyze the protein contents of the samples by coomassie brilliant blue staining (CBBS) and western blotting (WB).
    1. Dilute the BEVs samples from different fractions obtained in Step 7.8 and the samples left after UC defined in Step 6.2.9 using PBS and 5 × loading buffer to a final concentration of 0.5 or 1 µg/µL, if quantitation is needed, ensuring sufficient sample loading of 20 µL (10 µg or 20 µg) of each well in Step 8.5.2. Boil the samples at 95 °C for 10 min.
    2. Assemble the prefabricated polyacrylamide gel into the electrophoresis tank, and transfer the samples to the wells.
    3. Conduct vertical electrophoresis with the following parameters: 160 V for 50 min.
    4. Stain the gel in Coomassie brilliant blue solution for 1 h, rinse in deionized water until the blue background lightens, and capture images with a camera.
    5. Perform protein blotting procedures for other gels with the following parameters: 400 mA for 20 min.
    6. Block the blotting membranes with a 5% (w/v) BSA solution for 1 h at room temperature.
    7. Wash the membranes in TBST buffer three times for 5 min each.
    8. Incubate the membranes with primary antibodies (BEV markers: LPS, OmpA, LTA; EEV markers: CD63, CD9, TSG-101, Syntenin, Integrin β1; Other contamination markers: Flagellin, Calnexin) for 8-12 h at 4 °C.
    9. Wash the membranes in TBST buffer three times for 10 min each.
    10. Incubate the membranes with secondary antibodies for 1 h at room temperature.
    11. Perform Step 8.5.9.
    12. Develop the blotting using a chemiluminescence apparatus.
    13. Replace PBS/BEV solution (Step 6.1.1 and Step 6.2.2) with BEVs from Escherichia coli to establish a positive control (see Table of Materials for the procedure of isolating crude E. coli-BEVs), and conduct all the above experiments for characterizing BEVs.
      PAUSE POINT: Determine F6-F8 as the distribution of BEV-enriched fractions based on the results of the characterization described above for fecal BEVs, and utilize this narrowed range for subsequent analysis.
  6. Calculate the recovery rates in terms of particles and proteins to assess the efficiency of the two DGC modes. The results below are presented as percentages.
    1. Calculate the recovery rates of particles in the Top-down mode: total particles in F6-F8/particles after UC defined in Step 6.2.9.
    2. Calculate the recovery rates of particles in the Bottom-up mode: total particles in F6-F8/particles after UC defined in Step 6.2.9.
    3. Calculate the recovery rates of proteins in the Top-down mode: total protein contents in F6-F8 (µg)/protein content after UC defined in Step 6.2.9 (µg).
    4. Calculate the recovery rates of proteins in the Bottom-up mode: total protein contents in F6-F8 (µg)/protein content after UC defined in Step 6.2.9 (µg).
  7. Calculate the particle/protein ratio to assess the purity traditionally defined of UC and the two DGC modes, and the results below were all presented as percentages.
    1. Particle/protein ratio after UC defined in Step 6.2.9: particles after UC/protein content after UC (µg).
    2. Particle/protein ratio in the Top-down mode: total particles in F6-F8/protein content in F6-F8 (µg).
    3. Particle/protein ratio in the Bottom-up mode: total particles in F6-F8/protein content in F6-F8 (µg).
  8. Select the most suitable centrifugation mode (Top-down mode was selected in the protocol) for fecal BEV purification and future analysis.

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结果

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Determine the distribution of BEV-enriched fractions
To determine the distribution of bacterial extracellular vesicles (BEVs)-enriched fractions, a blank control was established to measure the absorbance values at OD 340 nm, and the density of each fraction was calculated based on the measurements and iodixanol guidelines (Step 8.1). Table 2 presents the density results, demonstrating that fractions F4 to F9 exhibited densities within the range typically associated with extracellular...

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讨论

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Bacterial extracellular vesicles (BEVs) are lipid-bilayer nanoparticles secreted by bacteria, carrying a wealth of proteins, lipids, nucleic acids, and other bioactive molecules, contributing to mediating the functional effects of bacteria20. BEVs derived from the gut have been verified to be involved in the development of diseases, such as inflammatory bowel disease, Crohn's disease, and colorectal cancer, and also affect general metabolism and mediate impaired cognitive function

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披露

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The authors declare no conflicting interests.

致谢

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This work was supported by the National Science Fund for Distinguished Young Scholars (82025024); the Key project of the National Natural Science Foundation of China (82230080); the National Key R&D Program of China (2021YFA1300604); the National Natural Science Foundation of China (81871735, 82272438, and 82002245); Guangdong Natural Science Fund for Distinguished Young Scholars (2023B1515020058); the Natural Science Foundation of Guangdong Province (2021A1515011639); the Major State Basic Research Development Program of Natural Science Foundation of Shandong Province in China (ZR2020ZD11); the Post-doctoral Science Foundation (2022M720059); the Outstanding Youths Development Scheme of Nanfang Hospital, Southern Medical University (2022J001).

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材料

本文使用的材料清单
姓名公司目录编号评论
1 % （w/v） 戊二醛（由去离子水中的 2.5% 储备溶液制备）ACMECAP1126步骤 8.3.3 使用 TEM 对 BEV 进行形态观察
1 % （w/v） 甲基纤维素（由去离子水中的原始粉末制备）Sigma-AldrichM7027在步骤 8.3.6 中使用 TEM 对 BEV 进行形态观察
1.5 % （w/v） 乙酸铀酰（由去离子水中的原始粉末制备）Polysciences21447-25在步骤 8.3.5 1000 &mu 使用 TEM 对 BEV 进行形态学观察
;L， 200 μL， 10 μL 移液管KIRGENKG1313， KG1212， KG1011转移溶液
5 % （w/v） 牛血清白蛋白溶液（由 TBST 缓冲液中的原始粉末制备）Fdbio scienceFD0030用于蛋白质印迹，用于步骤 8.5.6
5 & 次封闭 上样缓冲液Fdbio scienceFD006用于蛋白质印迹和考马斯亮蓝染色，步骤 8.5.1
75 % （v/v） 酒精LIRCONLIRCON-500 mL表面消毒
96 孔板RarA8096测量吸光度值 
抗钙联蛋白抗体Abcamab92573Western blotting（一抗）
抗 CD63 抗体Abcamab134045Western blotting（一抗）
抗 CD9 抗体Abcamab236630Western blotting（一抗）
鞭毛蛋白抗体翘神州40067-MM06Western blotting（一抗）
抗整合素 β 1 抗体Abcamab30394Western blotting（一抗）
抗 LPS 抗体Thermo FisherMA1-83152Western blotting（一抗）
抗 LTA 抗体Thermo Fisher MA1-7402Western blotting（一抗）
抗 OmpA 抗体CUSABIOCSB-PA359226ZA01EOD，https://www.cusabio.com/Western blotting（一抗）
抗 Syntenin 抗体Abcamab133267Western blotting（一抗）
抗 TSG101 抗体Abcamab125011Western blotting（一抗）
高压灭菌ZEALWAYGR110DP对实验中使用的用品和培养基进行灭菌
平衡梅特勒-托利多AL104平衡装有 PBS
二辛可宁酸分析Fdbio scienceFD2001在步骤 8.2 中测量 BEV 的蛋白质含量
BioRender BioRenderhttps://app.biorender.com制作图 1 中所示的 BEVs 分离和纯化示意图
海尔HR1200-II B2执行粪便样品处理程序
离心机 5810 R;转子 F-34-6-38Eppendorf5805000092; 5804727002， 适配器： 5804774000BEV 前处理（步骤 3）
化学发光仪BIO-OIOI600SE-MF用于蛋白质印迹，用于步骤 8.5.12
Cytation 5BioTekF01微孔板检测器，用于测量吸光度（步骤 8.1）和荧光（图 6）值 
Dil-labled 低密度脂蛋白ACMECAC12038成分分布的定义 
电泳设备Bio-rad1658033用于蛋白质免疫印迹，用于蛋白质分离和转印步骤 8.5.2、8.5.3、8.5.5
增强化学发光试剂盒 HRP Fdbio scienceFD8020用于步骤 8.5.12
Escherichia coli American Type Culture CollectionATCC8739分离 BEV 作为阳性对照。实验方案：将 25 g LB 粉末溶于 1 L 去离子水中，然后高压灭菌。转账 800 μL 的保存大肠杆菌加入培养基中。在 37 度培养;C 在培养箱摇床中。然后以 3， 000 &次离心;g 在 4 ° 下 20 分钟;C， 12， 000 & 次;g 在 4 &°g 下 30 分钟;C，过滤上清液，通过0.22 μm 膜，并以 160， 000 &次进行超高速离心;g 在 4 &°C 下放置 70 分钟。将定义为大肠杆菌粗 BEV 的沉淀悬浮在 1.2 mL PBS 中（步骤 3、4）。   
Falcon 管 50 mLKIRGENKG2811BEV 前处理（步骤 3）
Feto 蛋白染色缓冲液Absciab.001.50步骤 8.5.4 考马斯亮蓝色染色
纸BiosharpBS-TFP-070B步骤 8.3 使用 TEM 对 BEV 进行形态学观察（吸干溶液）
Formvar/Carbon 支撑铜网格 Sigma-AldrichTEM-FCF200CU50在步骤 8.3 使用 TEM 对 BEV 进行形态学观察
HEPES 粉末MeilunbioMB6078制备不同浓度的碘克沙醇缓冲液用于密度梯度离心
HRP AffiniPure 山羊抗小鼠 IgG （H+L）Fdbio 科学FDM007Western blotting（二抗）
HRP AffiniPure 山羊抗兔 IgG （H+L）FdbioscienceFDR007Western blotting （二抗）
培养摇床强文DHZ-L培养大肠杆菌 
Kimwipes&贸易;Delicate Task WipesKimtech Science34155在步骤 4.15 时擦拭超速离心管的内壁
LB 肉汤HopebioHB0128培养大肠杆菌 
低温冷冻机（-80 °C）海尔DW-86L338J储存样品
甲醇AlalddinM116118用于蛋白质印迹，在步骤 8.5.5 处激活 PVDF 膜
管 1.5 mLKIRGENKG2211回收密度梯度离心后的馏分
微量管 2 mLKIRGENKG2911回收密度梯度离心后的馏分
微量管 5 mLBBIF610888-0001回收密度梯度离心后的馏分
酶标仪 Thermo Fisher Multiskan MK3在步骤 8.2
Millipore 过滤器 0.22 &mu 测量 BEV 的蛋白质含量;mMerck millipore SLGP033RB过滤灭菌;材料：聚醚砜、PES
NaClGHTECH1.01307.040密度梯度离心液
NaOHGHTECH1.01394.068密度梯度离心液（pH调节）
优化&贸易;XPN-100Beckman CoulterA94469用于 BEV 分离的超速离心，步骤 4、7
OptiPrep™Serumwerk Bernburg AG1893密度梯度离心储备液
轨道摇YouningCS-100在第 2 步溶解粪便
磷酸盐缓冲盐ProcellPB180327第 2 步溶解粪便
移液器Eppendorf3120000267,3120000259转移溶液
塑料巴斯德移液器ABCbioABC217003-4步骤 3.4 的预处理中去除上清液
聚偏二氟乙烯 （PVDF） 膜MilliporeISEQ00010，IPVH00010在步骤 8.5.5 用于蛋白质转印的蛋白质印迹
预制聚丙烯酰胺凝胶，4–20% 15 孔ACEF15420凝胶用于蛋白质印迹，用于步骤 8.5.2、8.5.3
一抗稀释剂Fdbio scienceFD0040用于步骤 8.5.8
蛋白质分子量标准Fdbio 科学FD0672用于蛋白质印迹和考马斯亮蓝染色 步骤 8.5
快速蛋白质印迹液UBIOUW0500用于步骤 8.5.5 蛋白质转印的蛋白质印迹
转子 SW 32 Ti 水平转子Beckman Coulter369650用于步骤 4、7 的 BEV 分离的超速离心
注射器 20 mL、50 mL JetwayZSQ-20ML， YCXWJZSQ-50 mL转移缓冲液 amd 在预处理中去除上清液
TBS 粉末Fdbio scienceFD1021用于步骤 8.5 的蛋白质印迹
透射电子显微镜 （TEM）日立 H-7650步骤 8.3 中 BEV 的形态学观察
吐温-20Fdbio 科学FD0020用于步骤 8.5 的蛋白质印迹
超速离心管Beckman326823,355642步骤 4、7 中分离 BEV 的超
净工作台AIRTECHSW-CJ-2FD执行液体处理程序
水浴BluepardCU600用于在步骤 8.2.5 中测量 BEV 的蛋白质含量
ZetaViewParticle MetrixS/N 21-734， 软件 ZetaView （版本 8.05.14 SP7）纳米粒子跟踪分析 （NTA） 用于在步骤 8.4 中测量 BEV 的粒径和浓度
义器的试管样品  的信号检测 干扰滤 微量床 水 在 在

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