TMS è stato sostenuto dalla fiducia Harlan Fellowship (GWU) nel corso di questo studio. I finanziamenti per la raccolta sul campo e allevamento L. Fasciola e E. delphinii sono state fornite dal NSF concedere DEB 0642438 al JTL.

1. Preparazione del materiale <ol><li> Preparare gli aghi (1-2 micron e punta conica 3-4 mm) con tubi in vetro borosilicato capillari nonché di un estrattore micropipetta. Le impostazioni dello strumento: a terra: 561, velocità: 20; calore: 560; ritardo: 1; tiro: 100; Pressione: 500. </li><li> Preparare la soluzione di raccolta: 60% del Medio Grace (GM) integrato con 10% di siero bovino fetale (FBS) e 20% di tampone di anticoagulante (98 mM NaOH, 186 mM NaCl, 1,7 mM EDTA e 41 mM di acido citrico, pH 4,5 ). La suddetta soluzione è preparato fresco in condizioni sterili e mantenuto in ghiaccio in ogni momento. </li><li> Preparare la soluzione tampone di incubazione: 90% di GM integrato con 10% di FBS. Questa soluzione è anche preparato fresco e mantenuto in ghiaccio in ogni momento. </li><li> Preparare un totale di almeno 15 microlitri × il numero di insetti sia per la raccolta e le soluzioni di incubazione. </li></ol> 2. Iniezione di bruchi con Collection Solution <ol><li> Anestetizzare ins penultimelarve tar da raffreddamento in ghiaccio per 20 minuti prima dell&#39;estrazione hemocyte. </li><li> Montare la punta di un ago capillare (calibro 16) nel tubo che è collegato ad una siringa da 20 ml di vetro (Figura 2). Lasciare il tubo contenente la soluzione di raccolta aperto sul banco di laboratorio e riempire lentamente il capillare con il buffer. Il capillare è generalmente riempito con circa 10-15 ml di soluzione di raccolta, a seconda delle dimensioni del bruco (circa 1 ul di soluzione per 10 mg di massa corporea caterpillar totale = 0,01% della massa totale del corpo). Appoggiare l&#39;ago su una palla di argilla che si trova accanto al microscopio da dissezione. </li><li> Posizionare il bruco in una capsula di Petri. L&#39;insetto deve essere posizionato su un lato in modo che gli spiracoli sono chiaramente visibili (Figura 3). </li><li> Durante la visualizzazione sotto un microscopio da dissezione, tenere il bruco in posizione con una pinza e posizionare il ago vicino ad una delle spiracoli, in cui lacuticola setta è generalmente più dolce. Premere delicatamente l&#39;ago capillare contro il corpo dell&#39;insetto. Una volta che l&#39;ago è penetrato nella cuticola, iniettare il volume totale della soluzione nel bruco (Figura 3). E &#39;importante evitare l&#39;iniezione di bolle d&#39;aria nel bruco. Il corpo del bruco si gonfiano (ad esempio, aumento del volume del 20%) in seguito a iniezione, ma non dovrebbe scoppiare o trasudano. </li><li> Tornare il bruco al ghiaccio, posizionando l&#39;insetto dorsalmente per impedire tampone di fuoriuscire dalla ferita (Figura 3). </li><li> Ripetere i passaggi 2,1-2,4 per ogni caterpillar usato per l&#39;estrazione emolinfa. In questa fase, tutti gli insetti consentono di raffreddare in ghiaccio per 30 minuti prima di procedere. </li></ol> 3. Estrazione di emociti <ol><li> Posizionare il bruco in una capsula Petri sotto il microscopio da dissezione. </li><li> Fai un poco profondo (2-3 mm di lunghezza) incisione nella regione posteriore del bruco con un Scalpe sterile<html> l (Figura 3). Fare attenzione a non danneggiare l&#39;intestino o di altri organi interni, l&#39;obiettivo è quello di perforare la cuticola con attenzione e raccogliere il buffer / emolinfa mix contenente emociti dagli insetti individuali. </li><li> Premere il bruco delicatamente con pinze flessibili in plastica, mentre la raccolta del mix emolinfa (circa il 10-20 pl), con una punta di 10 microlitri pipetta, ancora una volta facendo attenzione a non danneggiare i tessuti interni (Figura 3). Emolinfa è raccolto dalla gocciolina spremuto dal corpo del bruco. Eliminare il caterpillar i resti. </li><li> Raccogliere emolinfa da ogni singolo bruco in distinte provette etichettate siliconato 0,5 ml microcentrifuga. </li><li> Aggiungere uguale volume della soluzione di incubazione (circa 10-20 microlitri) di ciascun campione emolinfa raccolti da un individuo bruco. Tenere campioni emolinfa su ghiaccio in ogni momento. </li><li> Ripetere i passaggi 3,1-3,5 per ogni caterpillar utilizzato per l&#39;esperimento. </li></ol>Le &quot;&gt; 4. Quantificazione di emociti <ol><li> Mescolare il campione e la soluzione emolinfa incubazione immediatamente prima emociti conteggio. Questo trattamento consente un&#39;adeguata separazione delle emociti e previene formazione di grumi. </li><li> Utilizzare una pipetta pulita per trasferire 10 microlitri di campione emolinfa miscelata con la soluzione di incubazione su un emocitometro. </li><li> Utilizzando un microscopio composto, contare e registrare il numero di emociti in almeno 15 dei quadrati grandi (area del quadrato uno: 0,04 mm 2, profondità: 0,1 mm) della griglia centrale del emocitometro (Figura 3). Aggiungere questi valori per ottenere una somma totale del numero di emociti nei 15 quadrati. </li><li> Sciacquare il emocitometro e la sua slitta coperchio a fondo utilizzando una bottiglia di lavaggio con acqua distillata e deionizzata e asciugare con Kimwipes (Kimberly-Clark) tra ogni campione. </li><li> Ripetere i passaggi 4,1-4,4 per ogni campione emolinfa. </li><li> Calcolare il numero medio di emociti per ml di emolinfa volume per ogni caterpillar, come segue: </li></ol> Il volume totale di ciascun quadrato della griglia emocitometro: (0,04 mm 2 zona) × (0,1 mm di profondità) = 0,004 mm 3 × 15 caselle = 0,06 mm 3 = il volume totale di tutti i 15 quadrati contati. (Somma del numero totale di emociti nei 15 quadrati contati / il volume totale di tutti i 15 quadrati contati) x 2 = fattore di diluizione del numero di emociti / mm 3 x 1.000 = il numero di emociti / ml di emolinfa.

<ol>
	<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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Metodi per l&#39;estrazione di emociti da insetti clinicamente importanti lepidotteri e insetti modello sono stati precedentemente segnalati 9, 14. Metodi di estrazione Hemocyte sono adattati a seconda delle specie di insetti, lo stadio di sviluppo dell&#39;insetto e le sue caratteristiche morfologiche. Per esempio, l&#39;isolamento emolinfa da larve Manduca può essere facilmente eseguita tagliandoli corno curvo vicino all&#39;estremità dell&#39;addome 15. Perché bruchi slug (Limacodida...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Micropipetta estrattore </td><td> Sutter Instruments </td><td> P-1000 </td><td></td></tr><tr><td> Tubi di vetro borosilicato </td><td> Sutter Instruments </td><td> B100-50-10 </td><td> OD: 1.0, ID: 0.50 mm </td></tr><tr><td> Media di Grace </td><td> Sigma </td><td> G8142 </td><td></td></tr><tr><td> Siero fetale bovino </td><td> Thermo Fisher Scientific </td><td> SH3007102 </td><td> Inattivato al calore </td></tr><tr><td> Dumont # 5 Pinze </td><td> Strumenti per le scienze Belle </td><td> 11252-40 </td><td></td></tr><tr><td> Neubauer emocitometro </td><td> Hausser scientifico </td><td> 3200 </td><td></td></tr><tr><td> Tubi di plasticang </td><td> Tri-Tech </td><td> TT-3-32OD </td><td> OD: 3/32&#39;&#39;, ID: 1/32 &#39; </td></tr><tr><td> Vetro medico siringa </td><td> Fortuna Optima </td><td> D-97.877 </td><td> 50 ml di volume </td></tr><tr><td> &#39;Ago Blunt </td><td> Piccole parti </td><td> NE-162PL-25 </td><td> Calibro 16 x 1 &quot;lunghezza </td></tr></tbody></table>

a simple protocol for extracting hemocytes from wild caterpillars

Emociti insetti (equivalente a mammiferi globuli bianchi) svolgono un ruolo importante in diversi processi fisiologici durante tutto il ciclo di vita di un insetto 1. Negli stadi larvali di insetti appartenenti agli ordini dei Lepidotteri (farfalle e falene) e Ditteri (mosche vere), emociti sono formate dalla ghiandola linfatica (un organo specializzato ematopoietiche) o cellule embrionali e possono essere trasportati fino alla fase adulta. Emociti embrionali sono coinvolti nella migrazione cellulare durante la regolazione dello sviluppo e chemiotassi durante l&#39;infiammazione. Hanno anche prendere parte a apoptosi delle cellule e sono essenziali per embriogenesi 2. Emociti mediare il braccio cellulare dell&#39;insetto risposta immunitaria innata che include diverse funzioni, come la cella di diffusione, aggregazione cellulare, formazione di noduli, la fagocitosi e l&#39;incapsulamento di invasori stranieri 3. Essi sono anche responsabili per orchestrare specifiche difese insetto umorali durante l&#39;infezione, come la produzione di peptidi antimicrobici e altre molecole effettrici 4, 5. Hemocyte morfologia e funzione sono principalmente stato studiato in modelli genetici insetti o fisiologico, compresa la mosca della frutta, Drosophila melanogaster 6, 7, le zanzare Aedes aegypti e Anopheles gambiae 8, 9 e il hornworm tabacco, Manduca sexta 10, 11. Tuttavia, esistono scarse informazioni attualmente circa la diversità, la classificazione, la morfologia e la funzione di emociti in non-modello di specie di insetti, in particolare quelli raccolti in natura 12. Qui si descrive un protocollo semplice ed efficiente per l&#39;estrazione di emociti da bruchi selvatici. Usiamo penultimo instar Lithacodes fasciola (giallo-spalle falena slug) (Figura 1) e Euclea delphinii (spinoso slug rovere) bruchi (Lepidoptera: Limacodidae) e mostrare che i volumi sufficienti di emolinfa (sangue degli insetti)può essere isolato e numeri hemocyte contati da larve individuale. Questo metodo può essere usato per studiare efficientemente tipi hemocyte in queste specie così come in altri bruchi di lepidotteri correlati raccolti dal campo, oppure può essere facilmente combinati con analisi immunologiche destinati a studiare la funzione hemocyte dopo infezione microbica o organismi parassitari 13.

Il protocollo qui descritto consente la raccolta di un volume minimo di 10-20 ml di emolinfa insetto da bruchi individuali. Emociti raccolti con questo metodo sono privi di aggregazione cellulare, difetti melanizzazione, frammenti di tessuto o altri contaminanti. Pertanto emociti possono essere facilmente osservati e contati al microscopio, e diversi insetti può essere osservato in un Hosur pochi. Abbiamo osservato che la maggior parte dei emociti nei nostri campioni erano plasmatocytes 3 ​​(emociti diff...

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

Emociti insetti svolgere molte funzioni importanti, sia immunitarie e non immunitarie, durante tutte le fasi di sviluppo degli insetti. La nostra attuale conoscenza dei tipi di hemocyte e la funzione viene da studi su insetti modelli genetici. Qui vi presentiamo un metodo per l&#39;estrazione, la quantificazione e la visualizzazione di emociti da bruchi selvatici.

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 ...

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16.1K Views.  The George Washington University. Emociti insetti svolgere molte funzioni importanti, sia immunitarie e non immunitarie, durante tutte le fasi di sviluppo degli insetti. La nostra attuale conoscenza dei tipi di hemocyte e la funzione viene da studi su insetti modelli genetici. Qui vi presentiamo un metodo per l'estrazione, la quantificazione e la visualizzazione di emociti da bruchi selvatici.

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.

Protocollo per la cura dei Mosquito (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 ...

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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 ...

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.

Protocollo per la coltura neuroni simpatico da Rat gangli cervicale superiore (SCG)

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

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).

A PCR-based metodo di genotipizzazione di distinguere tra le varietà selvatiche di tipo ornamentale e di 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 ...

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.

SIVQ-LCM protocollo per lo strumento 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 saggio biologico semplice per la valutazione dei fattori di crescita dell&#39;endotelio vascolare

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

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

L&#39;isolamento di filtrazione di acidi nucleici: DNA metodo di estrazione semplice e veloce

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

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 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 ...

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 semplice protocollo ad alta efficienza per l'isolamento delle isolozioni pancreatiche dai topi

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

Un semplice metodo di filtrazione da banco per isolare piccole vescicole extracellulari da cellule staminali mesenchimali umane

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...