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

This research explores the role of gut microbial extracellular vesicles in salt-sensitive hypertensive rats on a high-salt diet, examining how a high-salt diet alters vesicles and influence hypertensive and immune interactions. Recent advances in this field include understanding the role of gut microbial extracellular vesicles in hypertension, assessing the impact of high-salt diets on microbial diversity and implementing plant-based interventions targeting vesicles from metabolic and immune regulation. Current experimental challenges include isolating high priority extracellular vesicles, distinguish also derived vesicles from microbiome vesicles, minimizing sample loss in the standard dyeing protocols to ensure reproducibility across studies. This density grid and centrifugation protocol provides higher priority to extracellular vesicles with improved integrity and reproducibility compared to tradition methods, enabling more reliable gut microbiome extracellular vesicle research in hypertension studies.

[Narrator] To begin, transfer five grams of feces into a pre-weighed 50 milliliter centrifuge tube. Add 50 milliliters of preheated 37 degrees Celsius endotoxin-free PBS to the tube and rotate the tube for 30 minutes. Cool the high speed centrifuge to 4 degrees Celsius. Next place the sample symmetrically in the centrifuge and spin at 8,000G for 15 minutes. After centrifugation, aspirate the supernatant and transfer it into a new sterile centrifuge tube. After centrifuging again for 15 minutes, aspirate the supernatant carefully and use it for further analysis. Place a sterile 0.22 micrometer filter unit on ice. Transfer the supernatant onto the top of the filter and turn on the vacuum pump to collect the filtered samples. Transfer the filtrate into a centrifugal filter with a 10 kilodalton cutoff and a 15 milliliter capacity. Centrifuge at 4 degrees Celsius 3000G for 30 minutes to concentrate the sample to at least 1,400 microliters. Immediately place the sample on ice. Mix one volume of gradient buffer A with five volumes of density gradient medium to prepare the working solution. Prepare a 10% iodixanol solution by mixing one unit of working solution with four units of buffer B. Similarly, mix two units of working solution with three units of buffer B for a 20% iodixanol solution and four units of working solution with one unit of buffer B for a 40% iodixanol solution. Now mix the concentrated sample prepared earlier with seven milliliters of density gradient medium to make a 50% iodixanol solution. To prepare a density gradient, add trypan blue solution to the 40% and 10% iodixanol solutions. Transfer eight milliliters of 50% iodixanol solution to the bottom of a thin wall polypropylene centrifuge tube. Tilt the tube to 70 degrees and transfer eight milliliters of 40% iodixanol solution to the liquid surface. Next, add eight milliliters of 20% iodixanol solution, followed by seven milliliters of 10% iodixanol solution and two milliliters of endotoxin free PBS. Take the prepared density gradient tube and place it into a pre-cooled ultracentrifuge. Set the ultracentrifugation parameters to 100,000G 20 hours and 10 degrees Celsius and start the run. For manual gradient density separation for the collection, the density gradient is about 34 milliliters of liquid. Two milliliters of solution are slowly extracted from the center of the liquid surface, and each two milliliter solution is a gradient. So the density gradient solution is divided into 17 gradients. Finally, using a pipette, transfer the density fractions into sterile sample tubes and immediately placed them on ice. Nano particle tracking analysis revealed the size, distribution, and concentration profiles of extracellular vesicles collected from different density gradient fractions. The majority of extracellular vesicles across all fractions were concentrated within the 30 to 100 nanometer size range following a typical distribution pattern. The concentration of extracellular vesicles reached its highest level in fraction nine, peaking at approximately 3.85 times 10 to the power of nine particles per milliliter. The protein content, measured using a bicinchoninic acid kit, was also highest in fraction nine at 0.417 micrograms per microliter. Additionally, lipopolysaccharides expression levels, determined using an endotoxin detection kit, were significantly elevated in fractions nine and 10. Greater amounts of protein were also observed in fraction nine during sodium dodecyl sulfate poly acrylamide gel electrophoresis analysis. Transmission electron microscopy, or TEM images, revealed the extracellular vesicles possessed as circular membrane-like morphology.