Este trabalho foi suportado por concessões do National Science Foundation EUA (EAR-0920299 e 0745808-EAR); National Science Foundation EUA Leste Asiático e Pacífico Verão Institutos, a Sociedade Geológica da América Programa Bolsa Pesquisa e os Subsídios para Pesquisa de Pós-Graduação Alumni e Bolsa da Universidade Estadual de Ohio. Gostaríamos de agradecer ao editor e dois revisores anônimos por seus comentários perspicazes.

1. MTB Colecção <ol><li> Ao decidir sobre um local de água doce para coletar bactérias magnetotáticas (MTB), muitas vezes é melhor começar com uma lagoa ou de baixa movimentação de fluxo que tem uma camada de sedimento suave lamacenta. Nesta demonstração coletamos uma amostra na margem do rio Olentangy no campus da Universidade Estadual de Ohio (OSU), em Columbus, Ohio (EUA). Embora isso fosse um local conveniente para a demonstração, o protocolo aqui descrito é aplicável a qualquer localização aquática. Os materiais utilizados no presente protocolo podem ser encontrados na Tabela 1. Encontrar um local onde a profundidade da água está entre 10 e 100 cm. Em um local tão, você deve coletar a camada superior mais de sedimentos usando uma clara, recipiente com tampa. Colher o sedimento e de água para dentro do recipiente até que é preenchido com um terço até metade do sedimento e o volume restante com água. Manter o recipiente submersa até ser enchido com água e, em seguida, firmemente tampar o recipiente wom a sua tampa de rosca de topo. Não é necessário misturar o sedimento. Limpe a parte externa do recipiente com uma toalha seca e depois tirar a amostra para o seu laboratório. Não é necessário apressar a amostra de volta para seu laboratório. Deixamos amostras de BTT em recipientes de plástico no campo por vários dias antes de trazê-los de volta para o nosso laboratório. O MTB deve ser viável por várias semanas a meses, desde que você armazenar as amostras em um local fresco e com sombra no campo. </li><li> Uma vez que a amostra está no seu laboratório, soltar a tampa e deixe que cobre o recipiente para reduzir a quantidade de evaporação. Armazenar o recipiente à temperatura ambiente num quarto escuro, gaveta, ou cobrir completamente o recipiente com uma folha de alumínio. Permitir que o sedimento e partículas finas de completamente assentar no fundo do recipiente, deixando a amostra em repouso durante várias horas a vários dias. Não é necessário misturar o sedimento, MTB prefere um ambiente sem ser perturbado. As paredes claras do recipiente de plásticopermitirá que você para confirmar que as partículas foram para o fundo. Dependendo da sua amostra, MTB podem permanecer vivos na amostra por muitos meses. </li></ol> 2. Isolamento MTB <ol><li> Quando estiver pronto para isolar os ímanes MTB, local nos lados do recipiente de plástico a cerca de 1 cm acima da interface sedimento-água (Figura 1A). Ter cuidado para não perturbar o sedimento no fundo do recipiente. Coloque o pólo sul de uma barra magnética de um lado do recipiente e no lado norte de um outro íman em barra no lado oposto (Figura 1A). Quase todo o íman pode ser utilizado, tal como uma barra de agitação magnética ou ima grande. Tudo pode ser utilizado para suportar os ímans à altura correcta acima da interface água-sedimento. Descobrimos que os magnetos descansando sobre a parte superior de uma caixa de papelão ou de plástico é o melhor, no entanto, os ímans podem também ser gravados para o exterior do recipiente de plástico. Espere 30 minutos a várioshoras para as bactérias que nadar para o ímã. </li><li> Use uma pipeta estéril para recolher a água com cuidado de dentro do recipiente (Figura 1A), perto da posição do ímã de barra sul-pólos (para amostras coletadas no hemisfério Norte). Esta água deve conter o MTB que foram atraídos para o ímã de barra sul-pólo. Em seguida, uma pista de corrida capilar deve ser utilizada para enriquecer ainda mais o MTB. </li></ol> 3. Autódromo de MTB <ol><li> A fim de enriquecer a sua amostra com bactérias magnetotáticas, uma pista de corrida capilar é necessário (Figuras 1B e 1C). Estes necessitam de ser feita antes de isolar as células do recipiente transparente de plástico. </li><li> Use uma polegada 5,75 (146 mm) de vidro Pasteur pipeta para fazer uma pista de corrida. Use uma caneta de diamante ou arquivo para cortar a parte superior da pipeta, o comprimento da pipeta não é crucial, mas deve ser capaz de conter aproximadamente 1-2 ml de água. Em seguida, utilizar um bico de Bunsen para derreter a ponta de modo a quetorna-se selado (Figura 1C). A pipeta resultante deve ter uma extremidade aberta e uma extremidade fechada. </li><li> Fazer várias dessas pistas e, em seguida, autoclave. Além disso, você terá de autoclave algodão e várias agulhas de metal longos. </li><li> Adicionar água de amostra filtrada, recolhida a partir de perto da interface água-sedimento mostrado na Figura 1A, com uma pista de corrida autoclavado utilizando uma agulha de metal longa ligado a uma seringa filtrada. O tamanho dos poros do filtro deve ser 0,22 milímetros para eliminar os detritos e contaminantes da água. É importante ter a certeza absoluta de que não há bolhas de ar no capilar de vidro. </li><li> Ligue o fundo da pista com algodão estéril (Figura 1B). Utilize a agulha de metal para empurrar o algodão para a extremidade selada da pista de modo que é de 0,5 - 1 cm de distância entre a ponta fechada (Figura 1C). </li><li> Utilizando uma pipeta estéril, adicionar a água MTB contendo (a partir de Secção 2.2) para a amostra de reservoir (extremidade aberta) de uma pista de corridas de MTB preparada (Figura 1B). </li></ol> 4. Enriquecimento MTB <ol><li> Uma vez que a pista é preenchido com fluido amostra, colocá-lo em seu lado em uma superfície horizontal (por exemplo, uma bancada) e coloque o pólo sul de um ímã de barra (no hemisfério norte) próximo à ponta fechada da pista (Figuras 1B e 1C). </li><li> Esperar 5 a 30 minutos para o MTB para migrar através do algodão. Então, você deve coletar o líquido perto da ponta da pista. Espera demasiado longa pode introduzir contaminantes, tais como outras bactérias móveis, para a ponta do capilar. Opcionalmente, você pode usar um microscópio de luz para ver a ponta da pista e ver o MTB recolher na ponta da pista do. Isso permitirá que você para determinar quanto tempo leva o MTB para migrar através do tampão de algodão. </li><li> Gentilmente usar a faca de diamante para fazer um pequeno arranhão perto tampão de algodão e tirar fora o fim da pista. </li> &lt;li&gt; Usando uma seringa de 1 ml com uma agulha fina (25 ou calibre 27), para remover o líquido da ponta da pista. Esta amostra líquida agora deve conter o MTB enriquecido. </li></ol> 5. Observação MTB por microscopia de luz <ol><li> Colocar uma gota (10-20 ul) da amostra de MTB enriquecido para uma lamela. </li><li> Rapidamente virar a lamela mais para que a queda está agora virada para baixo e pendurado na lamela. </li><li> Coloque a lamela em um o-ring, que repousa sobre uma lâmina de vidro. O anel de vedação deve ter um diâmetro ligeiramente menor que a lamela (cerca de 1 cm), Figura 2. </li><li> Coloque essa queda pendurado em um estágio microscópio de luz e foco em uma borda da gota. Um objectivo 60X seco funciona muito bem, porque a maioria tem uma grande abertura numérica (NA; por exemplo, 0,93), mas não necessitam de óleo, o que é difícil de utilizar para o método de gota em suspensão (Figura 2). </li><li> Coloque a extremidade sul de um ímã de barra próximo ao d penduradorop e MTB começará a migrar em direcção ao bordo de queda mais próximo do íman (Figura 3). Dentro de alguns minutos MTB muitos devem estar na borda da gota (Figura 3). Provar-se que as bactérias são magnéticos, invertendo o pólo do íman e em seguida observar a bactéria nadar na direcção oposta. </li></ol> 6. MTB observação por Microscopia Eletrônica de Transmissão (TEM) <ol><li> Colocar uma gota (~ ul 20) do MTB enriquecido numa grelha de cobre e permitir que as bactérias que se contentar com 10 min. </li><li> Wick fora o excesso de água com um pedaço de papel de filtro limpo. </li><li> Opcionalmente, a grade pode ser negativamente coradas com acetato de uranilo a 2%, pH 2% de ácido fosfotúngstico 7,2, ou 2,5% de molibdato de sódio 7, 8, 9. Isto é feito através da colocação da grelha de cobre sobre uma gota de corante, imediatamente após a incubação da grade com o MTB enriquecido. Incubar a grade com a mancha negativa, os tempos podem variar, dependendo da rain utilizado, e, em seguida, para fora da mecha de fluido com um pedaço de papel de filtro limpo. </li><li> Observe o MTB usando microscopia eletrônica de transmissão (TEM, Figura 4). Para o trabalho descrito aqui MTB foram adsorvidos a Formvar estabilizado e revestidos de carbono 200 redes de malha de cobre (Ted Pella # 01800). As grelhas foram colocados com o lado de carbono para baixo sobre uma gota de suspensão de células para um máximo de 10 min, em seguida, imediatamente lavadas uma vez através da colocação da grade em uma gota de água durante 30 segundos. Para a coloração, as grades foram colocados numa gota de acetato de uranilo a 2% (Ted Pella # 19481) durante 30 segundos a 5 minutos, e depois secou-se completamente através de um pedaço de papel de filtro. As grades foram analisadas por MET usando um FEI Espírito Tecnai em 80kV ou FEI Tecnai F20 usando alto ângulo STEM campo escuro anular em 200KV. </li></ol>

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	<li>Blakemore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+bacteria.">Magnetotactic bacteria.</a> Science. 190, 377-379 (1975).</li><li>Blakemore, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+bacteria.">Magnetotactic bacteria.</a> Annual Reviews in Microbiology. 36, 217-238 (1982).</li><li>Bazylinski, D. A., Frankel, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Biomineralization+of+Magnetite+(Fe3O4)+and+Greigite+(Fe3S4)+in+a+Magnetotactic+Bacterium.">Controlled Biomineralization of Magnetite (Fe3O4) and Greigite (Fe3S4) in a Magnetotactic Bacterium.</a> Applied and Environmental Microbiology. 61, 3232-3239 (1995).</li><li>Lefevre, C. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=South-seeking+magnetotactic+bacteria+in+the+Northern+Hemisphere.">South-seeking magnetotactic bacteria in the Northern Hemisphere.</a> Science. 311, 371-374 (2006).</li><li>Wolfe, R., Thauer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+'capillary+racetrack'+method+for+isolation+of+magnetotactic+bacteria.">A 'capillary racetrack' method for isolation of magnetotactic bacteria.</a> FEMS Microbiology Letters. 45, 31-35 (1987).</li><li>Rodgers, F. G., Blakemore, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+structure+in+a+many-celled+magnetotactic+prokaryote.">Intercellular structure in a many-celled magnetotactic prokaryote.</a> Archives of Microbiology. 154, 18-22 (1990).</li><li>Moench, T. T., Konetzka, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+the+isolation+and+study+of+a+magnetotactic+bacterium.">A novel method for the isolation and study of a magnetotactic bacterium.</a> Archives of Microbiology. 119, 203-212 (1978).</li><li>Balkwill, D., Maratea, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+a+magnetotactic+spirillum.">Ultrastructure of a magnetotactic spirillum.</a> Journal of Bacteriology. 141, 1399-1408 (1980).</li><li>Lins, U., Freitas, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+homemade+apparatus+for+harvesting+uncultured+magnetotactic+microorganisms.">Simple homemade apparatus for harvesting uncultured magnetotactic microorganisms.</a> Brazilian Journal of Microbiology. 34, 111-116 (2003).</li><li>Jogler, C., Lin, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+Cloning+of+the+Magnetotactic+Metagenome:+Identification+of+Magnetosome+Island+Gene+Clusters+in+Uncultivated+Magnetotactic+Bacteria+from+Different+Aquatic+Sediments.">Toward Cloning of the Magnetotactic Metagenome: Identification of Magnetosome Island Gene Clusters in Uncultivated Magnetotactic Bacteria from Different Aquatic Sediments.</a> Applied and Environmental Microbiology. 75, 3972-3979 (2009).</li><li>Lin, W., Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+Isolated+but+Uncultivated+Magnetotactic+Bacterium+of+the+Phylum+Nitrospirae+from+Beijing,+China.">Newly Isolated but Uncultivated Magnetotactic Bacterium of the Phylum Nitrospirae from Beijing, China.</a> Applied and Environmental Microbiology. 78, 668-675 (2012).</li><li>Li, J., Pan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomineralization,+crystallography+and+magnetic+properties+of+bullet-shaped+magnetite+magnetosomes+in+giant+rod+magnetotactic+bacteria.">Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria.</a> Earth and Planetary Science Letters. 293, 368-376 (2010).</li><li>Oestreicher, Z., Valerde-Tercedor, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetosomes+and+magnetite+crystals+produced+by+magnetotactic+bacteria+as+resolved+by+atomic+force+microscopy+and+transmission+electron+microscopy.">Magnetosomes and magnetite crystals produced by magnetotactic bacteria as resolved by atomic force microscopy and transmission electron microscopy.</a> Micron. 43, 1331-1335 (2012).</li></ol>

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Bactérias magnetotáticas não são necessariamente encontrados em cada ambiente aquático 8, mas quando elas ocorrem, eles podem ser encontrados na ordem de 100 - 1000 células por mililitro 2. A fim de observar o MTB microscopia óptica, você vai precisar de cerca de 50 bactérias / ml em sua amostra 8. Se não houver nenhum ou poucos MTB em sua amostra, então você precisa selecionar um novo site ambiental para coletar suas amostras ou você terá de tentar uma ou mais das técnica...

<table border="1"><tbody><tr><td> Nome do item </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Lâminas de vidro </td><td> Fisher Scientific </td><td> S95933 </td><td></td></tr><tr><td> Pasteur pipetas de vidro </td><td> Fisher Scientific </td><td> 13-678-6A </td><td></td></tr><tr><td> O-ring </td><td> Loja de ferragens </td><td></td><td></td></tr><tr><td> Lamínulas </td><td> Fisher Scientific </td><td> 12-542B </td><td></td></tr><tr><td> Ímã de barra </td><td> Fisher Scientific </td><td> S95957 </td><td></td></tr><tr><td> Recipiente </td><td> Qualquer </td><td></td><td> Qualquer plastic ou recipiente de vidro que pode conter pelo menos 0,5 L e pode ser selada </td></tr><tr><td> Algodão </td><td> Qualquer </td><td></td><td></td></tr><tr><td> Microscópio com lente seca 60X </td><td> Zeiss </td><td></td><td> Uma lente de 60X a seco não é absolutamente necessária, mas isto apresenta uma NA elevada sem utilizar óleo </td></tr><tr><td> Diamante caneta </td><td> Fisher Scientific </td><td> 08-675 </td><td></td></tr><tr><td> Filtro de 0,22 milímetros </td><td> Fisher Scientific </td><td> 09-719C </td><td></td></tr><tr><td> 1 seringa ml </td><td> Fisher Scientific </td><td> NC9788564 </td><td></td></tr><tr><td> Microtubos </td><td> Fisher Scientific </td><td> 02-681-320 </td><td></td></tr><tr><td> Formvar / carbono200 mesh, grades de cobre </td><td> Ted Pella Inc. </td><td> 01800 </td><td></td></tr><tr><td> Acetato de uranilo </td><td> Ted Pella Inc. </td><td> 19481 </td><td></td></tr><tr><td> Tecnai Espírito TEM </td><td> FEI </td><td></td><td></td></tr><tr><td> Tecnai F20 S / TEM </td><td> FEI </td><td></td><td></td></tr></tbody></table>

collection, isolation and enrichment of naturally occurring magnetotactic bacteria from the environment

Bactérias magnetotáticas (MTB) são microorganismos aquáticos que foram pela primeira vez descritos nomeadamente em 1975 uma das amostras de sedimentos coletados em salinas de Massachusetts (EUA). Desde então MTB foram descobertos em água e sedimentos estratificados-colunas de todo o mundo 2. Uma característica comum a todos MTB é que eles contêm magnetossomos, que são intracelulares, nanocristais ligado à membrana magnéticas de magnetita (Fe 3 O 4) e / ou greigite (Fe 3 S 4) ou ambos os 3, 4. No hemisfério norte, MTB normalmente são atraídos para a extremidade sul de um ímã de barra, enquanto no hemisfério sul geralmente são atraídos para a extremidade norte de um ímã de 3,5. Esta propriedade pode ser explorada durante a tentativa de isolar a partir de amostras ambientais MTB. Uma das formas mais comuns de enriquecer MTB é a utilização de um contentor de plástico transparente de recolher os sedimentos e água a partir de uma fonte natural,tal como um tanque de água doce. No hemisfério norte, o sul de uma barra magnética é colocada contra a parte exterior do recipiente um pouco acima do sedimento na interface sedimento-água. Depois de algum tempo, a bactéria pode ser removido do interior do recipiente perto do magneto com uma pipeta e, em seguida, enriquecido ainda mais por meio de um capilar de pista 6 e um magnete. Uma vez enriquecido, a bactéria pode ser colocado sobre uma lamela de microscópio, utilizando um método de gota pendente e observados num microscópio de luz, ou depositado sobre uma grelha de cobre e observadas por microscopia electrónica de transmissão (TEM). Usando este método, isolado MTB pode ser estudada microscopicamente para determinar características tais como o tipo de comportamento de natação, e número de flagelos a morfologia das células, das células, a forma dos cristais magnéticos, número de magnetossomos, o número de cadeias de magnetossomos em cada célula, a composição de os cristais nanomineral e presença de vacúolos intracelulares.

Um íman é uma ferramenta eficaz que pode ser usado para isolar as bactérias magnetotáticas (MTB) contidas em amostras ambientais (Figura 1A). A pista de corrida capilar (Figura 1B) utiliza as propriedades magnéticas de MTB de atrair os através de um tampão de algodão, onde eles podem ser separados a partir de microrganismos não-magnetotáticas também contidos na amostra ambiental. <img alt="Figura 1" src="/files...

Watch this Scientific Journal Video about Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment at JoVE.com

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Nós demonstramos um método para recolher bactérias magnetotáticas (MTB), que podem ser aplicadas para águas naturais. MTB podem ser isolados e enriquecidos a partir de amostras de sedimento utilizando uma configuração relativamente simples que aproveita magnetismo da bactéria natural. Isolado MTB pode então ser analisada em detalhe utilizando luz e microscopia eletrônica.

Isolamento, coleta e Enriquecimento de bactérias naturais magnetotáticas do Meio Ambiente

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

collection-isolation-enrichment-naturally-occurring-magnetotactic

Research

JoVE Journal

Biology

22.9K visualizações.  The Ohio State University. Nós demonstramos um método para recolher bactérias magnetotáticas (MTB), que podem ser aplicadas para águas naturais. MTB podem ser isolados e enriquecidos a partir de amostras de sedimento utilizando uma configuração relativamente simples que aproveita magnetismo da bactéria natural. Isolado MTB pode então ser analisada em detalhe utilizando luz e microscopia eletrônica.

Vídeo: Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

Enriquecimento das células NK do sangue humano com o Kit RosetteSep de StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

Biologia

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

Coleta de sangue do caranguejo em ferradura americano, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

Isolamento de ARN a partir de pequenas não-codificante de Soro Humano

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. Birds are widely used subjects in biological research and are ideal for studies involving repeated sperm samples. However, few resources are currently available for those wishing to learn how to collect and extract DNA from avian sperm. Here we describe cloacal massage, a gentle, non-invasive manual technique for collecting avian sperm. Although this technique is established in the literature, it can be difficult to learn from the available descriptions. We also provide information for extracting DNA from avian semen using a commercial extraction kit with modifications. Cloacal massage can be easily used on any small- to medium-sized male bird in reproductive condition. Following collection, the semen can be used immediately for motility assays, or frozen for DNA extraction following the protocol described herein. This extraction protocol was refined for avian sperm and has been successfully used on samples collected from several passerine species (Passer domesticus, Spizella passerina, Haemorhous mexicanus, and Turdus migratorius) and one columbid (Columba livia).

This protocol describes cloacal massage, a non-invasive manual technique for collecting semen from birds, and DNA extraction from avian sperm using a commercial extraction kit with modifications.

Coleta de sêmen aviária por esfregaços massagem e isolamento do DNA de esperma

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ability to biomineralize magnetosomes, which are magnetic nanometer-sized crystals of magnetite (Fe3O4) or greigite (Fe3S4) surrounded by a lipid bilayer membrane, within their cytoplasm. For most known magnetotactic bacteria, magnetosomes are assembled in chains inside the cytoplasm, thereby conferring a permanent magnetic dipole moment to the cells and causing them to align passively with external magnetic fields. Because of these specific features, magnetotactic bacteria have a great potential for commercial and medical applications. However, most species are microaerophilic and have specific O2 concentration requirements, making them more difficult to grow routinely than many other bacteria such as Escherichia coli. Here we present detailed protocols for growing three of the most widely studied strains of magnetotactic bacteria, all belonging to the genus Magnetospirillum. These methods allow for precise control of the O2 concentration made available to the bacteria, in order to ensure that they grow normally and synthesize magnetosomes. Growing magnetotactic bacteria for further studies using these procedures does not require the experimentalist to be an expert in microbiology. The general methods presented in this article may also be used to isolate and culture other magnetotactic bacteria, although it is likely that growth media chemical composition will need to be modified.

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

We present a procedure for growing several strains of Magnetospirillum in two different types of growth media. Magnetospirillum gryphiswaldense strain MSR-1 is grown in both liquid and O2 concentration gradient semi-solid media while M. magneticum strain AMB-1 and M. magnetotacticum strain MS-1 are grown in liquid medium.

Crescimento de bactérias de Magnetotactic do género Magnetospirillum magnetotacticum: cepas MSR-1, AMB-1 e MS-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

Isolamento de células mioepiteliais das glândulas lacrimal e submandibular de Murina adulta

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Isolamento e enriquecimento de células progenitoras epiteliais pulmonares humanas para a cultura organoide

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Collection and Identification of Pollen from Honey Bee Colonies

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate pesticide exposure of bees via pollen. Described herein is an effective pollen-trapping method for collecting corbicular pollen from honey bees returning to their hives. This collection method results in large quantities of corbicular pollen that can be used for research purposes. Honey bees collect pollen from many plant species, but typically visit one species during each collection trip. Therefore, each corbicular pollen pellet predominantly represents one plant species, and each pollen pellet can be described by color. This allows the sorting of samples of corbicular pollen by color to segregate plant sources. Researchers can further classify corbicular pollen by analyzing the morphology of acetolyzed pollen grains for taxonomic identification. These methods are commonly used in studies related to pollinators such as pollination efficiency, pollinator foraging dynamics, diet quality, and diversity. Detailed methodologies are presented for collecting corbicular pollen using pollen traps, sorting pollen by color, and acetolyzing pollen grains. Also presented are results pertaining to the frequency of pellet colors and taxa of corbicular pollen collected from honey bees in five different cropping systems.

We describe methods for collecting corbicular pollen from honey bees as well as protocols for color sorting, acetolysis, and microscope slide preparation of pollen for taxonomic identification. In addition, we present pellet color and taxonomic diversity of corbicular pollen collected from five cropping systems using pollen traps.

Coleta e Identificação de Pólen das Colônias de Abelhas

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

Limpando o Ambiente Urbano - Um Pipeline para Amostragem e Detecção de SARS-CoV-2 de Reservatórios Ambientais

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Correção rápida in Vivo e isolamento de complexos translacionais de células eucarióticas

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...