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.

studi su animali invertebrati condotti nel presente documento non sono regolamentati negli Stati Uniti; pertanto il Comitato Cura e uso istituzionale degli animali non ha l&#39;autorità per la revisione di tali protocolli. Al posto di questi che non rientrano sotto la giurisdizione del regolamento negli Stati Uniti, gli autori dichiarano che questi studi sono stati condotti con sforzo sincero verso l&#39;uso etico, la cura e il trattamento di questi animali, il numero di individui è stato ridotto al minimo e questi sforzi sono coerenti con la Dichiarazione di Basilea e del Consiglio internazionale per Laboratory Animal Science (ICLAS) le linee guida etiche. 1. Doryteuthis pealeii (pealeii D.) Dissection <ol><li> Acquisto decapitato calamari D. pealeii del Dipartimento risorse marine al Marine Biological Laboratory a Woods Hole, MA o da qualsiasi mercato del pesce fresco. Non usare campioni che sono stati raccordati o precedentemente alterati al fine di applicare questa procedura. In particolare, il chromastrato tophore deve essere ancora intatta sul provino. <ol><li> Se gli animali non vengono utilizzati immediatamente, conservarli a -20 ° C fino al momento dell&#39;uso. Per scongelare i campioni, metterli in acqua a temperatura ambiente per 1 ora prima di dissezione. </li></ol></li><li> Utilizzando diritta di acciaio inossidabile forbici dissezione a punta fine, tagliare lungo il lato posteriore della animale. Iniziare alla base del mantello ventrale, e termina in corrispondenza della zona della pinna. </li><li> Rimuovere tutti gli organi interni (stomaco, Branchia, ghiandola inchiostro, organi riproduttivi, cuore sistemica, e cuore brachiale) sollevandole con dissezione forcipe e recidere il loro tessuto connettivo con le forbici dissezione. Scartare gli organi in un sacchetto campione. </li><li> Utilizzare dissezione T-pin per pin mantello (lato fin verso l&#39;alto) in uno standard 11-1 / 2 &quot;x 7-1 / 2&quot; x 1-1 / 2 &quot;pan dissezione di alluminio contenente un pad dissezione flessibile. Immergere il tessuto in 250 ml di acqua di mare che è stata filtrata attraverso un sistema di filtrazione a vuoto in polistirolo usa e gettacontenente 0,2 micron dimensioni dei pori. </li><li> Rimuovere accuratamente lo strato epidermico della pelle (colore opaco) con dissettore, mantenendo intatto lo strato di pelle chromatophore contenente. Una volta che lo strato epidermico viene rimosso, gettare in un sacchetto campione. </li><li> Sezionare piccoli (1 cm x 1 cm) sezioni dello strato chromatophore con pinze e forbici dissezione. Raccogliere campioni in provette da 1,5 ml micro-centrifuga (riempire la metà di ciascun tubo vuoto con il tessuto sezionato). </li><li> Aggiungere 500 ml di acqua di mare filtrata al tessuto contenente tubi. I campioni possono essere conservati a 4 ° C. </li></ol> 2. isolamento chromatophore pigmento Granuli <ol><li> Tissue Digestione <ol><li> Preparazione di papaina / collagenasi <ol><li> Pesare 30 mg di collagenasi e 5 mg di papaina utilizzando una &quot;x 4&quot; a basso azoto 4 pesare carta. Trasferire questi in una provetta da centrifuga in polipropilene da 50 ml con tappo a vite. </li><li> Misurare 10 ml di deionizacqua ed usando una pipetta graduata. Aggiungere direttamente al flacone contenente papaina / collagenasi. Vortex l&#39;impostazione più alta fino a quando la soluzione è chiara. </li></ol></li><li> La rimozione del tessuto extracellulare <ol><li> Centrifugare il tessuto chromatophore raccolti dal punto 1.7 per 5 minuti a 14.000 x g. Eliminare lo strato superiore di acqua marina in un secchio rifiuti di plastica. </li><li> Aggiungere 500 microlitri della soluzione di papaina / collagenasi al tessuto mediante un P1000 volume variabile singolo canale micropipetta. Vortex ogni campione per 1-2 minuti sulla posizione più alta. </li><li> Sonicare i campioni per 5 min ad una frequenza di 40 kHz dissociare le cellule dal tessuto circostante. </li><li> Centrifugare per 5 min a 14.000 x g. Eliminare il surnatante in un secchio di rifiuti di plastica. </li><li> Centrifugare il tessuto chromatophore raccolto per 5 min a 14.000 x g. Eliminare lo strato superiore di acqua marina in un secchio rifiuti di plastica. Ripetere ancora una volta. </li><li> rimuovere manualmente ogni residuo grande sec tessutozioni che non digeriscono in questo processo con forcipe prima di procedere alla fase successiva. </li></ol></li></ol></li><li> Isolando il pigmento Granuli <ol><li> Preparazione di omogeneizzazione Buffer <ol><li> Pesare 2,38 g di (4- (2-idrossietil) -1-acido piperazineethanesulfonic) (HEPES, 100 mM), 0,095 g di cloruro di magnesio (10 mM), 0,856 g di potassio-aspartato (50 mM), e 0,015 g di ditiotreitolo (1 mM). Trasferire in un contenitore di polistirolo da 150 ml con tappo a vite. Aggiungere una mini inibitore della proteasi tablet commerciale. </li><li> Misurare 100 ml di acqua deionizzata utilizzando un cilindro graduato. Aggiungere direttamente al contenitore contenente i sali di omogeneizzazione. Vortex fino a quando la soluzione è limpida. </li></ol></li><li> Omogeneizzazione dei granuli di pigmento <ol><li> Centrifugare ogni campione dal punto 2.1 a 14000 g per 5 min. Eliminare il surnatante in un secchio di rifiuti di plastica. Questo campione fosse nulla di tessuto extracellulare. </li><li> Aggiungere 500 &amp;# 181; l di tampone di omogeneizzazione a ciascuna provetta e vortex per 1-2 minuti ciascuno per mescolare accuratamente i campioni. Sonicare i campioni per 30 minuti (~ 40 kHz) e seguire con 5 minuti di centrifugazione a 14.000 x g. </li><li> Eliminare il surnatante in un secchio di rifiuti di plastica, e ripetere il passaggio precedente 2-3 volte per assicurare la completa digestione delle proteine ​​chromatophore sacculus e pastoie. Il residuo della soluzione deve contenere un surnatante giallo chiaro e una pastiglia di colore rosso contenente gli isolati granuli di pigmento. </li></ol></li></ol></li></ol> 3. pigmento Estrazione <ol><li> Preparazione di Metanolo acido (HCl-MeOH) <ol><li> Aggiungere con cautela 25 ml di concentrato (36,5-38% (w / w)) acido cloridrico (HCl) a 5 ml di metanolo (MeOH). Conservare la soluzione di HCl-MeOH in un tappo a vite fiala del campione di vetro. Agitare delicatamente per mescolare uniformemente la soluzione. NOTA: Il più acido della soluzione, la più pigmento verrà estratto durante la fiprima estrazione. </li></ol></li><li> Estrazione di pigmento <ol><li> Centrifugare la sospensione di pigmento granuli (da 2.2.2) per 5 min a 14000 g e scartare il surnatante in un secchio di rifiuti di plastica. </li><li> Aggiungere 500 ml di soluzione di HCl-MeOH e vortice ogni campione per ~ 3-4 min. Sonicare i campioni per 10 min (~ 40 kHz), quindi si centrifuga per 5 min a 14.000 x g. </li><li> Usando una pipetta di trasferimento, raccogliere il surnatante in un flaconcino tappo a vite in vetro 1 dram. Il supernatante dovrebbe essere di colore rosso scuro. </li><li> Ripetere la fase di estrazione 3.2.2 fino a quando non più di colore viene estratto (~ 4-5 lavaggi). NOTA: ogni estrazione sarà resa ~ 1,5 ml di pigmento. Il pellet incolore rimanente è il pigmento estratto granuli. NOTA: Entrambi i granuli incolori e pigmenti estratti possono essere conservati in acqua a 4 ° C fino all&#39;utilizzo. </li></ol></li></ol> 4. Caratterizzazione tutta Processo di estrazione <ol><li> scansione ElECTRON Microscopia <ol><li> Immagine granuli (non reagiti dal punto 2.2.1 e pigmento-estratta al passo 3.2.3) mediante microscopia elettronica a scansione (SEM) per verificare la purezza del processo di estrazione. </li><li> Per preparare i campioni granulari per l&#39;imaging, prima centrifuga sia granuli che non hanno reagito e pigmenti estratti granuli per 5 minuti a 14000 g, e scartare il surnatante in un secchio di rifiuti di plastica. Risospendere questi in 1 ml di etanolo, e deposito 2-3 gocce di sospensione granuli Onto mozziconi di alluminio SEM utilizzando una pipetta di trasferimento. </li><li> Dopo l&#39;essiccazione dei campioni durante la notte, sputtering cappotto con rivestimento in oro-platino per 3 minuti per ottenere un rivestimento 15 nm. </li><li> Immagine al SEM con un emettitore Schottky e un rivelatore di elettroni secondari. Utilizzare una tensione fascio 5-7 kV e una distanza di lavoro tra 8 e 9 mm di immagine questi materiali. </li></ol></li><li> Le misurazioni spettrofotometriche ultravioletto-visibile (UV-vis) <ol><li> Monitorare l&#39;assorbanza profile per tutte e 3 le condizioni (granuli che non ha reagito da 2.2.2, il pigmento estratto dalla 3.2.3, e pigmenti estratti da granuli 3.2.4) utilizzando uno spettrofotometro a doppio raggio 200-800 nm. </li><li> Raccogliere una misurazione vuoto utilizzando acqua deionizzata in una cuvetta 1 ml per i granuli non reagiti ei pigmenti estratti granuli. Raccogliere spettri di assorbimento UV-Vis per entrambi i campioni. </li><li> Raccogliere una misurazione vuoto utilizzando la soluzione di metanolo acido in una cuvetta 1 ml per il pigmento estratto. Raccogliere spettro di assorbimento UV-Vis per il campione. </li><li> Determinare la massima lunghezza d&#39;onda di ciascun campione identificando la lunghezza d&#39;onda alla quale il picco di assorbanza raggiunge la sua altezza relativa massima. </li></ol></li></ol>

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We have nothing to disclose.

Abbiamo dimostrato un metodo per estrarre i pigmenti da granuli chromatophore calamari. Prendendo di mira in particolare i granuli, il nostro obiettivo è quello di determinare il loro ruolo nella mediazione colorazione adattivo. Questo metodo si differenzia da precedenti relazioni destinate a caratterizzare i pigmenti di cefalopodi utilizzando campioni di tessuto di massa 14 o liofilizzato pelle 15. Mentre questo protocollo è efficace a estrarre pigmenti chromatophore...

Cefalopodi, come calamari, seppie, polpi e hanno la capacità di modificare dinamicamente il loro aspetto per camuffare e la segnalazione. 1-6 Questa capacità è sostenuto in parte dall&#39;espansione areale selettivo degli organi pigmentate noto come cromatofori. 4,7-9 Cromatofori sono attuatori morbidi che contengono una rete di granuli di pigmento nanostrutturati confinati all&#39;interno di un sacculus cytoelastic ancorato radialmente dalle fibre muscolari. 1,3 Come sono azionati, chromatophores espandono da 500% della superficie presentata distribuendo i granuli in tutto l&#39;organo. 3,7 , 10,11 Quando questa azione è concertata su un numero di cromatofori, la colorazione generale dell&#39;animale è cambiato. Mentre è noto che i granuli di pigmento contribuiscono a questo cambiamento di colore, la composizione rimane sconosciuta. Descriviamo una procedura per isolare e purificare pigmenti chromatophore che possono essere adattati per i futuri studi compositivi. 3,12 chromatophore tessuto contenente viene raccolto attraverso un&#39;attenta estirpazione dal cefalopode. Un processo di digestione e omogeneizzazione viene poi utilizzato per dissociare il tessuto circostante e separare le cellule chromatophore. I granuli nanostrutturati sono poi isolato e purificato dalle restanti cromatofore utilizzando ripetuta sonicazione e centrifugazione. Al momento di purificazione, i pigmenti vengono estratti dai granuli in un processo che è adattato da l&#39;estrazione del colore visibile da ali di farfalla con soluzioni di metanolo acide. 13 microscopia elettronica a scansione (SEM) e spettrofotometria vengono utilizzati per confermare che i pigmenti chromatophore vengono estratti con successo utilizzando questo processo. Questo metodo descrive l&#39;isolamento di granuli chromatophore che viene utilizzato per esplorare i contributi molecolari alla coloration in cefalopodi. 12 piccole estrazioni molecola di animali interi possono spesso essere un processo lungo e noioso. L&#39;obiettivo è quello di informare futuri ricercatori di un protocollo efficace e facile per l&#39;acquisizione dei pigmenti granuli nanostrutturati a cefalopodi.

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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>
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			<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>
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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.

Cromatofori sono sezionati dal D. pealeii dorsale del mantello (Figura 1A, 1B). Una volta che sono rimossi, chromatophores vengono lisate e purificati mediante centrifugazione e lavaggio cicli di isolare i granuli pigmentati (figure 2A, 2B). Soluzioni acide metanolo (HCl-MeOH) sono utilizzati per estrarre il pigmento dai granuli (Figura 2C), ottenendo un estratto di pigmenti solubili e insolubil...

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

Un protocollo per l&#39;estrazione dei pigmenti dai granuli nanostrutturati in Doryteuthis pealeii cromatofori calamaro è presentato.

Un metodo per estrarre pigmenti da Squid<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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A Method for Extracting Pigments from Squid Doryteuthis pealeii

9.3K Views.  University of New Hampshire.  Un protocollo per l'estrazione dei pigmenti dai granuli nanostrutturati in Doryteuthis pealeii cromatofori calamaro è presentato. 

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.

Estrazione del DNA da microbi intestinali della Termite (Zootermopsis Angusticollis) e visualizzare microbi intestinali

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 ...

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Biologia

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.

Pirosequenziamento: un metodo semplice per genotipizzazione accurata

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.

Metodo per la Cultura di Chick primi embrioni ex vivo (Nuova 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.

Metodo per la colorazione intero anticorpi Monte a Chick

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.

L&#39;uso di Rotorod come un metodo per l&#39;analisi qualitativa del Camminare 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 ...

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.

Un metodo HPLC lectina per arricchire Peptidi selettivamente glicosilata da campioni biologici complessi

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.

Ottenere ematociti concentrati dal hawaiano Bobtail Squid Euprymna scolopes L&#39;osservazione e la loro aderenza ai batteri simbionti e non Symbiotic

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 protocollo semplice per Estrazione emociti da bruchi selvaggi

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 metodo pratico e innovativo per estrarre il DNA genomico da kit di raccolta del sangue per Plasma Proteina 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 ...

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; Metodo di Cultura per il mouse embrionale del cuore

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

Un metodo per estrarre pigmenti da Squid

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