The authors acknowledge the support of the i3S Scientific Platforms &#34;Biointerfaces and Nanotechnology&#34;, and &#34;Histology and Electron Microscopy&#34;, a member of the national infrastructure Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122). We also thank Prof. J. A. Gonzalez-Reyes (University of C&#243;rdoba, Spain) for helping optimize the ultra-thin section staining protocol for TEM, and Dr. Cec&#237;lia Dur&#227;es and Dr. Ana Rita Pinto (University of Porto, Portugal) for the nanoparticle tracking analysis.
This work was funded by the US National Science Foundation (OCE-2049004 to SJB), by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal, 2020, and by Portuguese funds through Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia/Minist&#233;rio da Ci&#234;ncia, Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-029540 (PTDC/BIA-OUT/29540/2017 to PO). Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia is also greatly acknowledged for the PhD fellowship SFRH/BD/130478/2017 (SL), and FCT Investigator grant IF/00256/2015 (PO). M.C.M.-M. was supported by a Marie Sk&#322;odowska-Curie Individual Fellowship (Reintegration panel) within the Horizon 2020 Framework Programme (H2020-MSCA-IF-2018-RI-844891).

The authors declare no conflicts of interest.

General considerations 
The protocol (Figure 1) is presented as a set of options to highlight that there is no &#39;one-size-fits-all&#39; method for working with cyanobacterial extracellular vesicles. Interested researchers can utilize the sections of this protocol that are compatible and appropriate for their particular model organism, experimental questions/goals, and equipment availability. All vesicle isolation approaches involve trade-offs and will unavoidably result in some degree of bias. While one should seek to minimize this whenever possible, the most crucial consideration is to ensure that the detailed methodology used is reported according to appropriate MISEV (Minimal Information for Studies of Extracellular Vesicles) guidelines36.
Culture growth 
Cyanobacterial cultures can be readily obtained from one of the many culture collections available worldwide. A few examples are the Roscoff Culture Collection (Station Biologique de Roscoff, France), the Pasteur Culture Collection (Institute Pasteur, Paris, France), and the Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA, Maine, USA). The cyanobacterial strain of choice must be cultivated in the appropriate medium and environmental conditions, varying significantly among different strains. A list of commonly used media for cyanobacterial cultivation can be found at culture collection websites or other publications37,38,39.
Working with cyanobacterial extracellular vesicles presents some unique challenges compared with the methodologies reported for many typical model laboratory heterotrophs. Cyanobacterial cultures have vesicle concentrations of orders of magnitude lower than those found in other microbes such as Escherichia coli14,16,40. These differences, presumably arising from lower cell densities and/or vesicle production rates, mean that relatively large cultures (~1-20 L or more) may be required to yield sufficient material for bulk analyses. Thus, researchers are encouraged to test vesicle yields from smaller-scale cultures to determine how much material will be necessary to achieve their desired end goals. The importance of establishing whether the media used for the experiment has a detectable particulate background also needs to be stressed before beginning any experiment, as that material can potentially confound vesicle population quantification, reduce the sensitivity of vesicle concentration/size measurements, or contaminate the final vesicle preparations.
Another challenge for the field is that not all cyanobacteria grow well, or at all, in pure culture. Until rendered axenic, interpretations of physical vesicle characteristics, production rates, or contents cannot necessarily lead to clear conclusions about vesicles produced by any one strain in the community. Researchers are also advised to consider what other types of particles could be present and potentially confused with vesicles by specific nanoparticle analysis tools, which cannot necessarily discriminate between different particle types. For instance, it may be essential to verify that the strain being used lacks a prophage, either via genome sequencing, induction assays, or other means or does not release other types of particulate matter. In our experience, most cyanobacterial vesicles are &#60;0.2 &#181;m in diameter, but when looking at a new strain or growth condition, one should confirm whether using a 0.45 &#181;m pore-size filter alters the size distribution of purified particles.
Many aspects of culture conditions can influence vesicle production and its contents41,42. Thus, the physical and chemical conditions used for culture growth (including light irradiance, temperature, and media composition) need to be documented and controlled to the degree possible to ensure the reproducibility of results. Any chemical analysis of vesicle contents must account for background composition, particularly when carrying out &#39;-omics&#39; style high-throughput analyses. This can be especially critical when using undefined media, such as those based on a natural seawater background or supplemented with yeast extract or tryptone. Using a defined growth medium may be preferable depending on the experimental goals.
Researchers need to carefully monitor culture growth dynamics at regular intervals to ensure that they know where in the growth phase a given batch culture is and not just collect samples after an arbitrary amount of time. Vesicle composition can vary across growth phases, particularly between exponential and stationary phases41,42. For instance, at least some fraction of vesicles sampled in the stationary phase may arise from a different cellular mechanism, such as cell lysis, that will not occur during exponential growth. While this may still be of great biological interest, it is essential to know the sample. If a cyanobacterial culture reaches the desired growth phase at a time when it is not possible to proceed directly to sample concentration, separating the cells from the &#60;0.2 &#181;m fraction immediately (with centrifugation and/or direct 0.2 &#181;m filtration), and then storing the cell-free filtrate at 4 &#176;C is recommended. The material can be stored in this way for days with little to no noticeable impact on the concentration or size distribution of vesicles.
Vesicle purifications 
The frequent need to isolate vesicles from significant volume cultures is vital in the cyanobacterial vesicle isolation workflow. When working with larger volumes of material, the vesicles will need to be concentrated before downstream separation workflows. Concentrators (tangential flow filter membranes or centrifugal columns) with a nominal molecular weight limit of 100 kDa are generally advised, as they allow separation from soluble material with low molecular weight while keeping concentration times reasonable, but 30 kDa filters are also frequently used with success. While several non-ultracentrifugation-based methods of purifying vesicles (e.g., size exclusion chromatography, microfluidic-based systems, affinity-capture techniques, and precipitation-based approaches) are becoming popular in the extracellular vesicle field, in our experience, these approaches can result in decreased yields and are typically incompatible with the culture volumes needed.
Researchers need to consider the composition of the iodixanol background and the wash/resuspension buffers used during vesicle purification to ensure that they are compatible with the desired downstream applications. In many cases, the final vesicle sample can be resuspended in growth media or a defined buffer comparable in composition to the growth medium (e.g., natural vs. artificial seawater). However, this may not be possible with marine cyanobacterial vesicles, which will require further experimental manipulation for analysis, as high salt concentrations similar to seawater levels can inhibit many enzymatic reactions. In such cases, standard laboratory buffers such as 0.2 &#181;m filtered, 1x PBS typically work well for maintaining the stability of marine cyanobacterial vesicles and can be more compatible with downstream experimental processes.
Density gradient purification can be considered optional depending on the experimental goals and culture composition but is strongly recommended for producing a more rigorously pure and reproducible sample. EV populations are heterogeneous and can be found across a range of buoyant densities, which further vary by strain, growth conditions, and other factors4,5,6. The densities listed above represent those typically found for vesicles from cyanobacterial cultures and field samples in iodixanol, but results in other strains may vary. Other density gradient materials such as sucrose and CsCl can be utilized for vesicles, but they will migrate to different buoyant densities in these backgrounds. Different gradient media backgrounds can bias the recovery of lipid-enclosed viruses43&#160;and might potentially influence the recovery of vesicles from different strains.
Vesicles can be lost at multiple points through the vesicle isolation, and gradient purification process described here, reducing yields and increasing the amount of starting material needed to achieve a given final vesicle yield for downstream applications. Particular care should be taken when working with vesicle pellets following ultracentrifugation. While some cyanobacterial vesicles can have carotenoids or other compounds which may lend vesicle samples some amount of pigmentation (Figure 2), depending on the strain or amount of material, it is not necessarily expected to be able to visualize the vesicles pellet directly. Be aware of where the pellet is expected to be given the type of centrifuge rotor used. When possible, purified vesicle samples are suggested to be examined by electron microscopy to verify the composition of the final material recovered.
The impact of storage conditions on vesicles and their contents remains an open question. Though it is found that the storage does not have a notable effect on cyanobacterial vesicle size or concentration14, the functionality of isolated vesicle preparations may change over time44. While freeze/thaw cycles should be avoided whenever possible, the impact of freezing samples on overall vesicle numbers and sizes appears to be minimal. One should be aware of the potential for freeze-thaw cycles to influence the composition of vesicle contents, such as the length of vesicle-associated nucleic acids or the stability of proteins.
Vesicles from field samples 
Current methods for isolating extracellular vesicles from natural aquatic environments are conceptually and operationally similar to those described here for large-volume cultures. Still, they can require even greater volumes of material. Such field samples can involve collecting, filtration, and concentration of hundreds to thousands of liters of water to obtain sufficient material for analysis. Depending on the turbidity of the sample being used, incorporation of one or more pre-filtration steps before the 0.2 &#181;m filter may be needed. The specific TFF device used should be appropriate for working with such large volumes in a reasonable amount of time (ideally in the order of hours) and without putting excessive pressure on the sample. In practice, this often involves using a much greater total surface area than might be applied to culture-based samples, as well as larger-diameter tubing to facilitate increased flow rates. This increased filter surface area will likely lead to a marginal increase in particle losses compared to smaller TFF arrangements and a larger final volume of concentrate; however, these concerns must be balanced with considerations of total processing time. For situations such as an extended oceanographic cruise where samples will not return to a lab for many days post-sampling, we recommend carrying out initial 0.2 &#181;m filtration and TFF steps in the field. This smaller volume of concentrated material can then be stored at 4 &#176;C or -80 &#176;C on board (depending on availability and downstream analytical considerations) until it is returned to the lab for final processing.
Isolation and separation of extracellular vesicles from other small particles, both organic and inorganic, can be challenging, and methods for separating different particles are not yet perfect. For instance, iodixanol density gradients may not readily separate all classes of vesicles and viruses present in a given sample. As the types of confounding particles, and their physical properties, will vary between sampling sites, it is currently impossible to provide a protocol that will robustly partition all classes of small aquatic particles. Trial and error are essential, and experimentation with the iodixanol range used in the gradient and ultracentrifugation conditions will be required to maximize the separation; a collection of smaller volume, more finely resolved density fractions may also be required. Depending on the context, using CsCl gradients instead of iodixanol may help separate environmental particles45. Still, the change in osmotic conditions could lead to biases in the final recovered products, as discussed above.
Vesicle characterization 
Nanoparticle analysis instruments are not yet routinely available in microbiology laboratory settings but are becoming increasingly available. All methodologies have pros and cons, and we make no specific endorsement of one platform as being better than all others for cyanobacterial vesicle work; indeed, all have particular trade-offs concerning costs, resolution, ease of use, detection limits, compatibility with different growth media/buffer backgrounds, and data reproducibility. In addition to instruments based on nanoparticle tracking analysis described above, other approaches, including nanoflow cytometry, microfluidic resistive pulse sensing, and tunable resistive pulse sensing, can be applied46,47. Users should be careful to learn the details of their available instrumentation and verify that it works well with their system, as we have encountered difficulties when using some platforms with seawater-based media. We encourage the field to move toward quantitative characterization of vesicle size, concentrations, and production rates. Measuring vesicle concentrations on a fundamental particle per mL basis, and not in terms of protein content or other metrics, will allow for the integration of vesicles into more quantitative frameworks and enable intercomparisons among strains and conditions. Further efforts to improve the ability to calibrate concentration measurements for &#60;100nm particles are needed.
The fact that vesicle production rate measurements are done directly from 0.2 &#181;m filtered culture supernatant to minimize particle losses would be associated with other purification steps described above. However, this does mean that the approach does not necessarily discriminate between actual extracellular vesicles and other small particles found in the cultures. Only counting particles within specific size ranges (e.g., 50-250 nm diameter) may help exclude some outliers, but visual confirmation that pelleted &#60;0.2 &#181;m culture contents appear to be membrane-bound vesicles (by TEM or other approaches) is needed to be able to specifically claim that one is measuring the production of vesicles as opposed to the production of vesicle-like particles.
An essential factor in vesicle characterization is to ensure that the vesicle sample is being analyzed in the appropriate linear sensitivity range of the nanoparticle analysis device. When a sample is too concentrated,&#160;it is straightforward to dilute that material with a clean buffer and re-analyze it. On the other hand,&#160;the relatively low cell and/or vesicle density of some cyanobacterial cultures can sometimes yield vesicle preparations that are below some instruments&#39; detection limits. In cases where this occurs with bulk vesicle purifications, one can consider growing larger volume cultures, re-pelleting and resuspending the final material in a smaller volume, or evaluating whether there is a step in the isolation process where excessive losses are occurring and could be mitigated. When measuring vesicle production rates, the fixes are not necessarily so straightforward. Samples can be concentrated if necessary, but the first one should see whether adjustments to the media could result in higher cell densities or whether sufficiently concentrated samples could be obtained from later time points during the exponential phase where concentrations will be higher.
Limitations 
As with any protocol, vesicle isolation using these approaches has clear limitations. These approaches do not rely on their guarantee that an utterly pure preparation of vesicles will be isolated. Both cultures and field samples may contain other materials, which migrate similarly to vesicles in density gradients. Still, at a minimum, these types of additional purification methodologies are essential for ensuring rigor and reproducibility of vesicle analysis. While we describe vesicle isolation approaches in the context of cyanobacteria, cultures of many other microbes will also contain vesicles at comparatively low concentrations, and the procedures described here should be generally applicable. It is expected that these methods will serve not as a permanent protocol but instead as a starting point for spurring future advancements in working with extracellular vesicles from diverse microbes. Future efforts are required to merge these methods with other approaches such as size exclusion columns or asymmetric field flow fractionation to improve the discrimination and separation of different categories of small particles from cultures and environmental samples. We are also hopeful that these techniques can continue to evolve alongside improvements in nanoparticle characterization technologies to improve the ability to examine heterogeneity within vesicle populations, their contents, and their exact functional roles in the environment.

Extracellular vesicles (EVs) are spherical structures, ranging between ~20-400 nm in diameter, released by virtually all organisms into their surrounding environment1,2,3. Vesicles are delimited by a lipid bilayer and cannot replicate themselves. In Gram-negative bacteria, these structures are thought to primarily arise by &#39;blebbing&#39; of small portions from the outer membrane. Still, other processes, including flagellar motion, cell lysis, and secretion of both inner and outer membrane material, can produce vesicles as well4,5. EVs can contain various biomolecules, including lipids, soluble and membrane proteins, nucleic acids, and metabolites, and can transport this material between cells4,5,6. Given these features, EVs are being studied to understand their possible roles in a wide range of biological processes, including cellular communication, biofilm formation, horizontal gene transfer, host-phage dynamics, and nutrient exchange4,6.
Cyanobacteria are a large and diverse group of Gram-negative bacteria, including unicellular and filamentous organisms. They are of interest from many perspectives, including understanding their physiology and diversity7,8, the critical ecosystem functions they serve9,10, and their utility for biotechnology11,12. Cyanobacteria are found in a wide variety of habitats, either as free-living organisms in marine, freshwater, and terrestrial environments, or in symbiotic associations with mosses, ferns, plants, or in lichens and sponges13. They serve as crucial primary producers in aquatic ecosystems, producing oxygen and organic carbon through oxygenic photosynthesis9,10, and some are capable of fixing atmospheric nitrogen as well7. Marine and freshwater cyanobacteria, including Prochlorococcus, Synechococcus, and Synechocystis, produce EVs under laboratory conditions14,15,16, and cyanobacterial vesicles can also be found in natural environments14,17. The biological and ecological functions of cyanobacterial vesicles are unknown, but further research in this area is likely to provide new insights into questions on cyanobacterial physiology, differentiation, communication strategies, evolution, and trophic interactions. In addition, the ability of cyanobacterial EVs to carry diverse categories of biomolecules may have commercial applications18,19.
The present protocol describes methods for isolating and characterizing vesicles from cyanobacterial cultures and field samples to enable and encourage a broader examination of cyanobacterial extracellular vesicle biology. While the workflow described here is analogous to protocols for isolating and characterizing EVs from other bacteria, cyanobacterial cultures and field samples typically contain lower cell and vesicle concentrations than are commonly observed with host-associated or pathogenic model systems20,21,22. Thus, studies of cyanobacterial EVs require special considerations and optimizations for cultivation and vesicle isolation, which vary further between strains and media backgrounds. As no single protocol will work equally well for all strains, growth conditions, and downstream applications, we provide multiple options and discuss the trade-offs involved so that researchers may determine the approaches most appropriate for addressing their experimental questions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x Phosphate buffered saline (PBS), pH 7.4</td>
			<td>Home-made buffer</td>
			<td>---</td>
			<td>Standard wash/storage buffer which can be used with vesicles; can be made in lab or purchased commercially</td>
		</tr>
		<tr>
			<td>2% Uranyl acetate solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400-2</td>
			<td>Negative staining - TEM</td>
		</tr>
		<tr>
			<td>4x Laemmli Sample Buffer</td>
			<td>Bio-Rad</td>
			<td>161-0747</td>
			<td>Denaturation of vesicle sample prior to proteolysis, required for lipopolysaccharides (LPS) staining</td>
		</tr>
		<tr>
			<td>Amicon Ultra Centrifugal Filters (100 kDa)</td>
			<td>Merck</td>
			<td>UFC9100XX</td>
			<td>Alternative option for centrifugal ultrafiltration</td>
		</tr>
		<tr>
			<td>BG11 medium</td>
			<td>Home-made medium</td>
			<td>---</td>
			<td>Medium for cultivation of Synechocystis sp. PCC 6803</td>
		</tr>
		<tr>
			<td>Carbon Support Film 200 Mesh, copper. CF200-Cu</td>
			<td>Electron Microscopy Sciences</td>
			<td>71150</td>
			<td>Transmission electron microscopy (TEM) grid</td>
		</tr>
		<tr>
			<td>easiGlow Glow Discharge Cleaning System</td>
			<td>Ted Pella</td>
			<td>91000</td>
			<td>Commercial TEM grid glow discharger</td>
		</tr>
		<tr>
			<td>EMbed 812 epoxy resin</td>
			<td>Electron Microscopy Sciences</td>
			<td>14120</td>
			<td>Ultra-thin sections - TEM</td>
		</tr>
		<tr>
			<td>Filter holder for vacuum system</td>
			<td>Thermo Fisher Scientific</td>
			<td>300-4000</td>
			<td>Reusable units with filter membrane support plates</td>
		</tr>
		<tr>
			<td>Glutaraldehyde&#160; Grade I</td>
			<td>Sigma-Aldrich</td>
			<td>G5882-10X1ml</td>
			<td>Ultra-thin sections - TEM</td>
		</tr>
		<tr>
			<td>HEPES [N-(2-Hydroxyethyl)piperazine-N&#8242;-(2-ethanesulfonic acid) sodium salt]</td>
			<td>Sigma-Aldrich</td>
			<td>H7006</td>
			<td>Buffering agent for cyanobacterial growth media</td>
		</tr>
		<tr>
			<td>Lead Citrate, Trihydrate</td>
			<td>Electron Microscopy Sciences</td>
			<td>17800</td>
			<td>Stain for use in electron microscopy</td>
		</tr>
		<tr>
			<td>Macrosep Advance Centrifugal Devices with Omega Membrane 100K</td>
			<td>Pall</td>
			<td>MAP100C36</td>
			<td>For centrifugal ultrafiltration of small volume samples</td>
		</tr>
		<tr>
			<td>Millipore Pellicon 3 TFF Module</td>
			<td>EMD Millipore</td>
			<td>XX42P0060 or XX42P0080</td>
			<td>Alternative TFF option for concentrating large volume samples</td>
		</tr>
		<tr>
			<td>Milli-Q Reference Water Purification System</td>
			<td>Merck</td>
			<td>Z00QSV0WW</td>
			<td>Water purification system for obtaining type I ultrapure grade water</td>
		</tr>
		<tr>
			<td>NanoSight</td>
			<td>Malvern Panalytical</td>
			<td>LM14 or NS300</td>
			<td>Nanoparticle tracking analysis</td>
		</tr>
		<tr>
			<td>Optima L80 XP Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>L80 XP</td>
			<td>Ultracentrifuge (or similar model)</td>
		</tr>
		<tr>
			<td>OptiPrep / Iodixanol</td>
			<td>Sigma-Aldrich</td>
			<td>D1556</td>
			<td>Density gradient media</td>
		</tr>
		<tr>
			<td>Osmium Tetroxide Reagent Plus</td>
			<td>Sigma-Aldrich</td>
			<td>201030</td>
			<td>Ultra-thin sections - TEM</td>
		</tr>
		<tr>
			<td>Pall Centramate PE TFF holder</td>
			<td>Pall Corporation</td>
			<td>FS002K10</td>
			<td>TFF module good for concentrating 10s-100s of L of sample; requires additional 30 or 100 kDa filter modules to scale with your estimated volumes</td>
		</tr>
		<tr>
			<td>Peristaltic pump Masterflex L/S</td>
			<td>Cole-Parmer</td>
			<td>07559-07</td>
			<td>Pump for driving large-scale TFF modules</td>
		</tr>
		<tr>
			<td>Piston Gradient Fractionator</td>
			<td>Biocomp Instruments</td>
			<td>152-001</td>
			<td>Automated density gradient fractionation</td>
		</tr>
		<tr>
			<td>Polycarbonate tubes for the 70 Ti rotor</td>
			<td>Beckman Coulter</td>
			<td>355618</td>
			<td>Reusable ultracentrifuge polycarbonate aluminum tubes with cap assembly</td>
		</tr>
		<tr>
			<td>Pro99 medium</td>
			<td>Home-made medium</td>
			<td>---</td>
			<td>Medium for cultivation of Prochlorococcus</td>
		</tr>
		<tr>
			<td>Pro-Q Emerald LPS Gel Stain Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>P20495</td>
			<td>LPS staining</td>
		</tr>
		<tr>
			<td>SN medium</td>
			<td>Home-made medium</td>
			<td>---</td>
			<td>Medium for cultivation of cyanobacterial marine strains such as Synechococcus</td>
		</tr>
		<tr>
			<td>Sodium cacodylate trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C0250-100G</td>
			<td>Buffering agent in the preparation of vesicles samples for TEM</td>
		</tr>
		<tr>
			<td>SW32Ti swinging bucket rotor</td>
			<td>Beckman Coulter</td>
			<td>369650</td>
			<td>Ultracentrifuge rotor, holds 6x ~40 mL tubes; good for pelleting of bulk material</td>
		</tr>
		<tr>
			<td>SW60Ti swinging bucket rotor</td>
			<td>Beckman Coulter</td>
			<td>335650</td>
			<td>Ultracentrifuge rotor, holds 6x ~4.5 mL tubes; good for gradient purifications and final vesicle washes</td>
		</tr>
		<tr>
			<td>Syringe 1mL Luer</td>
			<td>BD Plastipak</td>
			<td>303172</td>
			<td>For vesicle isolation and loading samples into nanparticle tracking analysis (NTA) equipment</td>
		</tr>
		<tr>
			<td>Syringe filter 0.2 &#181;m</td>
			<td>Pall Corporation</td>
			<td>4602</td>
			<td>For vesicle isolation from cultures</td>
		</tr>
		<tr>
			<td>TAPS [N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid]</td>
			<td>Sigma-Aldrich</td>
			<td>T5130</td>
			<td>Buffering agent for cyanobacterial growth media</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>JEOL</td>
			<td>JEM-1400</td>
			<td>Or similar microscope</td>
		</tr>
		<tr>
			<td>Type 70 Ti Fixed-Angle Titanium Rotor</td>
			<td>Beckman Coulter</td>
			<td>337922</td>
			<td>Ultracentrifuge rotor, holds 8x ~39 mL tubes; good for pelleting of bulk material</td>
		</tr>
		<tr>
			<td>Type XIV protease from Streptomyces griseus</td>
			<td>Sigma-Aldrich</td>
			<td>P5147</td>
			<td>Enzyme for proteolysis of EVs proteins, required for LPS staining</td>
		</tr>
		<tr>
			<td>UltraClear tubes for the SW32Ti rotor</td>
			<td>Beckman Coulter</td>
			<td>344058</td>
			<td>Single use ultracentrifuge tubes</td>
		</tr>
		<tr>
			<td>UltraClear tubes for the SW60Ti rotor</td>
			<td>Beckman Coulter</td>
			<td>344062</td>
			<td>Single use ultracentrifuge tubes</td>
		</tr>
		<tr>
			<td>Ultramicrotome PowerTome XL, PT-PC</td>
			<td>RMC Products,&#160; Boeckeler Instruments</td>
			<td>75501</td>
			<td>Microtome for ultra-thin sections - TEM</td>
		</tr>
		<tr>
			<td>Universal 320 R centrifuge</td>
			<td>Hettich</td>
			<td>Z654736</td>
			<td>This or any similar general-purpose benchtop/floor standing centrifuge can be used for pelleting cells</td>
		</tr>
		<tr>
			<td>Vacuum apparatus</td>
			<td>KNF Neuberger</td>
			<td>N026.3 AT.18</td>
			<td>Or any similar vacuum pump and trap</td>
		</tr>
		<tr>
			<td>Vivaflow 200 100,000 MWCO PES</td>
			<td>Sartorius</td>
			<td>VF20P4</td>
			<td>TFF module, good for 2-3L. You can connect different modules for higher volume (figure 1).</td>
		</tr>
		<tr>
			<td>Whatman Polycap TC Capsule filter (0.2/0.2&#181;m)</td>
			<td>Cytiva</td>
			<td>6717-9502</td>
			<td>Capsule filter for filtering large volumes of liquid</td>
		</tr>
		<tr>
			<td>Zetasizer</td>
			<td>Malvern Panalytical</td>
			<td>Zetasizer Nano ZS</td>
			<td>Dynamic light scattering (DLS) instrument</td>
		</tr>
	</tbody>
</table>

isolation and characterization of cyanobacterial extracellular vesicles

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models. Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles -&#160;small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis. Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.

Figure 1 presents an overview of the cyanobacterial vesicle isolation process, highlighting key aspects of the protocol described here. Of particular note are the steps detailing separation of cyanobacterial biomass from the small particle (vesicle) fraction, concentrating the vesicle sample and vesicle isolation and purification (Figure 1, steps 2 to 4), which are critical to obtaining reproducible preparations of vesicles. Figure 2 presents representative results from the isolation and characterization of extracellular vesicles from the cyanobacterium Synechocystis sp. PCC 6803. The outer membrane of this cyanobacterium has been shown to contain carotenoids31, which confer a characteristic orange coloration to vesicles&#39; samples collected by direct pelleting through ultracentrifugation (Figure 2A). Once the vesicle pellet is resuspended, vesicles can be examined by transmission electron microscopy (TEM), either by negatively staining samples with uranyl acetate (Figure 2B) or by observation of ultrathin sections (Figure 2C). Vesicle size distribution and concentration can be assessed using dynamic light scattering (DLS) (Figure 2D) and nanoparticle tracking analyses (NTA) (Figure 2E). With the described protocol, typically ~3.5 &#177; 1.0 x 108 nanoparticles per mL were obtained in an exponentially growing culture of Synechocystis sp. PCC 6803 at an OD730 of 1.0. Biochemical analyses of isolated EVs can be carried out to complement the physical characterization of the isolated vesicles. As an example, purified&#160;Synechocystis sp. PCC 6803 vesicles were separated on an SDS-polyacrylamide gel and stained for lipopolysaccharides (LPS; Figure 2F). As LPS are specific to the outer membrane32, LPS detection can validate the presence of membrane-bound vesicles and serve as a marker for analyzing relative vesicle content between preparations from the same strain33. Inspection of the relative abundance of low vs. high molecular weight LPS can indicate changes in vesicle composition between samples34,35.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63481/63481fig01v2.jpg" /> 
Figure 1: Experimental procedures used for cyanobacterial EV isolation and characterization. Schematic representations of growing cultures (1) and separation of cyanobacterial biomass from the growth medium (2). Cell-free samples containing vesicles are concentrated (3), and then the EVs are isolated and purified (4). Depending on the application, vesicle preparations can be characterized using one or a combination of transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and lipopolysaccharide (LPS) profiling (5). RT, room temperature. <a href="https://www.jove.com/files/ftp_upload/63481/63481fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63481/63481fig02.jpg" /> 
Figure 2: Representative results of the extracellular vesicle isolation and characterization for the cyanobacterium Synechocystis sp. PCC 6803. (A) Photograph of the extracellular vesicle pellet (EVs pellet) obtained after ultracentrifugation of the concentrated cell-free extracellular medium from a 600 mL Synechocystis sp. PCC 6803 culture grown to an OD730&#160;of 1.0-1.5. Note the typical orange coloration of the vesicle pellet derived from the carotenoids found in the outer membrane. (B,C) Transmission electron microscopy (TEM) photographs of negatively-stained (B) and ultrathin sections (C) of Synechocystis sp. PCC 6803 vesicle samples. Scale bars: 200 nm and 50 nm, respectively. Samples were visualized on a transmission electron microscope operated at 80 kV. (D) Typical dynamic light scattering (DLS) plot depicting the distribution of vesicle volume as a function of vesicle diameter (in nm). Colored lines indicate data from three technical replicate measurements of the same sample. (E) Representative nanoparticle tracking analysis (NTA) data of Synechocystis extracellular vesicle size distribution (in nm). The black line indicates the mean of three technical replicates, and the red lines represent the standard error of the mean. (F) Lipopolysaccharides (LPS) profile was detected after separating the vesicle preparation by electrophoresis on a 16% (w/v) SDS-polyacrylamide gel and staining with a commercial LPS staining kit. Low- and high-molecular-weight LPS, corresponding to rough- and smooth-LPS forms, respectively, are detectable. <a href="https://www.jove.com/files/ftp_upload/63481/63481fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Equation to calculate r. The number of vesicles produced per cell per generation between two points during exponential growth (typically the beginning and end of the time course) is determined using this equation. <a href="https://www.jove.com/files/ftp_upload/63481/Supplementary File 1.docx" target="_blank">Please click here to download this File.</a>

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Isolation and Characterization of Cyanobacterial Extracellular Vesicles

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.

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential ...

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5.3K Views.  Wellesley College. 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.

Video: Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

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

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...

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

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

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

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.

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

Enhanced Bacterial Extracellular Vesicles Isolation for Microbiota Multi-Omics

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

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.

Author Spotlight: Accelerating Research on Bacterial Extracellular Vesicles Separation and Heterogeneity 

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.

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host ...

Enhanced Campylobacter Isolation from Meat

Detection and Isolation of Campylobacter spp. from Raw Meat

This article presents a rapid yet robust protocol for isolating Campylobacter spp. from raw meats, specifically focusing on Campylobacter jejuni and Campylobacter coli. The protocol builds upon established methods, ensuring compatibility with the prevailing techniques employed by regulatory bodies such as the Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA) in the USA, as well as the International Organization for Standardization (ISO) in Europe. Central to this protocol is collecting a rinsate, which is concentrated and resuspended in Bolton Broth media containing horse blood. This medium has been proven to facilitate the recovery of stressed Campylobacter cells and reduce the required enrichment duration by 50%. The enriched samples are then transferred onto nitrocellulose membranes on brucella plates. To improve the sensitivity and specificity of the method, 0.45 &#181;m and 0.65 &#181;m pore-size filter membranes were evaluated. Data revealed a 29-fold increase in cell recovery with the 0.65 &#181;m pore-size filter compared to the 0.45 &#181;m pore-size without impacting specificity. The highly motile characteristics of Campylobacter allow cells to actively move through the membrane filters towards the agar medium, which enables effective isolation of pure Campylobacter colonies. The protocol incorporates multiplex quantitative real-time polymerase chain reaction (mqPCR) assay to identify the isolates at the species level. This molecular technique offers a reliable and efficient means of species identification. Investigations conducted over the past twelve years involving retail meats have demonstrated the ability of this method to enhance recovery of Campylobacter from naturally contaminated meat samples compared to current reference methods. Furthermore, this protocol boasts reduced preparation and processing time. As a result, it presents a promising alternative for the efficient recovery of Campylobacter from meat. Moreover, this procedure can be seamlessly integrated with DNA-based methods, facilitating rapid screening of positive samples alongside comprehensive whole-genome sequencing analysis.

Author Spotlight: Development of an Enhanced Protocol for Rapid and Accurate Isolation of Campylobacter from Food Products

Campylobacter is the leading cause of bacterial foodborne gastroenteritis worldwide. Despite establishments enacting measures to reduce the prevalence throughout their facilities, contaminated products consistently reach consumers. The technique developed over the past twelve years addresses limitations of existing methods for isolating and detecting Campylobacter spp. from raw meat.

This article presents a rapid yet robust protocol for isolating Campylobacter spp. from raw meats, specifically focusing on Campylobacter jejuni and ...