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 ...

This protocol is significant as it not only demonstrates approaches for purifying vesicles from culture at different scales, trade offs, differences among methodologies and also consideration for working with field samples. Cyanobacteria presents unique vesicle sampling and analysis challenges. These techniques have successfully isolated and concentrated vesicles from culture and from environmental samples.To concentrate samples of less than 500 milliliters, start with rinsing the 15 to 20 milliliter ultrafiltration centrifugal concentrators with Type 1 ultrapure grade water. Load the concentrators into the centrifuge and spin at 4, 400 x g at four degrees Celsius. Discard the run through and repeat the step at least twice.Load the supernatant sample into a water rinsed concentrator and spin under the same conditions. Repeat this step until the sample is concentrated to a final volume of 15 to 30 milliliters. To concentrate a volume of more than 500 milliliters, perform tangential flow filtration by setting up the apparatus as described in the manuscript.Attach a peristaltic pump to the intake line and place adjustable clamps on the retentate line. Sanitize the tangential flow filtration as described in the manuscript, then flush the device with one liter of Type 1 ultrapure grade water. Add less than 0.2 micron filtrate into the sample reservoir.Slowly increase pump speed and back pressure levels on the retentate line to increase output from the filtrate lines. Continue to run the tangential flow filtration and refill the reservoir with culture supernatant as the material is removed. Stop concentrating the sample once the volume in the reservoir reaches the lowest possible amount needed to maintain flow into the intake line without introducing air bubbles.Close the outflow line with a clamp. Remove the back pressure on the retentate line. Recirculate the concentrated supernatant through the filter for 10 minutes, reducing the recirculation rate from 20 to 40 milliliters per minute to maximize the recovery.Move the retentate line into a clean vessel, remove the intake line from the sample and collect the concentrated material. Recover any remaining material in the sample reservoir using a pipette. Filter the concentrated supernatant through a 0.2 micron syringe filter.If needed, store the final concentrate at four degrees Celsius for approximately three weeks before moving to vesicle purification. To pellet the concentrated culture sample place it in a clean ultracentrifuge tube and add clean media or buffer to ensure that the tube is filled. After centrifugation, remove the supernatant with a pipette.Rewash the pelleted material with fresh culture media or wash buffer, such as 1X phosphate-buffered saline and repeat the centrifugation. Resuspend the final pellet in fresh culture by gentle pipetting with a one milliliter pipette and transfer it to a clean vessel. To perform density gradient ultracentrifugation, prepare iodixanol stocks as described in the manuscript.Mix one part of the 4X buffer with three parts of 60%iodixanol stock to make a 45%iodixanol solution. Dilute 45%iodixanol with 1X buffer to create stocks of gradient media of 40, 35, 30, 25, 20, 15 and 10%final iodixanol concentrations. Then, carefully overlay equal amounts of all these iodixanol gradient stocks into an ultracentrifuge tube, utilizing the entire tube volume.After centrifugation, collect the fractions of 0.5 milliliters each for approximately a 4.5 milliliter gradient by carefully pipetting or using a fraction collector. Determine the density of each fraction, in grams per milliliter, using an analytical balance and calibrated pipette. Remove the sample from the tube, determine the weight and return the sample to the tube.Dilute individual fractions with clean buffer in a new tube followed by a clean media or buffer wash. Carefully apply five microliters of vesicle and let it sit for five minutes. Remove the sample by touching the edge of a grid to a piece of clean filter paper.Pipette out 20 to 50 microliters of 2%uranyl acetate onto a flat surface covered by plastic film. Then place the grid floating on top of it for two minutes. Remove uranyl acetate using filter paper and float briefly on a drop of ultrapure water to wash.Fill the chamber with ultrapure water using a clean syringe and ensure no air bubbles. Click on start camera'and visualize the optimal region of the chamber around the thumbprint. Slide the screen gain'and camera level'sliders towards maximum, then decrease to the lowest level to see the dimmest particles.Adjust the focus as necessary to ensure that particles are roughly equally visible across the field of view and continue washing with ultra clean water until the chamber is clean. Add the vesicle sample to the chamber using a one milliliter syringe and confirm the acquisition settings. Select standard measurement'from the SOP drop down box and change the settings to collect at least three technical replicates of 60 second videos each.Press create'and run script'and follow the prompts and push approximately 100 microliters of sample into the chamber between replicates. Determine the particle parameters by clicking on analysis'and open experiment'and load the sample file. Select process selected files'and wait for the analysis to complete.The isolation and characterization of extracellular vesicles from the cyanobacterium Synechocystis sp. PCC6803, showed that the outer membrane contains carotinoids, which confer a characteristic orange colorization to vesicle samples collected by direct pelleting through ultracentrifugation. The resuspended vesicle pellet was examined by transmission electron microscopy either by negatively staining samples with uranyl acetate or by observing ultrathin sections.Vesicle size distribution and concentration were assessed using dynamic light scattering and nanoparticle tracking analyses. Purified Synechocystis sp. PCC6803 vesicles were separated on a sodium dodecyl sulfate-polyacrylamide gel and stained for lipopolysaccharides.Samples should be taken before and after the concentrated vesicle sample to ensure that the tangential flow filtration is working and the vesicles are getting concentrated. Then you need to measure both samples using the nanoparticle tracking analysis. Future efforts are required to merge this method with other approaches such as the size-exclusion chromatography or asymmetric field-flow fractionation to improve the discrimination and isolation of small particles from culture and environmental samples.This technique is helpful to study the cyanobacteria vesicles and the specific functional roles of these vesicles in cyanobacterial biology.

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Isolation and Characterization of Cyanobacterial Extracellular Vesicles

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