Este trabajo fue apoyado por becas de los EE.UU. National Science Foundation (EAR-0.920.299 y 0.745.808 EAR); EE.UU. National Science Foundation de Asia Oriental y el Pacífico Institutos de Verano, la Sociedad Geológica de América Programa de Investigación Grant y las Becas Alumni para la Investigación de Posgrado y Becas de la Universidad Estatal de Ohio. Nos gustaría dar las gracias al editor y dos revisores anónimos por sus perspicaces comentarios.

1. MTB Collection <ol><li> Al decidir sobre un sitio de agua dulce para recoger las bacterias magnetotácticas (MTB), a menudo es mejor comenzar con un estanque o arroyo de movimiento lento que tiene una capa suave sedimento fangoso. En esta demostración hemos recogido una muestra en el borde del río Olentangy en el campus de la Ohio State University (OSU), en Columbus, Ohio (EE.UU.). Mientras que esto era una ubicación conveniente para nuestra demostración, el protocolo aquí descrito es aplicable a cualquier ubicación acuático. Los materiales utilizados en este protocolo se pueden encontrar en la Tabla 1. Encontrar un lugar donde la profundidad del agua es de entre 10 y 100 cm. En tal situación, deberá recoger la capa superior, la mayoría de los sedimentos con un claro, con tapa de rosca contenedor. Sacar el sedimento y el agua en el recipiente hasta que se llena con un tercio a la mitad de los sedimentos y el volumen restante con agua. Mantener el envase sumergido hasta que se llena con agua y luego con fuerza tapar el recipiente with su tapa con tapa de rosca. No es necesario mezclar el sedimento. Limpie el exterior del recipiente en lugar seco con una toalla y luego tomar la muestra al laboratorio. No es necesario correr la muestra de nuevo a su laboratorio. Hemos dejado muestras de BTT en contenedores de plástico en el campo durante varios días antes de traer de vuelta a nuestro laboratorio. El CPTM debe ser viable por varias semanas o meses, siempre y cuando se almacenan las muestras en un lugar fresco y con sombra en el campo. </li><li> Una vez que la muestra está en su laboratorio, afloje la tapa y se deja que cubre el contenedor para reducir la cantidad de evaporación. Almacenar el contenedor a temperatura ambiente en una habitación oscura, cajón, o cubrir completamente el recipiente con papel de aluminio. Permitir el sedimento y las partículas finas se estabilice completamente a la parte inferior del recipiente, dejando la muestra en reposo durante varias horas a varios días. No es necesario mezclar el sedimento, MTB prefieren un ambiente sin perturbaciones. Las paredes transparentes del recipiente de plásticole permitirá confirmar que las partículas se han depositado en el fondo. Dependiendo de la muestra, MTB pueden permanecer vivos en la muestra durante muchos meses. </li></ol> 2. MTB Aislamiento <ol><li> Cuando esté listo para aislar los imanes, MTB lugar en los lados del recipiente de plástico de aproximadamente 1 cm por encima de la interfase agua-sedimento (Figura 1A). Tener cuidado de no perturbar el sedimento en el fondo del recipiente. Coloque el polo sur de un imán de barra en un lado del recipiente y el lado norte de otro imán de barra en el lado opuesto (Figura 1A). Casi cualquier imán se puede utilizar, tal como una barra de agitación magnética o imán refrigerador grande. Cualquier cosa puede ser utilizado para apoyar los imanes a la altura correcta por encima de la interfaz sedimento-agua. Hemos encontrado que los imanes descansando en la parte superior de una caja de cartón o de plástico es mejor, sin embargo, los imanes También se puede amarrar a la parte exterior del recipiente de plástico. Espere 30 minutos hasta varioshoras para las bacterias para nadar hacia el imán. </li><li> Utilizar una pipeta estéril para recoger cuidadosamente el agua del interior del recipiente (Figura 1A) cerca de la posición del imán de barra polo sur (para las muestras recogidas en el hemisferio norte). Esta agua debe contener la MTB que han sido atraídos por el imán de barra polo sur. A continuación, una pista de carreras capilar debe ser utilizada para enriquecer aún más la MTB. </li></ol> 3. MTB Hipódromo <ol><li> Con el fin de enriquecer la muestra con bacterias magnetotácticas, una pista de carreras capilar es necesario (Figuras 1B y 1C). Estos tienen que ser hecha antes de aislar las células desde el recipiente de plástico transparente. </li><li> Utilice un reductor de 5,75 (146 mm) pipeta Pasteur de vidrio para hacer una pista de carreras. Utilice una pluma de diamante o archivo para cortar la parte superior de la pipeta, la longitud de la pipeta no es crucial, pero debe ser capaz de contener aproximadamente 1-2 ml de agua. A continuación, utilice un quemador Bunsen para derretir la punta de modo quese convierte sellado (Figura 1C). La pipeta resultante debe tener un extremo abierto y un extremo cerrado. </li><li> Hacer varios de estos circuitos y autoclave entonces. Además, tendrá que autoclave algodón y varias agujas de metal largas. </li><li> Añadir agua filtrada muestra, recogida de agua cerca de la interfaz sedimento se muestra en la Figura 1A, a una pista de carreras autoclave usando una aguja larga de metal unida a una jeringa se filtró. El tamaño de poro del filtro debe ser de 0,22 mm para eliminar los desechos y contaminantes del agua. Es importante estar absolutamente seguro de que no hay burbujas de aire en el capilar de vidrio. </li><li> Conecte el fondo de la pista de carreras con algodón estéril (Figura 1B). Utilice la aguja de metal para empujar el algodón hacia el extremo sellado de la pista de carreras, así que es 0,5 a 1 cm de distancia de la punta de sellado (Figura 1C). </li><li> Con una pipeta estéril, añadir el agua que contiene MTB (de la sección 2.2) a la muestra reservoir (extremo abierto) de una pista de carreras de MTB preparada (Figura 1B). </li></ol> 4. MTB Enriquecimiento <ol><li> Una vez que la pista está llena de líquido muestra, yacía de costado en una superficie horizontal (por ejemplo, una mesa de trabajo) y coloque el polo sur de un imán de barra (en el hemisferio norte) al lado de la punta sellada de la pista (Figuras 1B y 1C). </li><li> Esperar 5 a 30 min para el MTB a migrar a través del algodón. Entonces usted debe recoger el líquido cerca de la punta de la pista. Esperar demasiado tiempo puede introducir contaminantes, tales como otras bacterias móviles, a la punta del capilar. Opcionalmente, se puede utilizar un microscopio de luz para ver la punta de la pista y ver el MTB recoger en la punta de la pista de carreras. Esto le permitirá determinar el tiempo que tarda el MTB a migrar a través del tapón de algodón. </li><li> Suavemente utilizar el bisturí de diamante para hacer un pequeño rasguño cerca de tapón de algodón y encaje el extremo de la pista. </li> &lt;li&gt; Usar una jeringa de 1 ml con una aguja delgada (25 o calibre 27) para retirar el líquido de la punta de la pista de carreras. Esta muestra de líquido debe contener ahora la MTB enriquecido. </li></ol> 5. Observación MTB por Microscopía de Luz <ol><li> Colocar una gota (10-20 l) de la muestra enriquecida MTB sobre un cubreobjetos. </li><li> Rápidamente la vuelta al cubreobjetos encima de lo que la caída está ahora mirando hacia abajo y colgando del cubreobjetos. </li><li> Colocar el cubreobjetos sobre un anillo o-que está descansando sobre un portaobjetos de vidrio. La junta tórica debe tener un diámetro ligeramente más pequeño que el cubreobjetos (alrededor de 1 cm; Figura 2). </li><li> Colocar esta gota colgante sobre una platina de microscopio de luz y se centran en un borde de la gota. Un objetivo seco 60X funciona muy bien porque la mayoría tienen una gran apertura numérica (NA; por ejemplo, 0,93), pero no requieren aceite, que es difícil de utilizar para el método de la gota colgante (Figura 2). </li><li> Coloque el extremo sur de un imán de barra cerca de la cortina drop y MTB comenzará a migrar hacia el borde de la gota más cercano al imán (Figura 3). En unos pocos minutos MTB muchos debería estar en el borde de la gota (figura 3). Demuéstrese a sí mismo que las bacterias son magnéticas mediante la inversión del polo del imán y luego observar las bacterias nadan en la dirección opuesta. </li></ol> 6. MTB observación por microscopía electrónica de transmisión (TEM) <ol><li> Colocar una gota (~ 20 l) de la MTB enriquecido en una rejilla de cobre y permitir que las bacterias que conformarse con 10 min. </li><li> Wick exceso de agua con un trozo de papel de filtro limpio. </li><li> Opcionalmente, la rejilla puede ser teñidas negativamente con acetato de uranilo al 2%, 2% pH ácido fosfotúngstico 7,2, o 2,5% de molibdato de sodio 7, 8, 9. Esto se realiza mediante la colocación de la rejilla de cobre sobre una gota de colorante inmediatamente después de la incubación de la cuadrícula con la MTB enriquecido. Incubar la cuadrícula con la tinción negativa, los tiempos pueden variar dependiendo de la stain utilizado y, a continuación mecha el fluido con un trozo de papel de filtro limpio. </li><li> Observe el MTB utilizando microscopía electrónica de transmisión (TEM, Figura 4). Para el trabajo descrito aquí MTB fueron adsorbidos en Formvar y estabilizado de carbono recubiertos con rejillas de cobre de malla 200 (Ted Pella # 01800). Las rejillas se colocaron con la cara hacia abajo de carbono en una gota de suspensión celular durante hasta 10 min, a continuación, inmediatamente se lava una vez mediante la colocación de la rejilla en una gota de agua durante 30 seg. Para la tinción, las rejillas se colocaron en una gota de acetato de uranilo al 2% (Ted Pella # 19481) durante 30 segundos a 5 minutos y después se secó completamente utilizando un trozo de papel de filtro. Las rejillas fueron analizadas por TEM utilizando un FEI Espíritu Tecnai a 80kV o un F20 FEI Tecnai con alto ángulo de campo oscuro anular STEM en 200kV. </li></ol>

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	<li>Blakemore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+bacteria.">Magnetotactic bacteria.</a> Science. 190, 377-379 (1975).</li><li>Blakemore, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+bacteria.">Magnetotactic bacteria.</a> Annual Reviews in Microbiology. 36, 217-238 (1982).</li><li>Bazylinski, D. A., Frankel, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Biomineralization+of+Magnetite+(Fe3O4)+and+Greigite+(Fe3S4)+in+a+Magnetotactic+Bacterium.">Controlled Biomineralization of Magnetite (Fe3O4) and Greigite (Fe3S4) in a Magnetotactic Bacterium.</a> Applied and Environmental Microbiology. 61, 3232-3239 (1995).</li><li>Lefevre, C. T., Menguy, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cultured+Greigite-Producing+Magnetotactic+Bacterium+in+a+Novel+Group+of+Sulfate-Reducing+Bacteria.">A Cultured Greigite-Producing Magnetotactic Bacterium in a Novel Group of Sulfate-Reducing Bacteria.</a> Science. 334, 1720-1723 (2011).</li><li>Simmons, S. L., Bazylinski, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=South-seeking+magnetotactic+bacteria+in+the+Northern+Hemisphere.">South-seeking magnetotactic bacteria in the Northern Hemisphere.</a> Science. 311, 371-374 (2006).</li><li>Wolfe, R., Thauer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+'capillary+racetrack'+method+for+isolation+of+magnetotactic+bacteria.">A 'capillary racetrack' method for isolation of magnetotactic bacteria.</a> FEMS Microbiology Letters. 45, 31-35 (1987).</li><li>Rodgers, F. G., Blakemore, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+structure+in+a+many-celled+magnetotactic+prokaryote.">Intercellular structure in a many-celled magnetotactic prokaryote.</a> Archives of Microbiology. 154, 18-22 (1990).</li><li>Moench, T. T., Konetzka, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+the+isolation+and+study+of+a+magnetotactic+bacterium.">A novel method for the isolation and study of a magnetotactic bacterium.</a> Archives of Microbiology. 119, 203-212 (1978).</li><li>Balkwill, D., Maratea, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+a+magnetotactic+spirillum.">Ultrastructure of a magnetotactic spirillum.</a> Journal of Bacteriology. 141, 1399-1408 (1980).</li><li>Lins, U., Freitas, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+homemade+apparatus+for+harvesting+uncultured+magnetotactic+microorganisms.">Simple homemade apparatus for harvesting uncultured magnetotactic microorganisms.</a> Brazilian Journal of Microbiology. 34, 111-116 (2003).</li><li>Jogler, C., Lin, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+Cloning+of+the+Magnetotactic+Metagenome:+Identification+of+Magnetosome+Island+Gene+Clusters+in+Uncultivated+Magnetotactic+Bacteria+from+Different+Aquatic+Sediments.">Toward Cloning of the Magnetotactic Metagenome: Identification of Magnetosome Island Gene Clusters in Uncultivated Magnetotactic Bacteria from Different Aquatic Sediments.</a> Applied and Environmental Microbiology. 75, 3972-3979 (2009).</li><li>Lin, W., Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+Isolated+but+Uncultivated+Magnetotactic+Bacterium+of+the+Phylum+Nitrospirae+from+Beijing,+China.">Newly Isolated but Uncultivated Magnetotactic Bacterium of the Phylum Nitrospirae from Beijing, China.</a> Applied and Environmental Microbiology. 78, 668-675 (2012).</li><li>Li, J., Pan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomineralization,+crystallography+and+magnetic+properties+of+bullet-shaped+magnetite+magnetosomes+in+giant+rod+magnetotactic+bacteria.">Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria.</a> Earth and Planetary Science Letters. 293, 368-376 (2010).</li><li>Oestreicher, Z., Valerde-Tercedor, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetosomes+and+magnetite+crystals+produced+by+magnetotactic+bacteria+as+resolved+by+atomic+force+microscopy+and+transmission+electron+microscopy.">Magnetosomes and magnetite crystals produced by magnetotactic bacteria as resolved by atomic force microscopy and transmission electron microscopy.</a> Micron. 43, 1331-1335 (2012).</li></ol>

No hay conflictos de interés declarado.

Magnetotácticas bacterias no se encuentran necesariamente en todos los ambientes acuáticos 8, pero cuando ocurren, pueden ser encontrados en el orden de 100 - 1.000 células por milímetro 2. Con el fin de observar el MTB por microscopía óptica, necesitará aproximadamente 50 bacterias / ml en la muestra 8. Si no hay ninguna o pocas MTB en la muestra, entonces usted tendrá que elegir un nuevo sitio ambiental para recoger sus muestras o usted tendrá que tomar una o varias de las té...

<table border="1"><tbody><tr><td> Nombre del elemento </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Portaobjetos de vidrio </td><td> Fisher Scientific </td><td> S95933 </td><td></td></tr><tr><td> Vidrio Pipetas Pasteur </td><td> Fisher Scientific </td><td> 13-678-6A </td><td></td></tr><tr><td> O-ring </td><td> Droguería </td><td></td><td></td></tr><tr><td> Cubreobjetos </td><td> Fisher Scientific </td><td> 12-542B </td><td></td></tr><tr><td> Bar imán </td><td> Fisher Scientific </td><td> S95957 </td><td></td></tr><tr><td> Contenedor </td><td> Cualquier </td><td></td><td> Cualquier plastic o recipiente de vidrio que pueda soportar al menos 0,5 litros y pueden sellarse </td></tr><tr><td> Algodón </td><td> Cualquier </td><td></td><td></td></tr><tr><td> Microscopio con lente de 60X seco </td><td> Zeiss </td><td></td><td> Una lente seca 60X no es absolutamente necesario, pero esto da una alta NA sin utilizar aceite </td></tr><tr><td> Diamond pluma </td><td> Fisher Scientific </td><td> 08-675 </td><td></td></tr><tr><td> 0,22 mm filtro </td><td> Fisher Scientific </td><td> 09-719C </td><td></td></tr><tr><td> 1 ml jeringa </td><td> Fisher Scientific </td><td> NC9788564 </td><td></td></tr><tr><td> Tubos de microcentrífuga </td><td> Fisher Scientific </td><td> 02-681-320 </td><td></td></tr><tr><td> Formvar / Carbon200 de malla, rejillas de cobre </td><td> Ted Pella Inc. </td><td> 01800 </td><td></td></tr><tr><td> Acetato de uranilo </td><td> Ted Pella Inc. </td><td> 19481 </td><td></td></tr><tr><td> Tecnai Espíritu TEM </td><td> FEI </td><td></td><td></td></tr><tr><td> Tecnai F20 S / TEM </td><td> FEI </td><td></td><td></td></tr></tbody></table>

collection, isolation and enrichment of naturally occurring magnetotactic bacteria from the environment

Bacterias magnetotácticas (MTB) son microorganismos acuáticos que fueron los primeros en particular descritos en 1975 1 a partir de muestras de sedimento recolectadas en las marismas de Massachusetts (EE.UU.). Desde entonces se han descubierto MTB en estratificado de agua y sedimentos columnas-de todo el mundo 2. Una característica común a todos MTB es que contienen magnetosomas, que son nanocristales intracelulares, unido a la membrana magnéticas de magnetita (Fe 3 O 4) y / o greigita (Fe 3 S 4) 3 o ambos, 4. En el hemisferio Norte, MTB suelen ser atraídos por el extremo sur de un imán de barra, mientras que en el hemisferio sur, por lo general son atraídos por el extremo norte de un imán de 3,5. Esta propiedad puede aprovecharse cuando se trata de aislar MTB partir de muestras ambientales. Una de las formas más comunes para enriquecer MTB es usar un recipiente de plástico transparente para recoger sedimentos y agua de una fuente natural,tal como un estanque de agua dulce. En el hemisferio norte, el extremo sur de un imán de barra se coloca contra la parte exterior del contenedor justo por encima del sedimento en la interfase agua-sedimento. Después de algún tiempo, la bacteria puede ser retirada desde el interior del recipiente cerca del imán con una pipeta y luego enriquecido aún más mediante el uso de una pista de carreras capilar 6 y un imán. Una vez enriquecido, las bacterias se pueden colocar en un portaobjetos de microscopio utilizando un método de la gota colgante y se observaron en un microscopio de luz o se depositan sobre una rejilla de cobre y se observaron mediante microscopía electrónica de transmisión (TEM). Usando este método, aislado MTB se puede estudiar al microscopio para determinar características tales como el comportamiento natatorio, tipo y número de flagelos morfología celular, de las células, la forma de los cristales magnéticos, número de magnetosomas, el número de cadenas de magnetosoma en cada célula, la composición de los cristales nanomineral, y la presencia de vacuolas intracelulares.

Un imán es una herramienta eficaz que se puede utilizar para aislar las bacterias magnetotácticas (MTB) contenidas en muestras ambientales (Figura 1A). Un circuito capilar (Figura 1B) utiliza las propiedades magnéticas de MTB para atraer a ellos a través de un tapón de algodón en el que se puede separar de no magnetotácticas microorganismos también contenidos dentro de la muestra ambiental. <img alt="Figura 1" src...

Watch this Scientific Journal Video about Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment at JoVE.com

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Se demuestra un método para recoger las bacterias magnetotácticas (MTB) que se pueden aplicar para aguas naturales. MTB puede ser aislado y enriquecido a partir de muestras de sedimento utilizando una configuración relativamente simple que toma ventaja de magnetismo de la bacteria natural. Aislado MTB pueden examinarse en detalle utilizando la luz y microscopía electrónica.

Recolección, aislamiento y enriquecimiento de bacterias naturales magnetotácticas del Medio Ambiente

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

collection-isolation-enrichment-naturally-occurring-magnetotactic

Research

JoVE Journal

Biology

22.9K vistas.  The Ohio State University. Se demuestra un método para recoger las bacterias magnetotácticas (MTB) que se pueden aplicar para aguas naturales. MTB puede ser aislado y enriquecido a partir de muestras de sedimento utilizando una configuración relativamente simple que toma ventaja de magnetismo de la bacteria natural. Aislado MTB pueden examinarse en detalle utilizando la luz y microscopía electrónica.

Video: Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

Enriquecimiento de las células NK a partir de sangre humana con el Kit de RosetteSep de Tecnologías de la StemCell

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

Investigación

Biología

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

Extracción de sangre del cangrejo herradura de América, Limulus polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

El aislamiento de los pequeños RNAs no codificantes de suero humano

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. Birds are widely used subjects in biological research and are ideal for studies involving repeated sperm samples. However, few resources are currently available for those wishing to learn how to collect and extract DNA from avian sperm. Here we describe cloacal massage, a gentle, non-invasive manual technique for collecting avian sperm. Although this technique is established in the literature, it can be difficult to learn from the available descriptions. We also provide information for extracting DNA from avian semen using a commercial extraction kit with modifications. Cloacal massage can be easily used on any small- to medium-sized male bird in reproductive condition. Following collection, the semen can be used immediately for motility assays, or frozen for DNA extraction following the protocol described herein. This extraction protocol was refined for avian sperm and has been successfully used on samples collected from several passerine species (Passer domesticus, Spizella passerina, Haemorhous mexicanus, and Turdus migratorius) and one columbid (Columba livia).

This protocol describes cloacal massage, a non-invasive manual technique for collecting semen from birds, and DNA extraction from avian sperm using a commercial extraction kit with modifications.

Colección de Semen aviar por masaje Cloacal y el aislamiento de ADN de esperma

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ability to biomineralize magnetosomes, which are magnetic nanometer-sized crystals of magnetite (Fe3O4) or greigite (Fe3S4) surrounded by a lipid bilayer membrane, within their cytoplasm. For most known magnetotactic bacteria, magnetosomes are assembled in chains inside the cytoplasm, thereby conferring a permanent magnetic dipole moment to the cells and causing them to align passively with external magnetic fields. Because of these specific features, magnetotactic bacteria have a great potential for commercial and medical applications. However, most species are microaerophilic and have specific O2 concentration requirements, making them more difficult to grow routinely than many other bacteria such as Escherichia coli. Here we present detailed protocols for growing three of the most widely studied strains of magnetotactic bacteria, all belonging to the genus Magnetospirillum. These methods allow for precise control of the O2 concentration made available to the bacteria, in order to ensure that they grow normally and synthesize magnetosomes. Growing magnetotactic bacteria for further studies using these procedures does not require the experimentalist to be an expert in microbiology. The general methods presented in this article may also be used to isolate and culture other magnetotactic bacteria, although it is likely that growth media chemical composition will need to be modified.

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

We present a procedure for growing several strains of Magnetospirillum in two different types of growth media. Magnetospirillum gryphiswaldense strain MSR-1 is grown in both liquid and O2 concentration gradient semi-solid media while M. magneticum strain AMB-1 and M. magnetotacticum strain MS-1 are grown in liquid medium.

Cultivo de bacterias magnétotactiques del género Magnetotacticum: cepas MSR-1, 1 AMB y MS-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

El aislamiento de las células mioepiteliales de la murina lagrimal adulta y las glándulas submandibulares

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Aislamiento y enriquecimiento de células progenitoras epiteliales pulmonares humanas para cultivo de organoides

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Collection and Identification of Pollen from Honey Bee Colonies

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate pesticide exposure of bees via pollen. Described herein is an effective pollen-trapping method for collecting corbicular pollen from honey bees returning to their hives. This collection method results in large quantities of corbicular pollen that can be used for research purposes. Honey bees collect pollen from many plant species, but typically visit one species during each collection trip. Therefore, each corbicular pollen pellet predominantly represents one plant species, and each pollen pellet can be described by color. This allows the sorting of samples of corbicular pollen by color to segregate plant sources. Researchers can further classify corbicular pollen by analyzing the morphology of acetolyzed pollen grains for taxonomic identification. These methods are commonly used in studies related to pollinators such as pollination efficiency, pollinator foraging dynamics, diet quality, and diversity. Detailed methodologies are presented for collecting corbicular pollen using pollen traps, sorting pollen by color, and acetolyzing pollen grains. Also presented are results pertaining to the frequency of pellet colors and taxa of corbicular pollen collected from honey bees in five different cropping systems.

We describe methods for collecting corbicular pollen from honey bees as well as protocols for color sorting, acetolysis, and microscope slide preparation of pollen for taxonomic identification. In addition, we present pellet color and taxonomic diversity of corbicular pollen collected from five cropping systems using pollen traps.

Recolección e identificación de polen de colonias de abejas melíferas

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

Hisopado del medio ambiente urbano - Una tubería para el muestreo y la detección del SARS-CoV-2 de los embalses ambientales

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Fijación rápida in vivo y aislamiento de complejos traslacionales de células eucariotas

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...