Name: Separation and Differential Characterization of Gut Microbial Extracellular Vesicles in Salt-Sensitive Rats under High-Salt Diet Conditions (Video) | JoVE Journal
Uploaded: 2025-06-06T15:45:00.000+00:00
Duration: 7 min 21 s
Description: Learn to isolate and characterize gut microbial extracellular vesicles from salt-sensitive rats using density gradient centrifugation. This method enables reliable profiling of vesicle size, morphology, and composition for hypertension research.

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5 minutes

This work was supported by the National Natural Science Foundation of China (82205240), the Natural Science Foundation of Sichuan Province (2025ZNSFSC1836), and the Clinical Basic Project of Sichuan Provincial Orthopedic Hospital (PY202414).

This animal experimental study complies with the relevant ethical guidelines and international standards. Studies involving animals were approved by the Laboratory Animal Welfare and Ethics Committee of Chengdu University of Chinese Medicine (institution: Chengdu University of Chinese Medicine; protocol number: 2018-21).
1. Animal preparation and diet regime
<ol>
	<li>Acclimatize 6-week-old male Dahl salt-sensitive rats, body weight 180-200 g, by rearing at 50% &#177; 10% humidity; 12 h / 12 h light cycle; 20.0 &#177; 2.0 &#176;C for 1 week under specific pathogen-free conditions.</li>
	<li>Subsequently, divide the rats equally into two groups (n = 6). Provide each group a different diet that lasting for 6 weeks: a normal salt diet (NSD) with 0.5% sodium chloride and an HSD with 8% sodium chloride, which is commonly used in published studies29,30,31. The mice had unlimited access to food and water throughout the study.</li>
</ol>
2. Monitoring of blood pressure
NOTE: Tail cuff plethysmography was used as a non-invasive method for blood pressure measurement, and volumetric pressure recording (VPR) was used when blood pressure was measured from tail blood volume.
<ol>
	<li>Place rats individually in small cages with sawdust, heated for 10 min at approximately 37 &#176;C and place in a custom rodent restraint with an adjustable nose and door for animal entry. Place rats on a heating pad in a prone position to keep their body temperature at 37 &#176;C.</li>
	<li>Connect the VPR sensor to the bottom of the tail and set at least 10 adaptation cycles to stabilize the BP28.
	<ol>
		<li>Fill the inflatable cuff on the tail root and place the plethysmographic sensor (or photoelectric sensor) below. Slowly inflate to above systolic pressure (usually 200 mmHg) and then slowly release. The instrument automatically detects the pressure value of pulse wave disappearance (systolic blood pressure) and recovery (diastolic blood pressure). Measure 3-5 consecutive times and take the average value.</li>
	</ol>
	</li>
	<li>Train all rats to accommodate the restraint procedure and perform this study in a quiet laboratory around 9 am to minimize the effects of circadian variation.</li>
	<li>Measure systolic blood pressure (SBP) and diastolic blood pressure (DBP) every other day before sacrifice. Estimate mean arterial pressure (MAP) from SBP and DBP 
	MAP = (SBP + 2 DBP)/3</li>
</ol>
3. Extraction of EVs
<ol>
	<li>Sample collection
	<ol>
		<li>Dip a sterile cotton swab in 75% alcohol and use it to stimulate the rat&#39;s anus. The stimulation was intended to promote intestinal peristalsis and relax the anal sphincter, thereby promoting defecation. After the feces are expelled, collect them using sterile forceps into appropriately sized sterile containers and immediately store them at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Sample preparation
	<ol>
		<li>Using a spoon, transfer 5 g of feces into a pre-weighed 50 mL centrifuge tube. Add 50 mL of pre-heated 37 &#176;C endotoxin-free PBS (maximum sample concentration 10%) to the tube and rotate for 30 min. Cool the high-speed centrifuge to 4 &#176;C.</li>
		<li>Centrifuge at 8,000 x g for 15 min after placing samples symmetrically to allow the centrifuge to equilibrate. After centrifugation, aspirate the supernatant and transfer it to a new sterile centrifuge tube.</li>
		<li>Centrifuge again at 8,000 x g for 15 min. Aspirate the supernatant and use it for further analysis.</li>
	</ol>
	</li>
	<li>Crude extract preparation
	<ol>
		<li>Use a sterile 0.22 &#181;m filter unit. Place the filter unit on ice and hold the vacuum pump firmly. Transfer the supernatant obtained to the top of the filter. Turn on the vacuum pump to collect the filtered samples.</li>
		<li>Transfer the filtrate to a centrifugal filter (10 kDa, 15 mL), centrifuge at 4 &#176;C, 3,000 x g for 30 min, and concentrate the sample to at least 1400 &#181;L.</li>
		<li>Collect the concentrated sample and, if necessary, dilute it to 1400 &#181;L using pre-cooled endotoxin-free PBS. Keep the sample on ice immediately after dilution. 
		&#8203;NOTE: The crude extract can be used immediately or stored at -80 &#176;C for several months.</li>
	</ol>
	</li>
	<li>Separation of EVs
	<ol>
		<li>Prepare the gradient buffer A and gradient buffer B as described below. Prepare the buffer one day in advance, as it takes several hours for the compound to dissolve completely. The prepared buffer can be stored at 4 &#176;C for 6 months.
		<ol>
			<li>Preparation of gradient buffer A: Dissolve 0.25 M sucrose, 6 mM EDTA, and 60 mM Tris in 800 mL of endotoxin-free water by magnetic mixer stirring, with the pH adjusted to 7.4 using hydrochloric acid. Dilute to 1 L with endotoxin-free water. Use a 0.22 &#181;m bottle-top filter to filter the buffer.</li>
			<li>Preparation of gradient buffer B: Dissolve 0.25 M sucrose, 1 mM EDTA, and 10 mM Tris in 800 mL of endotoxin-free water by magnetic mixer stirring, with pH adjusted to 7.4 with hydrochloric acid. Diluted to 1 L with endotoxin-free water. Use a 0.22 &#181;m bottle top filter to filter the buffer.</li>
		</ol>
		</li>
		<li>Mix 1 volume of gradient buffer A and 5 volumes of density gradient medium (the medium is 60% iodixanol solution) to prepare the working solution.</li>
		<li>Preparation of iodixanol solution (10%, 20%, 40%): To prepare a 10% iodixanol solution, mix 1 unit working solution and 4 units buffer B; for 20% iodixanol solution, mix 2 unit working solution and 3 units buffer B; for 40% iodixanol solution, mix 4 units working solution and 1 unit buffer B. Prepare each solution fresh and store on ice. 
		NOTE: Fresh working solutions should be prepared for each experiment, and an additional 10% volume of solutions can be prepared to account for the experimental error.</li>
		<li>Mix the solution prepared in step 3.3.3 with 7 mL of density gradient medium to make a 50% iodixanol solution.</li>
		<li>To prepare a density gradient, add trypan blue solution to 40% solution and 10% (wt/vol) iodixanol solution to provide a clear outline between the distinct layers.</li>
		<li>Transfer 8 mL of 50% iodixanol solution to the bottom of the thin-wall polypropylene centrifuge tube. Tilt the tube to 70&#176; and transfer 8 mL of 40% iodixanol solution to the liquid surface. Add 8 mL of 20% iodixanol solution, then 7 mL of 10% iodixanol solution, and finally 2 mL of endotoxin-free PBS. 
		NOTE: Preparing a density gradient requires practice. The quality of the density gradient has a great impact on the experimental results. Do not place the tube upright between different steps in the addition process, as this negatively impacts the quality of the density gradient.</li>
		<li>Precool the centrifuge in advance and place the prepared density gradient into the ultracentrifuge. Input parameters to start, parameter values: 100,000 x g, 20 h, 10 &#176;C.</li>
		<li>For manual gradient density separation, slowly draw 2 mL of the solution from the center of the surface of the system containing a total of 34 mL of liquid. Each 2mL is a gradient, thus dividing the density gradient solution into 17 gradients.</li>
		<li>Transfer the density fractions to a sterile sample tube using a pipette and immediately place them on ice. Clamp the centrifuge tube between the thumb and index finger to ensure that it remains upright.</li>
	</ol>
	</li>
	<li>Recovery of EVs
	<ol>
		<li>Using a fully automated EVs extraction system that applies negative pressure oscillation through a double coupling ultrasonic oscillation system on the ultrafiltration chip, remove nucleic acid and protein impurities in the sample through the nanopore and intercept EVs, leading to enrichment and purification of EVs.
		<ol>
			<li>Remove the filtrate in step 3.4.7 and add it to the ultrafiltration chip.</li>
			<li>Operate the instrument according to the instrument instructions and collect the filtered filtrate, namely the exosome-enriched solution. The main process is shown in Figure 1.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. EVs identification
<ol>
	<li>Particle size and concentration detection
	<ol>
		<li>Evaluate the size distribution and zeta potential using a resistive pulse sensing (RPS) -based nanocoulometer counter. Select nanopore chips with a measurement range of 60-200 nm for this experiment. Further, evaluate the particle size distribution and zeta potential of the EVs using a nanolibrary counter.
		<ol>
			<li>Take an appropriate amount of EVs solution and dilute it in (1/10, 1/100, 1/1000, 1/10000).</li>
			<li>Different dilutions of EVs solutions were placed on the instrument for testing.</li>
			<li>The dilution multiple with better stability was selected and tested again to obtain more accurate data.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>LPS and BCA assay
	<ol>
		<li>BCA assay: Lyse EVs using RIPA buffer and then separate the proteins. Determine the protein content of the EVs using the BCA protein concentration determination kit.</li>
		<li>LPS assay: Using the endotoxin detection kit, add samples, an endotoxin detector, and chromogenic according to the kit steps. Measure the absorbance at 545 nm and calculate the LPS expression of external vesicles according to the standard curve.</li>
	</ol>
	</li>
	<li>TEM evaluation: For negative staining and TEM analysis, apply samples to a luminescent copper grille coated with continuous carbon film and stain with 0.75% uranyl formate. For cryo-EM, absorb the EVs into a 300-mesh EM carbon grid with a hydrophilic surface and freeze in liquid nitrogen. Observe and analyze the grids using the Cryo-TEM and record the images at 22,000 magnifications32.</li>
	<li>Characterization of proteins: Lyse EVs using RIPA buffer and then separate the proteins. Determine protein concentration using the BCA protein assay kit, separate equal amounts of protein from each sample by SDS-PAGE, and stain with Coomassie blue dye solution after electrophoresis to visualize the protein profiles33,34.</li>
	<li>S sequencing and analysis: Extract genomic DNA from the fecal samples using a fecal DNA extraction kit. Assess the integrity and concentration of DNA by agarose gel electrophoresis and a spectrophotometer. Amplify the V3 - V4 region of the 16S rRNA gene with universal primers, purify the PCR products, quantify them, and sequence them on the Illumina platform. Perform data analysis using the QIIME2 pipeline35.
	<ol>
		<li>Classify amplicon sequence variants (ASVs) using DADA2 and match against the Silva database using the VSEARCH algorithm. Use &#945;-Diversity analysis to assess species richness and diversity within the samples, mainly using indices such as the Shannon index and the Chao1 index. &#946;-Diversity analysis compares the microbial community composition between samples and is usually visualized by principal coordinate analysis (PCoA). Use differential analysis methods, such as linear discriminant analysis effect size (LEfSe) and volcano plot analysis, to identify microbial taxa with significantly different conditions or between groups.</li>
	</ol>
	</li>
</ol>

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<li>Matsumoto, H., Haniu, H., Komori, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determination+of+Protein+Molecular+Weights+on+SDS-PAGE%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Determination of Protein Molecular Weights on SDS-PAGE.</a> Methods Mol Biol. 1855, 101-105 (2019).</li>
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

In this study, we focused on the gut microbiota EVs in salt-sensitive rats on an HSD and achieved a series of key achievements. First, an efficient extraction method based on density gradient centrifugation was successfully constructed to isolate EVs from salt-sensitive rat gut microbiota on HSD, and most non-EV components were isolated through a meticulous, standardized animal experimental manipulation and sample processing process. The EVs extraction method of density gradient centrifugation is highly efficient and rep...

The gut microbiota, also known as gut microbiota microflora or gut microecology, is a complex of tens of thousands of microorganisms located in the biological gastrointestinal tract and plays a crucial role in the maintenance of human health1. In recent years, with further research, it has been found that the gut microbiota can produce extracellular vesicles (EVs)2. EVs are small vesicles released by cells, which carry various molecules in the cell, such as proteins, nucleic acids, and lipids3,4. They can interact with other microbes5, intestinal epithelial cells, and even distant tissues and organs6, thus affecting the health of the human body7,8. There is a tight link between the EVs produced by these gut microbiota and diet9.
EVs produced by gut microbiota may be significant agents through which a high-salt diet (HSD) affects body health. HSD not only directly disrupts the balance of gut microbiota10, leading to a significant reduction in the number of beneficial bacteria (such as Lactobacillus)11, but also promotes the proliferation of harmful bacteria (such as Bacteroides, etc.)12. This imbalance reduces the intestinal barrier function and increases the risk of intestinal inflammation. In addition, an HSD also further affects the acid-base balance and nutrient absorption in the intestine by changing the metabolic activities13 of the gut microbiota, such as reducing the production of short-chain fatty acids14,15 with multiple physiological functions.
These changes not only impact intestinal health but may also indirectly regulate the production and release of EVs and alter the EV's composition and function. The high salt environment may affect the normal physiological functions of intestinal cells, including the release and transport of EVs, thereby disturbing the role of EVs in intercellular information transmission and immune regulation. At the same time, intestinal inflammation may promote a variety of EVs with special functions16 and spread to the whole body through the intestinal-organ axis and other ways17,18, which is closely related to the occurrence and development of hypertension19,20, cardiovascular21 and cerebrovascular diseases22,23, obesity24,25, diabetes26 and other chronic diseases.
Therefore, the overall goal of this study was to develop an efficient and reliable method to extract EVs from the gut microbiota of salt-sensitive rats fed an HSD and to systematically study their physical properties, composition, and functions. Due to the characteristics of the significant increase in blood pressure after a high-salt diet, salt-sensitive rats were selected and revealed the effect of HSD on gut microbiota EV by constructing an efficient extraction method. The method was based on density gradient centrifugation and combined various dynamic identification techniques such as particle size detection, LPS/BCA measurement, transmission electron microscopy, and proteomic analysis. The protocol aims to reveal the effects of HSD on gut microbiota EV and its mechanisms in cardiovascular disease. With its high efficiency, reproducibility, and broad applicability, this approach not only provides an important tool for exploring the mechanism of gut microbiota EVs in salt-induced hypertension but also lays the theoretical foundation for developing disease intervention strategies based on EVs. Through this study, we hope to open up new avenues for the prevention and treatment of cardiovascular diseases, such as hypertension27,28.

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separation and differential characterization of gut microbial extracellular vesicles in salt-sensitive rats under high-salt diet conditions

Separation and Differential Characterization of Gut Microbial Extracellular Vesicles in Salt-Sensitive Rats under High-Salt Diet Conditions

High salt intake is a major risk factor for hypertension, and its underlying mechanism may be closely linked to extracellular vesicles (EVs) secreted by gut microbiota. These EVs, produced by gut microbiota, carry various bioactive components that may play a crucial role in the development of hypertension induced by a high-salt diet (HSD). To investigate this mechanism, we developed an efficient extraction method based on density gradient centrifugation to isolate EVs from the gut microbiota of salt-sensitive rats fed an HSD. Through particle size analysis, transmission electron microscopy (TEM), and lipopolysaccharide (LPS) detection, we identified the gradient distribution of gut microbiota EVs and achieved precise extraction. Furthermore, 16S rRNA gene sequencing was employed to analyze the origin and compositional differences of EVs between the normal and HSD groups, revealing the impact of high salt intake on the genetic characteristics of gut microbiota EVs. This study provides valuable tools and scientific insights into the gut microbiota mechanisms underlying salt-induced hypertension and offers new perspectives for the prevention and treatment of related diseases.

EVs concentrations were determined in different fractions (Figure 2A). The experimental results showed that the concentration of the EVs exhibited a typical normal distribution pattern in a series of density gradient solutions (Figure 2B). Specifically, in fraction 9, the concentration of EVs reached its highest point (3.85 x 109), suggesting that the main distribution fraction of EVs may be 936.
For...

The protocol describes the isolation of gut microbial EVs from salt-sensitive rats fed HSD using density gradient centrifugation. EVs were characterized by nanoparticle tracking, TEM, LPS/BCA assays, and 16S rRNA sequencing to analyze the size, morphology, composition, and microbiota origin.

High salt intake is a major risk factor for hypertension, and its underlying mechanism may be closely linked to extracellular vesicles (EVs) secreted by ...

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JoVE Journal

Biochemistry

Source: Cian, M. B. et. al., Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii. J. Vis. Exp. (2020)This video demonstrates the separation of the inner and outer membrane fractions of Gram-negative bacteria using sucrose gradient ultracentrifugation. The membrane fractions are purified and stored at low temperatures for targeted analysis of membrane-specific components.

Separation of Inner and Outer Membrane Fractions of Gram-Negative Bacteria

Source: Watson, D. C., et.al. Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria. J. Vis. Exp. (2021)This video demonstrates the isolation and concentration of extracellular vesicles (EVs) derived from the outer membrane of genetically engineered bacteria. The process involves culturing the bacteria to release EVs, followed by sequential centrifugation and filtration steps to remove cells and debris. Finally, ultrafiltration is used to...

Isolation and Enrichment of Outer Membrane-Derived Extracellular Vesicles from Bacteria

In this video, we demonstrate a technique–size exclusion chromatography–to separate a heterogenous population of outer membrane vesicles or OMVs derived from bacteria. These vesicles are nanosized spherical structures and are separated into large and small subpopulations based on their size through Size Exclusion Chromatography.

Size Exclusion Chromatography Based Separation of Bacterial Extracellular Vesicles: A Technique to Separate Heterogenous Population of Gram-Negative Bacterial Outer Membrane Vesicles Based on Their Size

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

Bacterial vesicles play important roles in pathogenesis and have promising biotechnological applications. The heterogeneity of vesicles complicates analysis and use; therefore, a simple, reproducible method to separate varying sizes of vesicles is necessary. Here, we demonstrate the use of size exclusion chromatography to separate heterogeneous vesicles produced by Aggregatibacter actinomycetemcomitans.

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

The present protocol providesdetailed descriptions for isolation, concentration,and characterization of extracellular vesicles from cyanobacterial cultures. Approaches for purifying vesicles from cultures at different scales, trade-offs among methodologies, and considerations for working with field samples are also discussed.

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

The goal here is to outline a protocol to investigate the mechanisms of dysbiosis in cardiovascular disease. This paper discusses how to aseptically collect and transplant murine fecal samples, isolate intestines, and use the "Swiss-roll" method, followed by immunostaining techniques to interrogate changes in the gastrointestinal tract.

Murine Fecal Isolation and Microbiota Transplantation

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.

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

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations' survivability.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Learn to isolate and characterize gut microbial extracellular vesicles from salt-sensitive rats using density gradient centrifugation. This method enables reliable profiling of vesicle size, morphology, and composition for hypertension research.

Separation and Differential Characterization of Gut Microbial Extracellular Vesicles in Salt-Sensitive Rats under High-Salt Diet Conditions (Video) | JoVE Journal

Separation and Differential Characterization of Gut Microbial Extracellular Vesicles in Salt-Sensitive Rats under High-Salt Diet Conditions

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