TMS fue apoyado por la beca Harlan Trust (GWU) durante este estudio. La financiación para la recolección de campo y la cría de L. fasciola y E. delphinii fue proporcionado por la NSF subvención DEB 0642438 a JTL.

1. Preparación de materiales <ol><li> Preparar agujas (1-2 micras punta y el cono de 3-4 mm) utilizando tubos de vidrio de borosilicato capilares y un extractor de micropipeta. Ajuste del equipo: Rampa: 561; velocidad: 20; calor: 560; retardo: 1; tirón: 100; presión: 500. </li><li> Preparar la solución de recogida: 60% de medio de Grace (GM) suplementado con 10% de suero bovino fetal (FBS) y 20% de tampón de anticoagulante (98 mM de NaOH, 186 mM NaCl, 1,7 mM EDTA y 41 ácido cítrico mM, pH 4,5 ). La solución anterior se prepara fresco en condiciones estériles y se mantuvo en hielo en todo momento. </li><li> Preparar la solución tampón de incubación: 90% de GM suplementado con 10% de FBS. Esta solución también está preparado fresco y se mantuvieron en hielo en todo momento. </li><li> Preparar un total de al menos 15 l × el número de insectos para la colección y las soluciones de incubación. </li></ol> 2. La inyección de orugas con la solución Collection <ol><li> Se anestesia ins penúltimasalquitrán de larvas por enfriamiento en hielo durante 20 min antes de la extracción de hemocitos. </li><li> Colocar el extremo romo de una aguja limpia capilar (16-manométrica) en el tubo que está conectado a una jeringa de vidrio de 20 ml (Figura 2). Deje el tubo que contiene la solución de recopilación de abrir en el banco de laboratorio y lentamente llenar el capilar con el tampón. El capilar se llena generalmente de aproximadamente 10-15 l de solución de recogida, dependiendo del tamaño de la oruga (aproximadamente 1 l por solución de 10 mg de la masa corporal total oruga = 0,01% de la masa corporal total). Coloque la aguja en una bola de arcilla que se coloca al lado del microscopio de disección. </li><li> Coloque la oruga en una placa de Petri. El insecto debe ser colocado en un lado de manera que los espiráculos son claramente visibles (figura 3). </li><li> Mientras ve bajo un microscopio de disección, mantenga la oruga en su lugar con un par de pinzas y colocar la aguja cerca de uno de los espiráculos, donde el encutícula secta es generalmente más suave. Presione suavemente la aguja capilar contra el cuerpo del insecto. Una vez que la aguja ha penetrado en la cutícula, inyectar el volumen total de solución en la oruga (Figura 3). Es importante evitar la inyección de burbujas de aire en la oruga. El cuerpo de la oruga se hinchará (por ejemplo, aumento de volumen en un 20%) después de la inyección, pero no debe estallar o supurar. </li><li> Volver la oruga a la hielo, posicionando el insecto dorsalmente para prevenir tampón se escape fuera de la herida (Figura 3). </li><li> Repita los pasos 2.1-2.4 para cada oruga utilizados para la extracción de hemolinfa. En esta etapa, permitir que todos los insectos para enfriar en hielo durante 30 min antes de proceder. </li></ol> 3. Extracción de hemocitos <ol><li> Coloque la oruga en una placa de Petri bajo el microscopio de disección. </li><li> Hacer un poco profundo (2-3 mm de largo) incisión en la región posterior de la oruga utilizando un scalpe estéril<html> l (Figura 3). Tenga cuidado de no dañar los intestinos u otros órganos internos, el objetivo es perforar la cutícula con cuidado y recoger la mezcla de tampón / hemolinfa de los insectos que contiene hemocitos individuales. </li><li> Apriete la oruga suavemente con unas pinzas flexibles de plástico durante la percepción de la mezcla hemolinfa (aproximadamente 10-20 l) con una punta de pipeta de 10 l, de nuevo teniendo cuidado de no dañar los tejidos internos (Figura 3). La hemolinfa se obtiene de la gotita de exprimido de cuerpo de la oruga. Deseche los restos de la oruga. </li><li> Recoger hemolinfa de cada individuo oruga en distintos etiquetados siliconadas microcentrífuga de 0,5 ml. </li><li> Añadir un volumen igual de la solución de incubación (aproximadamente 10-20 l) a cada muestra de hemolinfa obtenida de un individuo oruga. Mantener muestras de hemolinfa en hielo en todo momento. </li><li> Repetir los pasos 3.1-3.5 para cada oruga usada en el experimento. </li></ol>le &quot;&gt; 4. Cuantificación de los hemocitos <ol><li> Mezclar la muestra de hemolinfa y la solución de incubación inmediatamente antes de hemocitos de conteo. Este tratamiento permite una separación adecuada de hemocitos y evita la formación de grumos. </li><li> Utilice una punta de pipeta limpia para transferir 10 l de muestra de hemolinfa se mezcla con la solución de incubación en un hemocitómetro. </li><li> El uso de un microscopio compuesto, contar y registrar el número de hemocitos en al menos 15 de los cuadrados grandes (área de punto de partida: 0,04 mm 2, profundidad: 0,1 mm) de la rejilla central del hemocitómetro (Figura 3). Agregue estos valores para obtener una suma total del número de hemocitos en los 15 cuadrados. </li><li> Enjuague el hemocitómetro y su tapa deslizante completamente utilizando una botella de lavado con agua destilada y desionizada y seque con Kimwipes (Kimberly-Clark) entre cada muestra. </li><li> Repita los pasos 4.1-4.4 para cada muestra de hemolinfa. </li><li> Calcular el número medio de hemocitos por ml de hemolinfa volume para cada oruga, como sigue: </li></ol> Volumen total de cada cuadrado de la cuadrícula de hemocitómetro: (0,04 mm 2 de área) × (0,1 mm de profundidad) = 0,004 mm 3 × 15 cuadrados = 0,06 mm 3 = el volumen total de todos los 15 cuadrados contados. (Suma del número total de hemocitos en los 15 cuadrados contados / el volumen total de todos los 15 cuadrados contados) x 2 = factor de dilución el número de hemocitos / mm 3 x 1.000 = el número de hemocitos / ml de hemolinfa.

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	<li>Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+cells+and+blood+cell+development+in+the+animal+kingdom.">Blood cells and blood cell development in the animal kingdom.</a> Annu. Rev. Cell Dev. Biol. 22, 677 (2006).</li><li>Wood, W., Jacinto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+embryonic+haemocytes:+masters+of+multitasking.">Drosophila melanogaster embryonic haemocytes: masters of multitasking.</a> Nat. Rev. Mol. Cell Biol. 8 (7), 542 (2007).</li><li>Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insect+cellular+immune+response.">The insect cellular immune response.</a> Insect Sci. 15 (1), 1 (2008).</li><li>Fauvarque, M. O., Williams, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+cellular+immunity:+a+story+of+migration+and+adhesion.">Drosophila cellular immunity: a story of migration and adhesion.</a> J. Cell Sci. 124 (9), 1373 (2011).</li><li>Nehme, N. T., Quintin, J., Cho, J. H., Lee, J., Lafarge, M. C., Kocks, C., Ferrandon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+roles+of+the+cellular+and+humoral+responses+in+the+Drosophila+host+defense+against+three+gram-positive+bacterial+infections.">Relative roles of the cellular and humoral responses in the Drosophila host defense against three gram-positive bacterial infections.</a> PLoS One. 6 (3), e14743 (2011).</li><li>Ulvila, J., Vanha-Aho, L. M., R&#228;met, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+phagocytosis+-+still+many+unknowns+under+the+surface.">Drosophila phagocytosis - still many unknowns under the surface.</a> APMIS. 119 (10), 651 (2011).</li><li>Moreira, C. G., Regan, J. C., Zaidman-Remy, A., Jacinto, A., Praq, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+hemocyte+migration:+an+in+vivo+assay+for+directional+cell+migration.">Drosophila hemocyte migration: an in vivo assay for directional cell migration.</a> Methods Mol. Biol. 769, 249 (2011).</li><li>Hillyer, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+immunity.">Mosquito immunity.</a> Adv. Exp. Med. Biol. 708, 218 (2010).</li><li>Castillo, J. C., Robertson, A. E., Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+hemocytes+from+the+mosquitoes+Anopheles+gambiae+and+Aedes+aegypti.">Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti.</a> Insect Biochem. Mol. Biol. 36 (12), 891 (2006).</li><li>Jiang, H., Vilcinskas, A., Kanost, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+in+lepidopteran+insects.">Immunity in lepidopteran insects.</a> Adv. Exp. Med. Biol. 708, 181 (2010).</li><li>Eleftherianos, I., Xu, M., Yadi, H., ffrench-Constant, R. H., Reynolds, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmatocyte-spreading+peptide+(PSP)+plays+a+central+role+in+insect+cellular+immune+defenses+against+bacterial+infection.">Plasmatocyte-spreading peptide (PSP) plays a central role in insect cellular immune defenses against bacterial infection.</a> J. Exp. Biol. 212 (12), 1840 (2009).</li><li>Ribeiro, C., Breh&#233;lin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+haemocytes:+what+type+of+cell+is+that.">Insect haemocytes: what type of cell is that.</a> J. Insect Physiol. 52 (5), 417 (2006).</li><li>Beetz, S., Brinkmann, M., Trenczek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+between+larval+and+pupal+hemocytes+of+the+tobacco+hornworm,+Manduca+sexta,+determined+by+monoclonal+antibodies+and+density+centrifugation.">Differences between larval and pupal hemocytes of the tobacco hornworm, Manduca sexta, determined by monoclonal antibodies and density centrifugation.</a> J. Insect Physiol. 50 (9), 805 (2004).</li><li>Beetz, S., Holthusen, T. K., Koolman, J., Trenczek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+hemocyte+counts+with+different+developmental+parameters+during+the+last+larval+instar+of+the+tobacco+hornworm,+Manduca+sexta.">Correlation of hemocyte counts with different developmental parameters during the last larval instar of the tobacco hornworm, Manduca sexta.</a> Arch. Insect Biochem. Physiol. 67 (2), 63 (2008).</li><li>Eleftherianos, I., Joyce, S., ffrench-Constant, R. H., Clarke, D. J., Reynolds, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+tri-trophic+interaction+between+insects,+nematodes+and+Photorhabdus.">Probing the tri-trophic interaction between insects, nematodes and Photorhabdus.</a> Parasitology. 137 (11), 1695 (2010).</li><li>Qayum, A. A., Telang, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Protocol+for+Collecting+and+Staining+Hemocytes+from+the+Yellow+Fever+Mosquito+Aedes+aegypti.">A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti.</a> J. Vis. Exp. (51), e2772 (2011).</li></ol>

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Los métodos para la extracción de hemocitos de los insectos de importancia médica y los insectos lepidópteros modelo han sido previamente informado de 9, 14. Métodos de extracción de hemocitos se adaptan de acuerdo con la especie de insecto, la etapa de desarrollo del insecto y sus características morfológicas. Por ejemplo, el aislamiento hemolinfa de las larvas de Manduca puede ser fácilmente realizada por cortando el cuerno curvado cerca del extremo del abdomen 15. Porque las or...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Micropipetas extractor </td><td> Sutter Instruments </td><td> P-1000 </td><td></td></tr><tr><td> Tubos de vidrio borosilicato </td><td> Sutter Instruments </td><td> B100-50-10 </td><td> OD: 1,0, ID: 0,50 mm </td></tr><tr><td> Medio de Grace </td><td> Sigma </td><td> G8142 </td><td></td></tr><tr><td> Suero Bovino Fetal </td><td> Thermo Fisher Scientific </td><td> SH3007102 </td><td> Inactivado por calor </td></tr><tr><td> Dumont # 5 Pinzas </td><td> Bellas herramientas científicas </td><td> 11252-40 </td><td></td></tr><tr><td> Hemocitómetro Neubauer </td><td> Hausser Scientific </td><td> 3200 </td><td></td></tr><tr><td> Plastic tubing </td><td> Tri-Tech </td><td> TT-3-32OD </td><td> OD: 3/32&#39;&#39;, ID: 1/32 &#39; </td></tr><tr><td> Cristal médico jeringa </td><td> Fortuna Optima </td><td> D-97877 </td><td> 50 ml de volumen </td></tr><tr><td> Aguja punta roma </td><td> Piezas pequeñas </td><td> NE-162PL-25 </td><td> Calibre 16 x 1 &quot;de largo </td></tr></tbody></table>

a simple protocol for extracting hemocytes from wild caterpillars

Hemocitos de insectos (equivalente a mamíferos células blancas de la sangre) juegan un papel importante en varios procesos fisiológicos durante el ciclo de vida de un insecto 1. En las fases larvarias de insectos pertenecientes al orden de los lepidópteros (polillas y mariposas) y dípteros (moscas verdaderas), hemocitos se forman a partir de la glándula linfática (un órgano hematopoyético especializado) o células embrionarias y se puede llevar hasta la etapa adulta. Hemocitos embrionarias están involucrados en la regulación de la migración celular durante el desarrollo y la quimiotaxis durante la inflamación. También participan en la apoptosis de células y son esenciales para la embriogénesis 2. Hemocitos mediar en el brazo celular de la respuesta inmune innata de insectos que incluye varias funciones, tales como la propagación de células de agregación celular, la formación de nódulos, la fagocitosis y la encapsulación de los invasores extraños 3. Ellos también son responsables de orquestar específicas defensas humorales de insectos durante la infección, como la producción de péptidos antimicrobianos y otras moléculas efectoras 4, 5. Hemocyte morfología y función han sido estudiada principalmente en modelos de insectos genéticas o fisiológicas, incluyendo la mosca de la fruta, Drosophila melanogaster 6, 7, el Aedes aegypti y Anopheles gambiae 8, 9 y el gusano del tabaco, Manduca sexta 10, 11. Sin embargo, existe poca información que existe actualmente sobre la diversidad, la clasificación, morfología y función de hemocitos en especies de insectos no-modelo, en especial los de origen silvestre 12. Aquí se describe un protocolo sencillo y eficiente para la extracción de hemocitos de larvas silvestres. Utilizamos penúltimo estadio Lithacodes fasciola (amarillo de hombros polilla slug) (Figura 1) y Euclea delphinii (espinosa babosa roble) orugas (Lepidoptera: Limacodidae) y muestran que los volúmenes suficientes de hemolinfa (sangre de insectos)puede ser aislado y números de hemocitos contados a partir de larvas individuales. Este método puede ser usado para estudiar eficientemente tipos de hemocitos en estas especies, así como en otras orugas de lepidópteros relacionados recolectadas desde el campo, o se puede combinar fácilmente con ensayos inmunológicos destinados a investigar la función de hemocitos después de la infección microbiana o con organismos parásitos 13.

El protocolo se describe en la presente memoria permite la recogida de un volumen mínimo de 10-20 l de hemolinfa de insectos de orugas individuales. Hemocitos recogidos mediante este método están libres de grumos de células, defectos melanización, restos de tejido u otros contaminantes. Por lo tanto hemocitos se puede observar fácilmente y contados bajo el microscopio, y varios insectos se puede observar dentro de un Hosur pocos. Hemos observado que la mayoría de los hemocitos en nuestras muestras consistieron de...

Watch this Scientific Journal Video about A Simple Protocol for Extracting Hemocytes from Wild Caterpillars at JoVE.com

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Hemocitos Insectos llevar a cabo muchas funciones importantes, tanto inmunes y no inmunes, en todas las etapas de desarrollo del insecto. Nuestro conocimiento actual de los tipos de hemocitos y la función proviene de estudios en modelos genéticos de insectos. A continuación, se presenta un método para la extracción, cuantificación y visualización de hemocitos de larvas silvestres.

Un protocolo simple para la extracción de hemocitos de orugas silvestres

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

a-simple-protocol-for-extracting-hemocytes-from-wild-caterpillars

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

16.1K vistas.  The George Washington University. Hemocitos Insectos llevar a cabo muchas funciones importantes, tanto inmunes y no inmunes, en todas las etapas de desarrollo del insecto. Nuestro conocimiento actual de los tipos de hemocitos y la función proviene de estudios en modelos genéticos de insectos. A continuación, se presenta un método para la extracción, cuantificación y visualización de hemocitos de larvas silvestres.

Video: A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

Protocolo para la cría de los mosquitos (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Investigación

Biología

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Un método simple para determinar el genotipo exacto

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

Protocolo para las neuronas simpáticas de rata cultivo Ganglios cervical superior (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

Un protocolo para la producción de KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Una basada en PCR genotipificación método para distinguir entre las variedades de tipo silvestre y ornamental de Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Técnica sencilla y eficaz para la preparación de suspensiones de células testiculares

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

SIVQ-LCM Protocol for the ArcturusXT Instrument

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an advanced, rapidly customizable laser dissection platform technology. In this report, we describe the integration of the image analysis software Spatially Invariant Vector Quantization (SIVQ) onto the ArcturusXT instrument. The ArcturusXT system contains both an infrared (IR) and ultraviolet (UV) laser, allowing for specific cell or large area dissections. The principal goal is to improve the speed, accuracy, and reproducibility of the laser dissection to increase sample throughput. This novel approach facilitates microdissection of both animal and human tissues in research and clinical workflows.

SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings.

Protocolo SIVQ-LCM para el Instrumento ArcturusXT

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

Un ensayo biológico simple para la Evaluación de Factores de Crecimiento Endotelial Vascular

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

A Method for Extracting Pigments from Squid Doryteuthis pealeii

A protocol for the extraction of pigments from the nanostructured granules in squid Doryteuthis pealeii chromatophores is presented.

Un método para extraer pigmentos de calamar<em&gt; Pealeii Doryteuthis</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Un protocolo simple para la extracción de hemocitos de orugas silvestres

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