The authors gratefully acknowledge the use of facilities at the University of New Hampshire including the University Instrumentation Center. This work was supported by the University of New Hampshire, Department of Chemistry.

los estudios en animales invertebrados realizadas en este documento no están regulados en los Estados Unidos; Por lo tanto, el Comité de Cuidado y Uso de Animales institucional no tiene autoridad para la revisión de dichos protocolos. En lugar de estos no cae bajo la jurisdicción de la regulación en los Estados Unidos, los autores declaran que estos estudios fueron realizados con un esfuerzo sincero hacia el uso ético, el cuidado y el tratamiento de estos animales, se redujo al mínimo el número de individuos y estos esfuerzos son consistentes con la Declaración de Basilea y el Consejo Internacional para la Ciencia Animal de Laboratorio (ICLAS) las directrices éticas. 1. Doryteuthis pealeii (pealeii D.) La disección <ol><li> Compra de calamar decapitado D. pealeii del Departamento de Recursos Marinos en el Laboratorio de Biología Marina en Woods Hole, MA o de cualquier mercado de pescado fresco. No utilice muestras que han sido previamente fileteados o alterados con el fin de aplicar este procedimiento. Específicamente, el cromacapa tophore todavía debe estar intacto en la muestra. <ol><li> Si los animales no se utilizan inmediatamente, almacenar a -20 ° C hasta su uso. Para descongelar las muestras, colocarlos en agua a temperatura ambiente durante 1 hora antes de la disección. </li></ol></li><li> El uso de acero inoxidable tijeras de disección de punta fina rectas, cortadas a lo largo de la parte posterior del animal. Comience en la base del manto ventral, y terminará en la región de la aleta. </li><li> Eliminar todos los órganos internos (estómago, las branquias, glándula de tinta, órganos reproductivos, corazón sistémico, y el corazón braquial) levantándolas con pinzas de disección y la ruptura de su tejido conjuntivo con tijeras de disección. Desechar los órganos en una bolsa de muestras. </li><li> Utilice la disección de T-alfileres para fijar el manto (lado de las aletas hacia arriba) en un estándar de 11-1 / 2 &quot;x 7-1 / 2&quot; x 1-1 / 2 &quot;pan disección de aluminio que contiene una almohadilla de disección flexible. Sumergir el tejido en 250 ml de agua de mar que se ha filtrado a través de un sistema de filtración a vacío desechable de poliestirenoque contiene 0,2 micras tamaños de poro. </li><li> Retirar con cuidado la capa epidérmica de la piel (color opaco) con pinza de disección, manteniendo intacta la capa de piel que contiene chromatophore. Una vez que se retira la capa epidérmica, desprenderse de ella en una bolsa de muestras. </li><li> Diseccionar pequeños (1 cm x 1 cm) de las secciones de la capa de chromatophore con unas pinzas y tijeras de disección. Recoger muestras en tubos de 1,5 ml de microcentrífuga (llene la mitad de cada tubo de vacío con el tejido diseccionado). </li><li> Añadir 500 l de agua de mar filtrada al tejido que contiene tubos. Las muestras se pueden almacenar durante la noche a 4 ° C. </li></ol> 2. Aislamiento de chromatophore pigmento gránulos <ol><li> La digestión del tejido <ol><li> Preparación de papaína / colagenasa <ol><li> Pesar 30 mg de colagenasa y 5 mg de papaína utilizando una &quot;x 4&quot; bajo nitrógeno 4 Documento de pesar. Transferirlas a un tubo de centrífuga de polipropileno de 50 ml con tapón de rosca. </li><li> Medir 10 ml de deionized agua usando una pipeta graduada. Añadir directamente al vial que contiene papaína / colagenasa. Vórtice a la máxima temperatura hasta que la solución es clara. </li></ol></li><li> La eliminación de tejido extracelular <ol><li> Centrifugar el tejido chromatophore recogido de la etapa 1.7 durante 5 minutos a 14.000 x g. Desechar la capa superior de agua de mar en un cubo de residuos plásticos. </li><li> Añadir 500 l de la solución de papaína / colagenasa al tejido usando una micropipeta de volumen variable solo canal P1000. Vortex cada muestra durante 1-2 minutos en la posición más alta. </li><li> Sonicar las muestras durante 5 min a una frecuencia de 40 kHz para disociar las células del tejido circundante. </li><li> Centrifugar durante 5 min a 14.000 x g. Descartar el sobrenadante en un cubo de residuos plásticos. </li><li> Centrifugar el tejido chromatophore recogido durante 5 min a 14.000 x g. Desechar la capa superior de agua de mar en un cubo de residuos plásticos. Repetir una vez más. </li><li> quitar manualmente cualquier tejido seg gran restantelas que no digieren en este proceso con unas pinzas antes de proceder al siguiente paso. </li></ol></li></ol></li><li> El aislamiento del pigmento gránulos <ol><li> Preparación de tampón de homogeneización <ol><li> Pesar 2,38 g de (4- (2-hidroxietil) -1-piperazinetanosulfónico) (HEPES, 100 mM), 0,095 g de cloruro de magnesio (10 mM), 0,856 g de potasio-aspartato (50 mM), y 0,015 g de ditiotreitol (1 mM). Transferir a un recipiente de poliestireno 150 ml con un tapón de rosca. Añadir un inhibidor de la proteasa de mini tableta comercial. </li><li> Medir 100 ml de agua desionizada usando un cilindro graduado. Añadir directamente al recipiente que contiene las sales de homogeneización. Vortex hasta que la solución es clara. </li></ol></li><li> La homogeneización de gránulos de pigmento <ol><li> Centrifugar cada muestra de la etapa 2.1 a 14.000 xg durante 5 min. Descartar el sobrenadante en un cubo de residuos plásticos. Esta muestra debe ser nula de tejido extracelular. </li><li> Añadir 500 y# 181; l de tampón de homogeneización a cada tubo y agitar durante 1-2 minutos cada uno para mezclar bien las muestras. Sonicar las muestras para 30 min (~ 40 kHz) y seguir con 5 min de centrifugación a 14.000 x g. </li><li> Descartar el sobrenadante en un cubo de residuos de plástico, y repetir el paso anterior 2-3 veces para asegurar la digestión completa de las correas de sujeción sáculo chromatophore y proteínas. La solución restante debe contener un sobrenadante de color amarillo claro y un sedimento de color rojo que contiene los gránulos de pigmento aisladas. </li></ol></li></ol></li></ol> 3. Pigmento Extracción <ol><li> Preparación de metanol ácido (HCl-MeOH) <ol><li> Añadir cuidadosamente 25 l de concentrado (36,5 a 38% (w / w)) de ácido clorhídrico (HCl) a 5 ml de metanol (MeOH). Almacenar la solución de HCl-MeOH en un vial de muestra de vidrio tapón de rosca. Agitar suavemente para mezclar uniformemente la solución. NOTA: La más ácida la solución, la más pigmento se extrajo durante la fiextracción de primera. </li></ol></li><li> Extracción de Pigmento <ol><li> Centrifugar la suspensión de gránulos de pigmento (de 2.2.2) durante 5 min a 14.000 xg y descartar el sobrenadante en un cubo de residuos de plástico. </li><li> Añadir 500 l de la solución de HCl-MeOH y agitar cada muestra para ~ 3-4 min. Sonicar las muestras para 10 min (~ 40 kHz), centrifugar durante 5 min a 14.000 x g. </li><li> Usando una pipeta de transferencia, recoger el sobrenadante en un vial de vidrio con tapa de rosca 1 dram. El sobrenadante debería ser de color rojo oscuro. </li><li> 3.2.2 Repetir la etapa de extracción hasta que ya no se extrae el color (~ 4-5 lavados). NOTA: Cada extracción producirá ~ 1,5 ml de pigmento. El sedimento restante es incoloro los gránulos de pigmento extraído. NOTA: Los dos gránulos incoloros y pigmentos extraídos se pueden almacenar en agua a 4 ° C hasta su uso posterior. </li></ol></li></ol> 4. Caracterización largo proceso de extracción <ol><li> El Escaneoectron Microscopía <ol><li> La imagen de gránulos (sin reaccionar de la etapa 2.2.1 y pigmento extraído de la etapa 3.2.3) usando microscopía electrónica de barrido (SEM) para verificar la pureza del proceso de extracción. </li><li> Para preparar las muestras de los gránulos para formación de imágenes, primero centrífuga tanto gránulos que no han reaccionado y los gránulos de pigmento se extrajo durante 5 minutos a 14.000 xg, y descartar el sobrenadante en un cubo de residuos de plástico. Vuelva a suspender estos en 1 ml de etanol, y depósito de 2-3 gotas de la suspensión de gránulos Onto trozos de aluminio SEM utilizando una pipeta de transferencia. </li><li> Después de secar las muestras durante la noche, revestir con recubrimiento de oro-platino durante 3 minutos para lograr un revestimiento de 15 nm. </li><li> Imagen utilizando un SEM con un emisor Schottky y un detector de electrones secundarios. Utilice un voltaje del haz de 5-7 kV y una distancia de trabajo entre 8 y 9 mm a imagen de estos materiales. </li></ol></li><li> Las mediciones espectrofotométricas ultravioleta-visible (UV-VIS) <ol><li> Supervisar el pro absorbanciaarchivos para las 3 condiciones (gránulo sin reaccionar de 2.2.2, 3.2.3 pigmento extraído de, y pigmentos extraídos de gránulos 3.2.4) utilizando un espectrofotómetro de doble haz de 200-800 nm. </li><li> Se recoge una medida en blanco usando agua desionizada en una cubeta de 1 ml para los gránulos sin reaccionar y los gránulos de pigmento extraído. Recoger los espectros de absorción UV-Vis para ambas muestras. </li><li> Se recoge una medida en blanco utilizando la solución de metanol ácido en una cubeta de 1 ml para el pigmento extraído. Recoger espectro de absorción UV-Vis para la muestra. </li><li> Determinar la máxima longitud de onda de cada muestra mediante la identificación de la longitud de onda a la que el pico de absorbancia alcanza su altura máxima relativa. </li></ol></li></ol>

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We have nothing to disclose.

Hemos demostrado un método para extraer pigmentos de gránulos cromatóforos calamar. Al dirigirse específicamente a los gránulos, nuestro objetivo es determinar su papel en la mediación de la coloración de adaptación. Este método difiere de informes anteriores diseñados para caracterizar los pigmentos de cefalópodos usando muestras de tejido a granel 14 o liofilizado piel 15. Mientras que este protocolo es eficaz en la extracción de pigmentos cromatóforos, s...

Cefalópodos como el calamar, la sepia, el pulpo y tienen la capacidad de alterar su apariencia dinámica para camuflar y señalización. 1-6 Esta capacidad es apoyado en parte por la expansión de área selectiva de órganos pigmentadas conocidas como cromatóforos. 4,7-9 Cromatóforos son actuadores blandos que contienen una red de gránulos de pigmento nanoestructurados confinados dentro de un sáculo cytoelastic que está anclado radialmente por las fibras musculares. 1,3 a medida que se accionan, cromatóforos expanden por 500% de la superficie presentado la distribución de los gránulos de todo el órgano. 3,7 10,11 Cuando esta acción es concertada a través de una serie de cromatóforos, se cambia la coloración general del animal. Aunque se sabe que los gránulos de pigmento contribuyen a este cambio de color, su composición sigue siendo desconocido. Se describe un procedimiento para aislar y purificar los pigmentos cromatóforos que pueden ser adaptados para futuros estudios de composición. 3,12 chromatophore tejido que contiene se cosecha a través de la extirpación cuidado del cefalópodo. Un proceso de la digestión y la homogeneización se utiliza a continuación para disociar el tejido circundante y separar las células cromatóforos. Los gránulos nanoestructurados son entonces aislado y purificado a partir de los restantes cromatóforos utilizando sonicación y centrifugación repetidas. Tras la purificación, los pigmentos se extraen de los gránulos en un proceso que es una adaptación de la extracción de color visible de alas de mariposa usando soluciones de metanol ácidos. Microscopía electrónica 13 de barrido (SEM) y espectrofotometría se utilizan para confirmar que los pigmentos cromatóforos se extraen con éxito utilizando este proceso. Este método describe el aislamiento de gránulos cromatóforos que se utiliza para explorar las contribuciones moleculares acoloration en cefalópodos. 12 extracciones de moléculas de animales enteros a menudo puede ser un proceso largo y tedioso. El objetivo es informar a los futuros investigadores de un protocolo eficaz y fácil para la adquisición de los pigmentos de los gránulos nanoestructurados en los cefalópodos.

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			<td height="42" style="height:42px;width:216px;">Collagenase</td>
			<td style="width:71px;">Alfa Aesar</td>
			<td style="width:119px;">9001-12-1</td>
			<td style="width:205px;">No hazard</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dithiothreitol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">3482-12-3</td>
			<td>Irritant, acute toxicity</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HEPES</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">7365-45-9</td>
			<td>Mild irritant</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hydrochloric acid</td>
			<td style="width:71px;">EMD Chemicals</td>
			<td style="width:119px;">7647-01-0</td>
			<td>Corrosive</td>
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			<td height="42" style="height:42px;width:216px;">k-Aspartate</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">1115-63-5</td>
			<td>Reacts violently with oxidants</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Magnesium Chloride</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">7786-30-3</td>
			<td>Mild eye irritant</td>
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			<td height="42" style="height:42px;width:216px;">Methanol</td>
			<td style="width:71px;">Pharmco-AAPER</td>
			<td style="width:119px;">67-56-1</td>
			<td>Highly flammable</td>
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			<td height="42" style="height:42px;width:216px;">Mini tablet prtoease inhibitor</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">469315-90-01</td>
			<td>Corrosive to metal and skin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Papain</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">9001-73-4</td>
			<td>Irritant&#160;</td>
		</tr>
	</tbody>
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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.

Cromatóforos se disecan de la D. pealeii dorsal manto (Figura 1A, 1B). Una vez que se quitan, cromatóforos se lisan y se purificaron usando los ciclos de centrifugación y lavado para aislar los gránulos pigmentados (Figuras 2A, 2B). Soluciones de metanol ácido (HCl-MeOH) se utilizan para extraer el pigmento de los gránulos (Figura 2C), produciendo un extracto de pigmento soluble y gránulo...

Watch this Scientific Journal Video about A Method for Extracting Pigments from Squid Doryteuthis pealeii at JoVE.com

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Se presenta un protocolo para la extracción de pigmentos a partir de los gránulos nanoestructurados en Doryteuthis pealeii cromatóforos calamar.

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 ...

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Biology

A Method for Extracting Pigments from Squid Doryteuthis pealeii

9.3K vistas.  University of New Hampshire.  Se presenta un protocolo para la extracción de pigmentos a partir de los gránulos nanoestructurados en Doryteuthis pealeii cromatóforos calamar. 

Video: A Method for Extracting Pigments from Squid Doryteuthis pealeii

Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and species-rich microbial population that assists in the degradation of lignocellulose predominantly into acetate, the key nutrient fueling termite metabolism (Odelson &amp; Breznak, 1983).  Within these microbial populations are bacteria, methanogenic archaea and, in some ("lower") termites, eukaryotic protozoa. Thus, termites are excellent research subjects for studying the interactions among microbial species and the numerous biochemical functions they perform to the benefit of their host.  The species composition of microbial populations in termite guts as well as key genes involved in various biochemical processes has been explored using molecular techniques (Kudo et al., 1998; Schmit-Wagner et al., 2003; Salmassi &amp; Leadbetter, 2003). These techniques depend on the extraction and purification of high-quality nucleic acids from the termite gut environment.  The extraction technique described in this video is a modified compilation of protocols developed for extraction and purification of nucleic acids from environmental samples (Mor  et al., 1994; Berthelet et al., 1996; Purdy et al., 1996; Salmassi &amp; Leadbetter, 2003; Ottesen et al. 2006) and it produces DNA from termite hindgut material suitable for use as template for polymerase chain reaction (PCR).

Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes

This video illustrates the technique for extracting DNA from the species of microbes resident in the termite hindgut. The preparation of a wet mount slide, which is useful for visualizing the gut microbial community is also illustrated, and a tour through the species-rich gut environment is given.

Extracción de ADN de los microbios del intestino de las termitas (Zootermopsis Angusticollis) y visualizar microbios intestinales

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and ...

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 ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Método para la Cultura de primeros embriones del polluelo ex vivo (Nueva Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Método para la tinción de anticuerpos enteros en el monte del polluelo

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

El uso de Rotorod como un método para el análisis cualitativo de la Ruta de la rata

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

Una lectina Método HPLC para enriquecer péptidos selectivamente glicosilada de muestras biológicas complejas

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made significant contributions to our understanding of the co-evolution of eukaryotes with their microbiota. The symbiotic association between the Hawaiian bobtail squid, Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri has been utilized as a model system for understanding the effects of beneficial bacteria on animal development. Recent studies have shown that macrophage-like hemocytes, the sole cellular component of the squid host's innate immune system, likely play an important role in mediating the establishment and maintenance of this association. This protocol will demonstrate how to obtain hemocytes from E. scolopes and then use these cells in bacterial binding assays. Adult squid are first anesthetized before hemolymph is collected by syringe from the main cephalic blood vessel. The host hemocytes, contained in the extracted hemolymph, are adhered to chambered glass coverslips and then exposed to green fluorescent protein-labeled symbiotic Vibrio fischeri and non-symbiotic Vibrio harveyi. The hemocytes are counterstained with a fluorescent dye (Cell Tracker Orange, Invitrogen) and then visualized using fluorescent microscopy.

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

This video will demonstrate how to obtain hemocytes (blood cells) from the Hawaiian bobtail squid, Euprymna scolopes for use in cell biological and bacterial adhesion assays. Hemocytes will be stained with a fluorescent dye and exposed to GFP-labeled bacteria.

La obtención de hemocitos de la hawaiana Squid Bobtail Euprymna scolopes Y la observación de su adhesión a las bacterias simbióticas y no simbióticas-

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

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 Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

Un método práctico y novela para extraer el ADN genómico de los kits de extracción de sangre para la Preservación Plasma Protein

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Método de la cultura para el corazón de embriones de ratón

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Un método para extraer pigmentos de calamar

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