Este estudio fue, en parte o en conjunto, apoyados por la Fundación de Ciencias naturales nacionales de China (31472187 y 31672550) y la ciencia agrícola y tecnología programa de innovación de la Academia China de ciencias agrícolas.

Todos los experimentos en animales fueron aprobados por el Comité de ética local de Shanghai Instituto de investigación veterinaria, Academia China de ciencias agrícolas (permiso número: SHVRIAU-14-0101).
1. in Vitro cultura de esquistosomas
Nota: Esquistosomas representan un riesgo biológico. Los trabajadores deben usar guantes de látex en todo momento cuando manejo de gusanos, schistosomal suspensiones u otras relacionadas con los materiales biológicos. Conejos de Nueva Zelandia infectaron con ~ 2.000 cercariae vía abdominal la exposición. Se recolectaron esquistosomas en la etapa hígada de conejos infectados con S. japonicum cercariae en 28 días post-infección por la perfusión del hígado y las venas mesentéricas. Los procedimientos de colección de gusano han sido referenciados en publicaciones anteriores, informes de estudios realizados en ratones18.
<ol><li>Recoger parásitos en un vaso de precipitados de 500 mL. Lávelos cuidadosamente y suavemente 3 x con 200 mL de precalentado (37 ° C) PBS (pH 7.4).</li>
	<li>Separar ~ 200 pares de gusano para cada plato de Petri de 90 mm. Luego lave bien los parásitos y suavemente 2 x con 50 mL de precalentado (37 ° C) medio de cultivo RPMI-1640 que contiene 100 U de penicilina y 100 mg/mL de estreptomicina.</li>
	<li>Mantener parásitos en 40 mL de medio de cultivo RPMI-1640 precalentado sin antibióticos a 37 ° C debajo del 5% de CO2 a una densidad de gusano ~ 5 pares/ml para 2 h en 90 mm Petri dish. Notas: El Medio RPMI 1640 no debe contener suero, de lo contrario se introducirán exógeno vesículas extracelulares.</li>
</ol>2. tratamiento previo de la condición Media
<ol><li>Recoge el medio de cultivo y luego centrifugar el medio a 4 ° C a 2.000 x g por 30 min eliminar huevos y desechos tegmental del gusano.</li>
	<li>Transfiera el sobrenadante a un tubo nuevo y luego filtrar la solución con un filtro de jeringa de 0,22 μm.</li>
	<li>Recoger la solución filtrada en un bolso del dializado (corte de peso molecular: 3.5kD) y luego dializarse la solución utilizando 8% PEG8000 solución a 4 ° C durante la noche. Notas: Este paso es importante para enriquecer el SjEVs para aumentar el rendimiento de aislamiento de SjEVs.</li>
</ol>3. aislamiento de SjEVs
<ol><li>Transferir la solución de dializado a un tubo nuevo y añadir 0,5 volúmenes de reactivo de aislamiento total exosomas.</li>
	<li>Mezcle la solución bien por Vortex o pipeteando arriba y abajo para asegurar la homogeneidad de la solución.</li>
	<li>Incubar la mezcla a 2 ° C a 8 ° C durante la noche.</li>
	<li>Centrifugar la mezcla a 15.000 × g durante 1 h a 2 ° C a 8 ° C.</li>
	<li>Aspirar y descartar el sobrenadante. Notas: EVs están contenido en el sedimento en el fondo del tubo (no visible en la mayoría de los casos).</li>
	<li>Resuspender el precipitado de EVs en 2 mL de PBS y luego almacenar la solución de EVs a-80 ° C hasta su posterior análisis.</li>
</ol>4. Caracterización de los vehículos eléctricos por Western Blot
<ol><li>Determinar la concentración de proteína de la muestra SjEVs, por ejemplo, por análisis de la BCA.</li>
	<li>Preparar 10-20 μg de EVs en 22,5 μl de tampón de RIPA. Luego agregar 7.5 μl de 4 x cargando buffer y calor durante 5 minutos a 95 ° C.</li>
	<li>Proteínas de SjEVs separadas por gel de SDS-PAGE de 12%.</li>
	<li>Transferencia de las proteínas a membranas PVDF y luego probe con anticuerpos anti-CD63 (1:1, 000 dilución), seguidos por sondeo con anti-conejo IgG-HRP (1:5, 000 dilución).</li>
	<li>Utilizar el reactivo de detección ECL para el desarrollo de la membrana y detectar las señales de una sistema de imagen de la quimioluminescencia.</li>
</ol>5. Caracterización de los vehículos eléctricos por microscopía electrónica de transmisión
<ol><li>Añadir 2 μl de SjEVs aisladas a rejillas de formvar recubierto de malla 200 y luego deje que se seque a temperatura ambiente.</li>
	<li>Mancha con el 2% de ácido fosfotúngstico durante 1 minuto y luego deje que se seque a temperatura ambiente.</li>
	<li>Carga de las rejillas sobre el portamuestras de microscopio electrónico de transmisión y expuesto a un haz de electrones kV 80 para la captura de la imagen.</li>
</ol>6. etiqueta SjEVs con PKH67 y análisis de absorción de la célula
Notas: Todos los pasos posteriores se realizaron a temperatura ambiente (20-25 ° C), a menos que se indique lo contrario.
<ol><li>Transferencia 5 μg solución SjEVs en un tubo de polipropileno de fondo cónico y ajustar el volumen de 12 mL mediante la adición de Medio RPMI 1640.</li>
	<li>Centrifugue el SjEVs en 110.000 × g durante 90 minutos a 4 ° C para SjEVs de pellets.</li>
	<li>Después de la centrifugación, cuidadosamente aspirar el sobrenadante. Nota: El tinte de etanol de PKH67 no se debe agregar directamente a la SjEV de la pelotilla, como esto daría lugar a la coloración heterogénea y viabilidad celular reducida.</li>
	<li>Preparar una solución de suspensión EV 2 x añadiendo 1 mL de diluyente C a la pelotilla SjEV; para asegurar la completa dispersión, resuspender con pipeteo suave.</li>
	<li>Justo antes de la tinción en diluyente C, preparar un fresco 2 x solución colorante agregando 2 μl de la solución de tinte etanólico de PKH67 a 1 mL de diluyente C en un tubo de centrífuga de polipropileno y mezcla bien para dispersar.</li>
	<li>Rápidamente añadir 1 mL de 2 x EV suspensión (paso 6.4) a 1 mL de 2 x solución de colorante (paso 6.5) e inmediatamente mezcle la muestra mediante pipeteo.</li>
	<li>Incubar la mezcla a temperatura ambiente durante 4 minutos.</li>
	<li>Detener la coloración añadiendo 4 mL de SBF y luego incubar durante 1 minuto.</li>
	<li>Añadir medio RPMI1640 para ajustar el volumen de 12 mL y centrifugar luego a 110.000 × g a 4 ° C durante 90 minutos.</li>
	<li>Quite el sobrenadante y añadir medio RPMI1640 para Resuspender el PKH67 etiquetada SjEV pellets.</li>
	<li>Centrifugue a 110.000 × g a 4 ° C durante 90 minutos a lavar una vez y luego añadir PBS para volver a suspender los pellets de SjEVs.</li>
</ol>7. ensayo de absorción de célula de SjEVs
<ol><li>Las células mamíferas de la semilla como NCTC clonación células de 1469 o células HEK293T en placas de 6 pocillos (2 × 105 células por pocillo) y las células de la cultura durante la noche.</li>
	<li>Añadir 5 μg SjEVs etiqueta de PKH67 y las células a 37 ° C para 2-4 h después de la cultura.</li>
	<li>Después de la incubación, retirar el medio y lavar las células con PBS dos veces.</li>
	<li>Fijar las células con solución de formaldehído 4% durante 15 minutos y lavar dos veces con PBS.</li>
	<li>Se tiñen los núcleos de célula con 4′, 6-diamidino-2-phenylindole (DAPI).</li>
	<li>Observar las células utilizando microscopía de fluorescencia.</li>
</ol>

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Los autores no tienen nada que revelar.

Estudios recientes sobre EVs han demostrado eso schistosome que EVS juegan un papel importante en huésped-patógeno interacciones3,9,12,16. Atender más sus funciones reguladoras es esencial aislar EVs de esquistosomas. Aquí, describimos un método alternativo para el aislamiento de SjEV. Este método produce un tamaño de amplia gama de SjEVs, de 100 nm a 400 nm, en adultos S. japonicum<...

Las vesículas extracelulares (EVs) son una población de pequeñas vesículas de membrana-limita con varias proteínas, lípidos y ácidos nucleicos. Estudios recientes demostraron que EVs desempeñan un papel crucial en la comunicación de la célula y participan en la regulación de numerosos procesos fisiológicos, incluyendo el desarrollo de las células, regulación inmune, angiogénesis y migración de célula2, 3,4,5. La acumulación de pruebas indica que EVs, circulación de exosomas y su carga de miRNA representan potenciales biomarcadores de ciertas enfermedades6.
Varios protozoos como Trichomonas vaginalis, Trypanosoma cruziy Leishmania spp., han demostrado ser capaces de secretar EVs; helmintos, además se han encontrado secretar EVs en vida alberga7. EVs parasitarias han demostrado para estar implicado en el mantenimiento de la infección, patogenicidad8y9de la regulación inmune. Recientes estudios en esquistosomas ambos Schistosoma mansoni (S. mansoni) 10,11 y S. japonicum12 han indicado que los esquistosomas adultos secretan exosomas como vesículas que pueden ser involucrados en las normas funcionales de procesos biológicos específicos.
Hasta la fecha, se han utilizado varios métodos para aislar las vesículas extracelulares, como la ultracentrifugación, ultrafiltración13, el uso de reactivos basados en polímeros, cromatografía por exclusión de tamaño y aislamiento de inmunoafinidad. Estos métodos poseen sus propias ventajas y limitaciones de la14. Generalmente, la ultracentrifugación se considera el método estándar de oro para el aislamiento de la vesícula. Sin embargo, este método adolece de la limitación del potencial de agregación EV14.
De esquistosomas, han divulgado un par de métodos para el aislamiento de EV: Estos incluyen ultracentrifugación11,12,10 y el uso de comerciales EV aislamiento kit16 para varias etapas (huevos16, esquistosómula11, esquistosomas adultos10,12,17). Dado que microvesículas son de una amplia gama de tamaño de varios cientos nanómetros a mil nanómetros, desarrollamos un método alternativo que combina con el uso de la centrífuga de banco, ultrafiltración y un kit de aislamiento comercial de EV para aislar el servicio voluntario europeo de adultos Schistosoma japonicum. El servicio voluntario europeo aislado típicamente poseía un diámetro de 100-400 nm y con éxito fueron internalizado por las células del receptoras.

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Total Exosome Isolation Reagent (from cell culture media)</td><td>ThermoFisher SCIENTIFIC</td><td>4478359</td><td>For isolating S. japonicum extracellular vesicles</td></tr><tr><td>PKH67</td><td>Sigma-aldrich</td><td>MINI67</td><td>For labeling Evs</td></tr><tr><td>RPMI 1640 Medium</td><td>ThermoFisher SCIENTIFIC</td><td>11875119</td><td>For parasite culture</td></tr><tr><td>Penicillin-Streptomycin</td><td>ThermoFisher SCIENTIFIC</td><td>15140122</td><td></td></tr><tr><td>0.22&amp;mu;m syringe filter</td><td>Merck</td><td>SLGP033RB</td><td></td></tr><tr><td>SnakeSkin Dialysis Tubing</td><td>Thermo SCIENTIFIC</td><td>68035</td><td></td></tr><tr><td>PEG8000</td><td>Sangon Biotech</td><td>A100159&amp;nbsp;</td><td></td></tr><tr><td>RIPA</td><td>Beyotime</td><td>P0013B</td><td></td></tr><tr><td>EMD Millipore&amp;trade; Immobilon&amp;trade; Western Chemiluminescent HRP Substrate (ECL)</td><td>Fisher scientific</td><td>WBKLS0100&amp;nbsp;</td><td></td></tr><tr><td>DAPI</td><td>Cell Signaling Technology</td><td>4083</td><td></td></tr><tr><td>BCA kit</td><td>Beyotime</td><td>P0010</td><td></td></tr><tr><td>CD63 antibodies</td><td>Sangon Biotech</td><td>D160973-0025</td><td></td></tr><tr><td>PVDF</td><td>Bio-Rad</td><td>1704273</td><td></td></tr><tr><td>Formvar/Carbon 400 mesh</td><td>Ted Pella</td><td>01754-F</td><td></td></tr><tr><td>Phosphotungstic Acid</td><td>Ted Pella</td><td>19402</td><td></td></tr><tr><td>anti-mouse IgG-HRP</td><td>CWBIO</td><td>CW0102</td><td></td></tr><tr><td>NCTC clone 1469 cells</td><td>ATCC</td><td>ATCC&amp;reg; CCL-9.1&amp;trade;</td><td></td></tr><tr><td>FBS</td><td>HyClone</td><td>SV30087.02</td><td></td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GE chemoluminescance imaging system</td><td>GE</td><td>ImageQuant LAS4000mini</td><td></td></tr><tr><td>Transmission electron microscopy</td><td>Hitachi</td><td>H-7600</td><td></td></tr><tr><td>Milli-Q water</td><td>Milli-Q</td><td>Milli-Q Elix</td><td></td></tr><tr><td>Eppendor Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>Centrifuge 5804R</td><td></td></tr><tr><td>Zetasizer Nano</td><td>Malvern</td><td>Zetasizer Nano ZS&amp;nbsp;</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td>Optima L-100 XP Ultracentrifuge</td><td></td></tr><tr><td>Incubator</td><td>ESCO</td><td>CCL-107B</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Zeiss Axin Observer Z1</td><td></td></tr></tbody></table>

isolation and characterization of extracellular vesicles from adult schistosoma japonicum

Las vesículas extracelulares (EVs) son vesículas membranosas por una variedad de células en el microambiente extracelular. EVs representan una población de vesículas heterogéneas, cuyo rango de tamaño entre 40 y 1.000 nm. Evidencia acumulada indica que EVs desempeñan importantes funciones reguladoras en las interacciones patógeno-host. Una profunda comprensión del schistosome EVs debe proporcionar penetraciones en los mecanismos subyacentes a las interacciones host esquistosoma, permitir el desarrollo de nuevas estrategias contra la esquistosomiasis. Aquí, nuestro objetivo es profundizar el estudio funciones de EVs en esquistosomas presentando un protocolo para el aislamiento y la caracterización de los vehículos eléctricos de adultos Schistosoma japonicum (S. japonicum). EVs se aislaron en vitro el medio de cultivo utilizando centrifugación combinada con un kit de aislamiento comercial exosomas. Los aislados de S. japonicum EVs (SjEVs) por lo general poseen un diámetro de 100-400 nm y se caracterizan por microscopía electrónica de transmisión y el borrar occidental. El uso de PKH67 SjEVs de marcado tinte ha demostrado que SjEVs es internalizados por las células del receptoras. En general, el protocolo proporciona un método alternativo para aislar EVs de esquistosomas adultos; el SjEVs aislado puede ser adecuado para el análisis funcional.

Para cuantificar el rendimiento de SjEVs aislados usando el protocolo descrito, se utilizan análisis de proteína BCA para acceder a la concentración de proteína de la SjEVs aislada de esquistosomas adultos de 28 días. Como se muestra en la tabla 1, la concentración de proteína de SjEV sido 208 μg 250 μg por 100 mL de medio (tabla 1). El tamaño de partícula, según lo determinado por análisis de nanopartículas de Malvern, indica que el aislado...

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Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Aislamiento y caracterización de las vesículas extracelulares de adultos Schistosoma japonicum

Aquí, presentamos un protocolo para describir el aislamiento de las vesículas extracelulares (EVs) en medio de cultivo en vitro de adultos Schistosoma japonicum.

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

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Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

7.8K vistas.  Ministry of Agriculture. Aquí, presentamos un protocolo para describir el aislamiento de las vesículas extracelulares (EVs) en medio de cultivo en vitro de adultos Schistosoma japonicum.

Video: Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

Transformación y cercarias In vitro El cultivo de la Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

Investigación

Inmunología e Infección

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

Con óvulos de<em&gt; Schistosoma mansoni</em&gt; Como un<em&gt; En vivo</em&gt; Modelo de la inflamación pulmonar inducida por helmintos

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and mammalian final host, respectively. The ability to isolate and sustain the different life cycle stages under in vitro culture conditions has greatly facilitated investigations of the cellular, biochemical and molecular mechanisms regulating parasite growth, development and host interactions. Transmission of schistosomiasis requires asexual reproduction and development of multiple larval stages within the snail host; from the infective miracidium, through primary and secondary sporocysts, to the final cercarial stage that is infective to humans. In this paper we present a step-by-step protocol for mass hatching and isolation of Schistosoma mansoni miracidia from eggs obtained from livers of infected mice, and their subsequent introduction into in vitro culture. It is anticipated that the detailed protocol will encourage new researchers to engage in and broaden this important field of schistosome research.

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

This article describes a protocol for the large-scale axenic isolation and collection of free-swimming miracidia of the human blood fluke Schistosoma mansoni and their subsequent processing for introduction into in vitro culture.

Total aislamiento y cultivo In Vitro de Intramolluscan etapas de la humano platija de la sangre Schistosoma Mansoni

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and ...

Biología del Desarrollo

An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase &#921;) with tissue, filtration through a 70 &#181;m cell strainer, differential ultracentrifugation, and filtration through a 0.22 &#181;m membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.

Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

Un método de enriquecimiento para pequeñas vesículas extracelulares derivadas del tejido de cáncer de hígado

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's ...

Investigación sobre el cáncer

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

Cromatografía de exclusión de tamaño para separar vesículas extracelulares de medios de cultivo celular condicionados

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain ...

Neurociencias

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

Aislamiento y absorción de células dendríticas de pequeñas vesículas extracelulares de Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

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.

Purificación y análisis de las vesículas extracelulares de Caenorhabditis elegans

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

Biología

<meta charset="utf8"></head><body>Técnicas de refinamiento para el aislamiento de exosomas derivados del bazo murino

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed that Exo derived from humoral or cell-supernatant are promising targets for novel diagnostic or prognostic biomarkers, underscoring their significant role in disease pathogenesis. Tissue-derived Exo (Ti-Exo) have attracted increasing attention due to its ability to accurately reflect tissue specificity and the microenvironment. Ti-Exo, present in interstitial space, play crucial roles in intercellular communication and cross-organ signaling. Despite their recognized value in elucidating disease mechanisms, isolating Ti-Exo remains challenging due to the complexity of tissue matrices and variability in extraction methods. In this study, we developed a practical protocol for isolating exosomes from mice spleen tissue, providing a reproducible technique for subsequent identification analysis and functional studies. We used Type I collagenase digestion combined with differential ultracentrifugation to isolate spleen-derived Exo. The characteristics of isolated Exo were determined through electron microscopy, the nano-flow cytometer, and the western blot. The isolated spleen-derived Exo displayed the typical morphology of lipid bilayer vesicles, with particle sizes ranging from 30 nm to 150 nm. In addition, the expression profile of exosome markers confirmed the presence and purity of exosomes. Taken together, we successfully established a practical protocol for isolating spleen-derived Exo in mice.

<meta charset="utf8"></head><body>Aislamiento y caracterización de exosomas derivados de tejidos de bazo de ratón

Here, we present a protocol to isolate mouse spleen-derived exosomes using a combination of digestion with collagenase type I and ultracentrifugation.

Aislamiento y caracterización de exosomas derivados de tejidos de bazo de ratón

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed ...

Aislamiento y caracterización de las vesículas extracelulares de adultos Schistosoma japonicum

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Transformación y cercarias In vitro El cultivo de la Schistosoma mansoni Schistosomules

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Aislamiento y caracterización de exosomas derivados de tejidos de bazo de ratón

Aislamiento y caracterización de exosomas derivados de tejidos de bazo de ratón