TMS foi apoiado pela confiança Harlan Fellowship (UGT) durante este estudo. O financiamento para a coleta de campo e criando L. fasciola e E. delphinii foi fornecido pela concessão do NSF DEB 0642438 para JTL.

1. Preparação de material <ol><li> Prepare agulhas (1-2 ponta fim e cone 3-4 mm) de tubos de vidro de borosilicato capilares e um extrator micropipeta. Configurações do instrumento: rampa: 561, velocidade: 20; calor: 560; atraso: 1; tração: 100; pressão: 500. </li><li> Preparar a solução de recolha: 60% de meio de Grace (GM), suplementado com 10% de soro fetal bovino (FBS) a 20% de Tampão de anticoagulante (98 mM NaOH, 186 mM de NaCl, 1,7 mM de EDTA e 41 mM de ácido cítrico, pH 4,5 ). A solução acima é preparada em condições estéreis e mantido em gelo em todos os momentos. </li><li> Preparar a solução de tampão de incubação: 90% do GM suplementado com 10% de FBS. Esta solução também é preparada e mantida em gelo em todos os momentos. </li><li> Preparar um total de, pelo menos, 15 ul × o número de insectos para a recolha e as soluções de incubação. </li></ol> 2. Injeção de Lagartas com solução de coleta <ol><li> Anestesiar ins penúltimaslarvas de alcatrão por arrefecimento com gelo durante 20 minutos antes da extracção hemocitária. </li><li> Coloque a extremidade romba de uma agulha limpa capilar (calibre 16) para dentro do tubo que está ligado a uma seringa de vidro de 20 ml (Figura 2). Deixar o tubo que contém a solução de recolha de abrir na bancada do laboratório e lentamente encher o capilar pelo tampão. O capilar é normalmente preenchido com aproximadamente 10-15 uL de solução de recolha, dependendo do tamanho da lagarta (cerca de 1 ul de solução por 10 mg de lagarta massa corporal total = 0,01% da massa corporal total). Descanse a agulha em uma bola de argila que é colocado ao lado do microscópio de dissecação. </li><li> Coloque a lagarta em uma placa de Petri. O insecto deve ser posicionada para o lado, de modo que os espiráculos são claramente visíveis (Figura 3). </li><li> Durante a visualização sob um microscópio de dissecação, manter a lagarta no local com um par de pinças e colocar a agulha perto de um dos espiráculos, em que o emcutícula seita é geralmente mais macia. Pressione suavemente a agulha capilar contra o corpo do inseto. Uma vez que a agulha penetrou a cutícula, injectar o volume total da solução para a lagarta (Figura 3). É importante evitar a injecção de bolhas de ar dentro da lagarta. Do corpo da lagarta vai inchar (por exemplo, aumento do volume em 20%) após a injeção, mas não deve estourar ou vaza. </li><li> Retorne a lagarta para o gelo, colocando o inseto dorsalmente para evitar tampão de vazar para fora da ferida (Figura 3). </li><li> Repita os passos de 2,1-2,4 para cada lagarta usada para extração de hemolinfa. Nesta fase, permitir que todos os insectos para arrefecer em gelo durante 30 min antes de prosseguir. </li></ol> 3. Extração de Hemócitos <ol><li> Coloque a lagarta em uma placa de Petri com o microscópio de dissecação. </li><li> Faça uma incisão (2-3 mm de comprimento) rasa na região posterior da lagarta utilizando um scalpe estéril<html> l (Figura 3). Tome cuidado para não danificar o intestino ou outros órgãos internos, o objetivo é perfurar a cutícula cuidadosamente e recolher o mix de buffer / hemolinfa contendo hemócitos de insetos individuais. </li><li> Aperte a lagarta delicadamente usando uma pinça de plástico flexíveis ao coletar a mistura hemolinfa (aproximadamente 10-20 ul) com uma ponta de pipeta 10 jul, novamente tomando cuidado para não ferir os tecidos internos (Figura 3). Hemolinfa é coletado da minúscula gotinha do corpo da lagarta. Descartar restos da lagarta. </li><li> Colete hemolinfa de cada indivíduo em separado lagarta rotulados siliconizado 0,5 microtubos ml. </li><li> Adicionar um volume igual de solução de incubação (cerca de 10-20 ul) de cada uma das amostras recolhidas a partir da hemolinfa de uma lagarta individual. Manter as amostras de hemolinfa sobre gelo em todos os momentos. </li><li> Repita os passos de 3,1-3,5 para cada lagarta utilizados no experimento. </li></ol>le &quot;Quantificação&gt; 4. dos hemócitos <ol><li> Misturar a amostra e hemolinfa a solução imediatamente antes da incubação hemócitos contagem. Este tratamento permite uma adequada separação de hemócitos e impede aglomeração. </li><li> Usar uma pipeta para transferir limpo 10 ul de amostra de hemolinfa misturado com a solução de incubação para um hemocitómetro. </li><li> Usando um microscópio composto, contar e registar o número de hemócitos em pelo menos 15 dos grandes quadrados (área de um quadrado de 0,04 mm 2, a profundidade: 0,1 mm) da grelha central do hemocitómetro (Figura 3). Adicionar esses valores para obter uma soma total do número de hemócitos nos 15 quadrados. </li><li> Lave o hemocitômetro e seu slide cobertura, cuidadosamente, com um frasco de lavagem com água destilada e deionizada e seque com Kimwipes (Kimberly-Clark) entre cada amostra. </li><li> Repita os passos de 4,1-4,4 para cada amostra de hemolinfa. </li><li> Calcula-se a média do número de hemócitos por ml de hemolinfa volume para cada uma lagarta, como se segue: </li></ol> O volume total de cada quadrado da grelha de hemocitómetro: (0,04 mm 2 de área) x (0,1 mm de profundidade) = 0,004 mm 3 × 15 quadrados = 0,06 mm 3 = o volume total de todos os 15 quadrados contados. (Soma de o número total de hemócitos em 15 quadrados contados / volume do total de todos os 15 quadrados contados) x factor de diluição 2 = o número de hemócitos / mm 3 x 1,000 = o número de hemócitos / ml de hemolinfa.

<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>

Não há conflitos de interesse declarados.

Métodos para a extração de hemócitos de insetos de importância médica e insetos lepidópteros modelo foram relatados 9, 14. Métodos de extração de hemócitos são adaptados de acordo com as espécies de insetos, o estágio de desenvolvimento do inseto e suas características morfológicas. Por exemplo, o isolamento a partir da hemolinfa de larvas Manduca pode ser prontamente realizada cortando o chifre curvo perto do fim do abdómen 15. Porque lagartas Slug (Limacodidae) não poss...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Micropipeta extrator </td><td> Sutter Instruments </td><td> P-1000 </td><td></td></tr><tr><td> Tubos de vidro de borossilicato </td><td> Sutter Instruments </td><td> B100-50-10 </td><td> OD: 1.0, ID: 0,50 milímetros </td></tr><tr><td> Meio de Grace </td><td> Sigma </td><td> G8142 </td><td></td></tr><tr><td> Soro Fetal Bovino </td><td> Thermo Fisher Scientific </td><td> SH3007102 </td><td> Inactivado por calor </td></tr><tr><td> Dumont n º 5 Fórceps </td><td> Belas ferramentas da ciência </td><td> 11252-40 </td><td></td></tr><tr><td> Câmara de Neubauer </td><td> Hausser Scientific </td><td> 3200 </td><td></td></tr><tr><td> Plástico tubing </td><td> Tri-Tech </td><td> TT-3-32OD </td><td> OD: 3/32&#39;&#39;, ID: 1/32 &#39; </td></tr><tr><td> Médicas de vidro seringa </td><td> Fortuna Optima </td><td> D-97877 </td><td> Volume de 50 ml </td></tr><tr><td> Agulha de ponta romba </td><td> Peças pequenas </td><td> NE-162PL-25 </td><td> Calibre 16 x 1 comprimento &quot; </td></tr></tbody></table>

a simple protocol for extracting hemocytes from wild caterpillars

Hemócitos de insetos (equivalente a mamíferos células brancas do sangue) têm um papel importante em vários processos fisiológicos ao longo do ciclo de vida de um inseto 1. Em estágios larvais de insectos pertencentes à ordem dos lepidópteros (borboletas e traças) e Diptera (moscas verdadeiras), são formados a partir de hemócitos do gânglio linfático (um órgão especializado hematopoiética) ou células embrionárias e pode ser carregado até à fase adulta. Hemócitos embrionários estão envolvidos na migração de células durante o desenvolvimento e a quimiotaxia de regulação durante a inflamação. Eles também participam de apoptose das células e são essenciais para a embriogênese 2. Hemócitos mediar o braço celular da resposta imune inata de insectos, que inclui várias funções, tais como a agregação de células de células difusão, formação de nódulos, e a fagocitose de encapsulamento de invasores estranhos 3. Eles também são responsáveis ​​por orquestrar específicas de insectos defesas humorais durante a infecção, tais como a produção de péptidos antimicrobianos e outras moléculas efectoras 4, 5. Hemócitos morfologia e função têm sido principalmente estudados em modelos genéticos ou fisiológica de insectos, incluindo a mosca da fruta, Drosophila melanogaster 6, 7, os mosquitos Aedes aegypti e Anopheles gambiae 8, 9 e o hornworm tabaco, Manduca sexta 10, 11. No entanto, pouca informação existe atualmente sobre a diversidade, classificação, morfologia e função de hemócitos em espécies não-modelo de insetos, especialmente aqueles coletados de 12 a selvagem. Aqui nós descrevemos um protocolo simples e eficiente para extrair hemócitos de lagartas selvagens. Usamos penúltimo estádio Lithacodes fasciola (amarelo Traça lesma) (Figura 1) e Euclea delphinii (spiny lesma carvalho) lagartas (Lepidoptera: Limacodidae) e mostram que os volumes suficientes de hemolinfa (sangue do inseto)pode ser isolado e os números contados a partir de hemócitos larvas individual. Este método pode ser utilizado de forma eficiente para estudar tipos de hemócitos nestas espécies, bem como em outras lagartas relacionados colhidas a partir do campo, ou pode ser facilmente combinado com ensaios imunológicos concebidos para investigar a função hemocitária após infecção com micróbios ou parasitas 13.

O protocolo aqui descrito permite a recolha de um volume mínimo de 10-20 ul de hemolinfa de larvas de insectos individuais. Hemócitos recolhidas utilizando este método são livres de agregação de células, defeitos de melanização, restos de tecido ou outros contaminantes. Portanto hemócitos podem ser facilmente observados e contados sob o microscópio, e vários insectos pode ser observado no prazo de uma Hosur poucos. Observou-se que a maioria dos hemócitos em nossas amostras consistiu de 3 (plasmat...

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

Hemócitos de insetos realizar muitas funções importantes, tanto imunes e não-imunes, em todas as fases do desenvolvimento do inseto. Nosso conhecimento atual de tipos de hemócitos e função vem de estudos sobre insetos modelos genéticos. Aqui, apresentamos um método para extrair, quantificar e visualizar hemócitos de lagartas selvagens.

Um protocolo simples para extrair Hemócitos de Lagartas selvagens

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

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

Research

JoVE Journal

Biology

16.1K visualizações.  The George Washington University. Hemócitos de insetos realizar muitas funções importantes, tanto imunes e não-imunes, em todas as fases do desenvolvimento do inseto. Nosso conhecimento atual de tipos de hemócitos e função vem de estudos sobre insetos modelos genéticos. Aqui, apresentamos um método para extrair, quantificar e visualizar hemócitos de lagartas selvagens.

Vídeo: A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Protocol for Mosquito Rearing (A. gambiae)

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

Protocol for Mosquito Rearing A. gambiae

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

Protocolo para a Criação 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 ...

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.

Pyrosequencing: Um método simples para genotipagem Accurate

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

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

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

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

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

Protocolo para Neurônios Cultivar Sympathetic de Gânglios Superior Rat Cervical (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).

Um método de genotipagem baseados em PCR para distinguir entre variedades do tipo selvagem e Ornamental de Imperata cylindrica</em

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

SIVQ-LCM Protocol for the ArcturusXT Instrument

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

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

Protocolo SIVQ-LCM para o Instrumento ArcturusXT

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

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

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

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

A Bioensaio simples para a avaliação dos fatores de crescimento endotelial vascular

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.

Isolamento de filtração de Ácidos Nucleicos: Um Método de extracção de DNA simples e rápido

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.

Um método para extrair Pigmentos de 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.

Um protocolo de alta eficiência simples para isolamento de Islet pancreático de camundongos

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.

Um Método Simples de Filtração de Bancada para Isolar Pequenas Vesículas Extracelulares de Células-Tronco Mesenquimais Humanas

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