This work was supported by grants from the Institute of Science and Technology of the Federal District (ICyTDF, 64/2012 to EO) and the Mexican Council for Science and Technology (Conacyt, 179895 to MS).

1. Estabelecimento e Manutenção de E. Culturas histolytica <ol><li> Crescer com culturas puras (totalmente livre de todos os outros organismos contaminantes) 1 x 10 5 trofozoítos de Entamoeba histolytica HML tensão: IMSS clonar um 18 em 16 x 125 mm tubos de cultura com tampas de rosca de teflon forro (ou 1 x 10 6 trofozoítos em um descartável T- 25 frascos) e 15 ml (ou 50 ml em frascos T-25) de TYI-S-33 médio (caldo de TYI, suplementado com 3% de vitamina mistura diamante, de soro de bovino adulto inactivado pelo calor 10%, a 0,5 UI / ml de penicilina e 35 ug / ml de estreptomicina) 19 dentro de uma incubadora a 37 ° C. </li><li> Trofozoítos da colheita durante a fase de crescimento logarítmica, geralmente com intervalos de 48-96 hr (Figura 1) por arrefecimento dos tubos de cultura durante 5-10 min num banho de gelo-água para libertar trofozoítos ligados ao tubo de cultura de vidro. </li><li> Transferir a cultura para um tubo cônico e invertê-lo várias vezes para dispersar o cells. Determinar o número de células usando um hemocitômetro (câmara de Neubauer), e transferir um inóculo em um tubo de cultura contendo meio TYI-S-33 fresco. <ol><li> Use baixo número de amebas por períodos mais longos de incubação (~ 3 x 10 5 células para ~ 5 dias) e um número maior de períodos mais curtos (~ 1 x 10 6 células para ~ 1 dia). Enquanto inóculos contados são desejáveis, culturas estabelecidas se torna previsível, de modo que os volumes estimados de inóculos são viáveis. </li><li> Titular da quantidade de trofozoítos de otimizar o número de células para cada experimento. </li><li> Manter uma cultura duplicado paralelo para ter um back-up em caso de contaminação acidental ou ruptura do tubo. </li></ol></li><li> Vedar bem os tubos e incubar a 37 ° C numa atmosfera de 5 ° inclinado ângulo horizontal. </li><li> Verificar culturas por inspecção visual regular, uma vez que um excessivo crescimento da cultura pode conter muitas células lisadas. </li></ol> 2. Estabelecimento e Manutenção de MDCK Cultura &lt;/ P&gt; <ol><li> Crescer MDCK (rim canino de Madin Darby), as células do tipo I (de alta resistência) no descartáveis ​​frascos T-75 com 10 ml de meio DMEM suplementado com penicilina (100 UI / mL), estreptomicina (100 mg / ml), insulina (0,08 L / ml) e 10% de soro de bezerro recém-nascido, em uma atmosfera humidificada de 5% de CO 2 e 95% de ar a 37 ° C. </li><li> Para dividir as células, vê-los em um microscópio invertido. Dividir células quando elas são ~ 85-100% confluentes. </li><li> Use um aspirador a vácuo em uma capa estéril e uma pipeta de Pasteur de vidro estéril para remover a mídia da cultura. Para evitar raspar células, vire o frasco de cabeça para baixo e mídia aspirado a partir do canto inferior. </li><li> Dispensar 10 ml de PBS pré-aquecido (140 mM de NaCl, 2,7 mM KCl, 10 mM de Na 2 HPO 4, 1,8 mM de KH 2 PO 4, pH 7,4) num frasco T-75 (5 ml no caso de um frasco T-25 ) e balançar suavemente o frasco para lavar as células. Remover PBS por aspiração a vácuo. Extensa lavagem é necessária uma vez que o soro inibe tripsina. </li><li> Dispensar 1,5 ml de tripsina a 0,05% para um frasco T-75 (para T-25 balão de utilizar 0,5 ml) e agitar suavemente o frasco para cobrir a camada de células com tripsina. </li><li> Incubar por 5-10 minutos na incubadora (tempo exato depende da atividade de tripsina). </li><li> Verifique se há desprendimento de células, mantendo frasco contra a luz e olhar para as células fluindo. Descolamento celular (células arredondadas) também pode ser verificada usando um microscópio. Muitas vezes, as células liberam mais rápido com uma rápida, suave tapa para o lado do balão. </li><li> Adicionar 15 ml de DMEM suplementado com um T-75 frascos (5 mL para um frasco T-25). Volte a suspender as células por pipetagem cima e para baixo 4 ou 5 vezes. </li><li> Para propagar células, diluir 1:5 com suspensão de células fresco suplementado DMEM. O número de células também pode ser determinada, neste ponto usando um hemocitómetro, mas este é geralmente apenas necessário se a divisão de células em formatos especiais para determinados ensaios. </li></ol> 3. Preparação de Trofozoíto total Lisados <ol><li> Retire trofozoítos de cutubos LTURE arrefecendo o tubo durante 5-10 min num banho de gelo-água. Transferir assepticamente a cultura para um tubo cónico e centrifugar a 360 xg durante 10 min a 4 ° C. Cuidadosamente descarte do sobrenadante por decantação, adicionar gelo-PBS frio ao sedimento, inverter o tubo várias vezes para dispersar as células e centrifugar novamente a 360 x g durante 10 min. Repita esta etapa duas vezes para remover todos os vestígios de médio TYI-S-33. </li><li> Determinar o número trofozoítos usando um hemocitômetro. </li><li> Lyse trofozoítos por ciclos de congelamento e descongelamento. Snap-congelar um inoculo medido de trofozoítos (600.000 trofozoítos / ml) diluído em PBS gelado, em azoto líquido durante 1 minuto. Descongelar a amostra a 4 ° C e vórtex vigorosamente. Repetir a congelação e descongelação de 3 vezes para completar a lise das células. </li><li> Activar a proteases presentes nos lisados ​​com 0,02% β-mercaptoetanol. </li></ol> 4. Preparação de Trofozoíto secretada produtos <ol><li> Realize os passos de 3,1-3,2. </li><li> Determinarnúmero de células e viabilidade neste ponto usando um hemocitómetro e a aplicação de um teste de exclusão com azul de tripano 20: <ol><li> Diluir 9 x 10 6 em trofozóitos de 3 ml de PBS gelado e as células de transferência para um tubo de 50 ml. Tome 10 ml desta suspensão e adicionar 1 ml de 0,4% tripan solução estoque azul pH 7.2. </li><li> Use uma câmara de Neubauer para contagem de células em baixa ampliação. </li><li> Contagem de todas as células e separar o número de células azuis, uma vez que as células azuis tomaram-se o corante e devem ser consideradas mortas. </li><li> Calcula-se a percentagem de células viáveis ​​através da divisão do número de células viáveis ​​pelo número total de células e multiplicando por 100. </li></ol></li><li> Transferir o tubo cónico contendo a suspensão trofozóito na incubadora a 37 ° C durante 2 horas numa atmosfera de 5 ° inclinado ângulo horizontal. </li><li> Para a coleta de produtos secretados, tubos de centrifugação a 360 xg por 10 min. Use uma seringa descartável e estéril e cuidadosamente recolher o supernatant, evitando a contaminação de amostras com trofozoitos da pelota. Eliminar quaisquer células transferidas pela passagem do sobrenadante através de uma membrana de acetato de celulose de 0,22 um. Activar proteases com 0,02% β-mercaptoetanol. </li><li> Para descartar a contaminação indesejada putativo com moléculas liberadas a partir de células mortas, reaplicar o teste do azul de tripan para a viabilidade celular. Número viável e total de células deve ser o mesmo que o obtido no passo 4.2. Se este não for o caso, o sobrenadante foi recolhido pode conter não só proteínas secretadas e deve ser descartado. </li></ol> 5. Interação de células MDCK com Trofozoítos, Trofozoíto Lysates ou secretados produtos <ol><li> Incubar as monocamadas de células MDCK confluentes com trofozoítos vivas (uma relação de MDCK-a-amiba de 01:01), trofozóito lisados ​​(um rácio de MDCK-a-amiba de 1:2) ou produtos de secreção (um rácio de MDCK-a-amiba de 1 : 10) a 37 ° C durante 2 e 30 minutos (Figura 2A). </li><li> Lavar as células epiteliais cinco vezes com PBS gelado para eliminar as moléculas não ligadas ou trofozoítos. </li></ol> 6. Preparação de Amostras para Imunofluorescência <ol><li> Células MDCK Cultura em lamínulas estéreis colocados dentro de 24 com vários poços de cultura de células prato. Inspeccionar as células no microscópio invertido até atingirem 100% de confluência. Em seguida, substitui meio com 1 ml de meio DMEM suplementado fresco e transferir a placa para uma incubadora a 37 ° C durante 24 horas mais. </li><li> Remover o meio utilizando um aspirador de vácuo e uma pipeta de Pasteur de vidro estéril e adicionar 1 ml de PBS quente a cada poço. Remover PBS e repita a lavagem com PBS fresco. Tomar cuidado para não raspar as células a partir do fundo do poço com a pipeta de vidro. </li><li> Adicionar diferentes condições de trofozoítos diluído em 1 ml de PBS quente: trofozoítos vivos (t), lisados ​​totais trofozoíto (LT) e produtos secretados (SP). <ol><li> Colocar a placa de cultura na incubadora durante 2 ou 30 minutos. </li><li> Prepare duas condições de controle: um time chamado 0 min, onde as células MDCK não devem ser incubados com E. histolytica, mas apenas com 1 ml de PBS quente; e uma outra condição como controlo anticorpos secundários, neste caso incubar MDCK com T, LT ou SP durante 2 ou 30 minutos e omitir a incubação com os anticorpos primários. </li></ol></li><li> Lave as células epiteliais cinco vezes com PBS frio para eliminar moléculas não ligadas ou trofozoítos. </li><li> Fix e permeabilizar as células com 1 ml de etanol a 96% durante 30 min a -20 ° C. </li><li> Para remover o etanol, executar três lavagens rápidas com 1 ml de PBS, à temperatura ambiente. </li><li> Execute as seguintes etapas em câmara úmida para evitar a secagem de amostras: <ol><li> Use qualquer caixa plana disponível que pode ser fechado e cheio com uma toalha de papel húmido no qual as amostras podem ser colocadas com segurança. Por exemplo, uma caixa de ponteira vazio serve a esse propósito bem. </li><li> Dentro da câmara, coloque uma camada uniforme Parafilm e pipeta de 25 mL de blocosolução (0,5% BSA) ing para cada lamela. </li><li> Tome lamelas para fora dos poços com uma pinça fina, remover cuidadosamente o excesso de líquido a partir da ponta com um filtro de papel e colocar em lamela a gota com solução de bloqueio (BSA a 0,5%) com as células de frente para a solução de bloqueio. Incubar as amostras durante 1 h à temperatura ambiente. </li></ol></li><li> Prepara-se uma mistura de anticorpos primários em PBS: IgM de ratinho anti-EhCPADH112 (mα-EhCPADH112; diluição de 1:10) e IgG de coelho anti-ZO-1 (pα-ZO-1, diluição 1:100). </li><li> Coloque uma folha nova de Parafilm na caixa úmido e pipeta de 25 ml de solução de anticorpos para cada lamela. </li><li> Levantar as lamelas a partir da solução de bloqueio, secar o excesso de líquido nos bordos utilizando um papel de filtro, e colocá-las em gotas com as células de frente para a solução de anticorpo. Incubar as amostras durante a noite a 4 ° C. </li><li> Coloque cada lamela em um poço de um 24 com vários poços (lado da célula na parte superior) e adicionar 1 ml de PBS. <ol><li&gt; Lavar cinco vezes com 1 ml de PBS para remover as moléculas não ligadas. </li></ol></li><li> Prepara-se uma mistura de anticorpos secundários fluorescentes em PBS: rato de cabra anti-IgM acoplado a FITC (diluição 1:100) e de cabra anti-IgG de coelho acoplado a TRITC (diluição 1:50). </li><li> Colocar uma nova folha de Parafilm na caixa húmido e pipeta 25 ul da solução de anticorpos secundários para cada lamela. </li><li> Transferir as amostras como em 6.10 e incubar durante 1 hora à temperatura ambiente, no escuro, para evitar o branqueamento dos corantes fluorescentes. Repita o passo 6.11.1. </li><li> Para coloração nuclear, incubar durante 3 minutos com 200 ul de uma solução 0,05 mM de DAPI a temperatura ambiente e protegido da luz. Repita o passo 6.11.1. </li><li> Para conservar os corantes fluorescentes, coloque as lamelas (células virada para baixo) em 5 mL Vecta Escudo Antifade solução de montagem em lâminas de vidro. Vedar as lamínulas usando unha polonês para evitar a secagem da amostra. </li><li> Para o armazenamento a longo prazo, manter os slides de umacaixa de lâmina de microscópio a -20 ° C. </li><li> Analisar os preparativos por microscopia confocal através de seções Z-stack e XZ-aviões (Figura 2A). </li></ol> 7. Incubação de Trofozoítos com inibidores da protease ou um anticorpo específico <ol><li> Repita os passos 3.1 a 3.2. Para cada condição experimental, incubar 1 x 10 5 trofozoítos (ressuspensas em 50 ul de PBS) com inibidores de protease (1 mM completos e 40 ug / ml de E-64) ou 30 ug de anticorpos monoclonais contra EhCPADH112 (mαEhCPADH112) 21 durante 20 min a 4 ° C (Figura 2B). Usar estas preparações para ensaios de co-incubação, tal como descrito na etapa 8.5. </li></ol> 8. Medição da resistência elétrica transepitelial (TER) <ol><li> Culturas de células MDCK em Transwell suportes permeáveis ​​esterilizados (0,4 um de tamanho de poro). No compartimento superior, colocar 100 ul de suspensão celular e no compartimento inferiorcolocar 600 ul de meio DMEM suplementado. <ol><li> Verifique o nível médio periodicamente. Meio fresco pode ser adicionado como requerido. </li><li> Inspecionar as células em um microscópio invertido até atingirem 100% de confluência. Mudança de meio cada 2-3 dias. </li><li> Para melhorar a fixação das células (se necessário), os filtros Transwell equilibrar durante a noite a 37 ° C em DMEM antes da adição da suspensão de células. </li></ol></li><li> Esterilizar eléctrodos STX2 na capa por imersão em etanol e, em seguida, em PBS esterilizado durante 15 min cada, sob luz UV. Conecte os eletrodos a uma epitelial voltohmmeter EVOM e selecionar o modo de resistência. </li><li> Recolhe meio do compartimento superior de cada transwell e reuni-los. Tome 50 ul desta mistura mídia e combiná-lo com 50 ul de trofozoítos suspensão (1 x 10 5 trofozoítos em PBS) ou com apenas PBS (controle). Use uma mistura semelhante para cada transwell. </li><li> Adicionar misturas à câmara superior de cada containin transwellg as células MDCK. As condições experimentais incluem células MDCK sem trofozoítos (controlo), um co-cultura com trofozoítos ou com trofozoítos pré-incubadas com inibidores da protease ou mαEhCPADH112. Para eliminar a resistência proporcionada pelos filtros, usar transwells incubadas apenas com meio. Para cada condição experimental uso de pelo menos três transwells. </li><li> Imediatamente, medir TER mergulhando os eletrodos Stx2 em cada transwell. <ol><li> Certifique-se de que o eléctrodo mais toca no fundo da câmara inferior e que o eléctrodo mais curto é coberto por meio do compartimento superior (ver Figura 2B). </li><li> Certifique-se de manter os eletrodos perpendicularmente a cada vez. Isto irá melhorar a reprodutibilidade, de forma significativa. Evite mover ou inclinar o eletrodo durante as medições, pois isso levará a variação dos dados. </li></ol></li><li> Esta medida deve ser considerado o valor inicial TER. Em seguida, monitorar o TER durante a seguinte30 min. </li><li> Para calcular o valor final TER, subtrair o valor de TER apenas filtros. Para comparar os resultados das diferentes experiências de normalizar cada ponto de dados para os valores iniciais, tendo estes como 100% (Figura 2B). </li></ol>

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Appendix 3, (2001).</li><li>Arroyo, R., Orozco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+and+identification+of+an+Entamoeba+histolytica+adhesin.">Localization and identification of an Entamoeba histolytica adhesin.</a> Mol Biochem Parasitol. 23, 151-158 (1987).</li><li>Diamond, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axenic+cultivation+of+Entamoeba+hitolytica.">Axenic cultivation of Entamoeba hitolytica.</a> Science. 134, 336-337 (1961).</li><li>Diamond, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+of+axenic+cultivation+of+Entamoeba+histolytica+Schaudinn,+1903+and+E.+histolytica-like+amebae.">Techniques of axenic cultivation of Entamoeba histolytica Schaudinn, 1903 and E. histolytica-like amebae.</a> J Parasitol. 54, 1047-1056 (1968).</li><li>Dukes, J. D., Whitley, P., Chalmers, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MDCK+variety+pack:+choosing+the+right+strain.">The MDCK variety pack: choosing the right strain.</a> BMC Cell Biol. 12, (2011).</li><li>Espinosa-Cantellano, M., Martinez-Palomo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+intestinal+amebiasis:+from+molecules+to+disease.">Pathogenesis of intestinal amebiasis: from molecules to disease.</a> Clin Microbiol Rev. 13, 318-331 (2000).</li><li>Martinez-Palomo, 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=Structural+bases+of+the+cytolytic+mechanisms+of+Entamoeba+histolytica.">Structural bases of the cytolytic mechanisms of Entamoeba histolytica.</a> J Protozool. 32, 166-175 (1985).</li><li>Martinez-Lopez, C., 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=The+EhADH112+recombinant+polypeptide+inhibits+cell+destruction+and+liver+abscess+formation+by+Entamoeba+histolytica+trophozoites.">The EhADH112 recombinant polypeptide inhibits cell destruction and liver abscess formation by Entamoeba histolytica trophozoites.</a> Cell Microbiol. 6, 367-376 (2004).</li><li>Ocadiz-Ruiz, 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=Effect+of+the+silencing+of+the+Ehcp112+gene+on+the+in+vitro+virulence+of+Entamoeba+histolytica.">Effect of the silencing of the Ehcp112 gene on the in vitro virulence of Entamoeba histolytica.</a> Parasit Vectors. 6, 248 (2013).</li><li>Johnson, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+imaging+techniques+to+studies+of+epithelial+tight+junctions.">Applications of imaging techniques to studies of epithelial tight junctions.</a> Adv Drug Deliv Rev. 57, 111-121 (2005).</li></ol>

The authors have nothing to disclose.

Para o estudo in vitro interações patógeno-hospedeiro durante a infecção epitelial pelo E. histolytica, é fundamental para trabalhar com culturas bem estabelecidas de ambas as células epiteliais e trofozoítos. Por exemplo, antigamente, E. culturas histolytica geralmente tinha sido estabelecida em associação com certas espécies de bactérias ou tripanossomatídeos 22,23. No entanto, a co-cultura de E. culturas histolytica é contraproducente para...

Entamoeba histolytica é um protozoário única célula responsável da amebíase humano, uma infecção intestinal causando inflamação e diarréia. E. histolytica infecta até 50 milhões de pessoas por ano, mas apenas cerca de 10% das pessoas infectadas desenvolvem os sintomas associados a amebíase 1. A infecção ocorre após a ingestão de alimentos contaminados ou água contendo E. cistos histolytica. No intestino, os cistos produzir trofozoítos vivos que aderem a mucina do cólon e proliferam 2. Trofozoítos geralmente formam cistos que são excretados via fezes. Em outros casos, e por razões ainda desconhecidas, trofozoítos quebrar a camada epitelial intestinal e invadir os tecidos subjacentes. No pior dos casos, eles entram na corrente sanguínea e afetar outros órgãos como o fígado 3. Quebrando a barreira epitelial requer rompimento de estruturas epiteliais transmembranares que mantêm as células unidas. Célula epitelialos contactos são formados pelo complexo de junções de apicais consistindo apertado (TJ) e conexões aderentes (AJ), e 4 desmossomas. As junções mais apicais são TJ e, por isso, eles são a primeira barreira afrontado por E. histolytica e alguns outros agentes patogénicos durante a invasão de host. TJ são compostos de receptores de adesão transmembranares como claudinas, occludin e moléculas de adesão juncional (JAM) que se envolvem em interações heterofílicos homo ou com os receptores da célula vizinha. Eles são intracelularmente ligados por moléculas de andaime dos occludens zonula (ZO) da família de receptores de adesão que se ligam ao citoesqueleto de actina para proporcionar mais resistência mecânica ao epitélio. TJ são responsáveis ​​pela vedação do tecido intestinal do lúmen intestinal, evitando excesso de água e vazamento de soluto. Assim, depois de TJ são interrompidos pelo parasita, os tecidos são invadidos. E. histolytica segrega várias moléculas, tais como: (i) aqueles que estão envolvidos na adesão de amebas para alvet células 5; (Ii) fatores de membrana ativa participação em morte de células hospedeiras por exocitose, por exemplo, os peptídeos que formam os canais de íon denominado amebaporos 6,7; e (iii) as proteinases que degradam as proteínas da matriz extracelular e tecido mediam desintegração 5,8,9. A protease de cisteína EhCP112 e a molécula de adesão EhADH112 que em conjunto formam o complexo EhCPADH112 são dois E. histolytica virulência proteínas que desempenham um papel importante na desmontagem de TJ 10. Trofozoítos vivo, seus lisados ​​totais e produtos secretados induzir alterações moleculares no TJ distúrbio complexo e funcional da barreira epitelial. Neste estudo, demonstra-se que EhCP112 e EhADH112 interagir com ocludina e claudina-1 de proteínas que conduzem à internalização e a degradação das proteínas celulares, facilitando, assim, E. entrada histolytica através da via paracelular. Os dados e os óf outros grupos 11-17 sugerem fortemente a necessidade de interações patógeno-hospedeiro específicas que permitem a invasão do parasita. Desvendando as bases moleculares destas interações é de extrema importância para uma melhor compreensão da patogênese amebíase. Perturbação selectiva dos TJ por trofozoítos, caracterizado pelo aumento da permeabilidade paracelular, pode ser medido por uma diminuição da resistência eléctrica transepitelial (TER). A transferência de proteínas de parasitas no sentido epitélios host podem ser determinada por imunofluorescência e por microscopia confocal a laser, um método que também pode revelar a co-localização de factores de virulência ameba com marcadores juncionais epiteliais indicando possíveis interacções directas. Neste artigo, vamos descrever em detalhes como células epiteliais e trofozoítos são cultivadas, colhidas e manipuladas para examinar as interações patógeno-hospedeiro e suas consequências.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Entamoeba histolytica HM1:IMSS, Clone A&#160;</td>
			<td>IMSS Hospital, Mexico</td>
			<td>Without/number</td>
			<td>Virulent trophozoites18&#160;</td>
		</tr>
		<tr>
			<td rowspan="8">TYI broth</td>
			<td rowspan="8">Becton, Dickinson and Company 
			Merck 
			Merck 
			Merck 
			J.T. Baker 
			Reproquifin 
			SIGMA-Aldrich 
			SIGMA-Aldrich</td>
			<td rowspan="8">211862 
			K35625437 626 
			21578 
			4873 
			3252-01 
			CAS 50-81-7 
			C7880 
			F-5879</td>
			<td rowspan="8">3.45% BBL Biosate peptone 
			58 mM glucose 
			39 mM NaCl 
			5 mM KH2PO4 
			6.5 mM K2HPO4 
			16.3 mM ascorbic acid 
			8.1 mM L-cysteine 
			&#160;0.1 mM ferric ammonium citrate, adjust pH 6.819</td>
		</tr>
		<tr>
			<td>Bovine serum adult</td>
			<td>Microlab , Labs., Mex.</td>
			<td>SU146</td>
			<td>Use at 10% and inactivated to 56&#176; C for 30 min</td>
		</tr>
		<tr>
			<td>Diamond&#160; vitamin&#160; mixture- Tween 80</td>
			<td>In vitro</td>
			<td>SR-07</td>
			<td>Use at 3%</td>
		</tr>
		<tr>
			<td>Penicillin&#160;</td>
			<td>Lakeside,&#160; M&#233;x.</td>
			<td>34564SSA IV</td>
			<td>0.5 IU/mL</td>
		</tr>
		<tr>
			<td>Streptomycin&#160;</td>
			<td>Lakeside,&#160; M&#233;x.</td>
			<td>75757SSA IV</td>
			<td>35 &#181;g/ mL</td>
		</tr>
		<tr>
			<td>Pyrex 15 mL screw cap culture tubes with PTFE lined phenolic caps</td>
			<td>Corning-Pyrex</td>
			<td>9826-16X</td>
			<td>16x125 mm, capacity 15 mL and caps fabricated from special formula resistant to effects of temperature</td>
		</tr>
		<tr>
			<td>Cell culture plates, 6 Well</td>
			<td>Corning-Costar</td>
			<td>3516</td>
			<td>Sterile plates, well diameter 34.8 mm and growth area 9.5 cm2.&#160; Rings on lid prevent cross-contamination</td>
		</tr>
		<tr>
			<td>25cm2 cell culture flask</td>
			<td>Corning-Costar</td>
			<td>430168</td>
			<td>Canted neck flasks</td>
		</tr>
		<tr>
			<td>MDCK (Madin Darby canine kidney) type I</td>
			<td>American Type Culture Collection</td>
			<td>CCL34</td>
			<td>Kidney epithelial cells grown between the 60th and 90th passage</td>
		</tr>
		<tr>
			<td>DMEM medium&#160;</td>
			<td>Gibco&#160;</td>
			<td>12800-017</td>
			<td>Dulbecco&#39;s Modified Eagle Medium with high glucose.</td>
		</tr>
		<tr>
			<td>Neonate Calf Serum</td>
			<td>In vitro</td>
			<td>S-02</td>
			<td>Use at 10%.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin mixture&#160;</td>
			<td>In vitro&#160;</td>
			<td>A-01</td>
			<td>Stock solution 10,000 U/&#181;g/mL</td>
		</tr>
		<tr>
			<td>Insulin&#160;&#160;</td>
			<td>AMSA</td>
			<td>398MJ94SSA IV</td>
			<td>Stock solution 100 IU/mL</td>
		</tr>
		<tr>
			<td>Trypsin solution&#160;</td>
			<td>In vitro</td>
			<td>EN-005</td>
			<td>0.05% enzyme solution without calcium and magnesium</td>
		</tr>
		<tr>
			<td>75cm2 cell culture flask</td>
			<td>Corning-Costar</td>
			<td>430720</td>
			<td>Canted neck flasks for trophozoite culture in TYI-S-33 medium</td>
		</tr>
		<tr>
			<td>Transwell permeable supports</td>
			<td>Corning-Costar</td>
			<td>3470</td>
			<td>0.4. &#181;m polyster membrane, 6.5 mm insert in 24 well plate, growth area 0.3 cm2</td>
		</tr>
		<tr>
			<td>24 well cell culture dish&#160;&#160;</td>
			<td>Corning-Costar</td>
			<td>3524</td>
			<td>Clear polystyrene, treated for optimal cell attachment, sterilized by gamma radiation and certified non-pyrogenic</td>
		</tr>
		<tr>
			<td>Complete Mini</td>
			<td>Roche</td>
			<td>11836 153 001</td>
			<td>Protease inhibitor cocktail inhibits a broad spectrum of serine, cysteine and metallo-proteases. Final concentration 1 mM&#160;</td>
		</tr>
		<tr>
			<td>Trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane (E-64)</td>
			<td>SIGMA-Aldrich</td>
			<td>E3132</td>
			<td>Cystein protease inhibitor, final concentration 40 &#181;g/mL</td>
		</tr>
		<tr>
			<td>p&#945;ZO-1&#160;</td>
			<td>Invitrogen</td>
			<td>402200</td>
			<td>IgG rabbit policlonal&#160; antibody&#160; against&#160; a synthetic peptide in the middle region of the ZO-1 human protein</td>
		</tr>
		<tr>
			<td>m&#945;EhCPADH112</td>
			<td>Homemade antibody</td>
			<td>Without/ Number</td>
			<td>IgM mouse monoclonal antibody&#160; against&#160; 444-601 epitope located at C-terminal of EhCPADH11221,27</td>
		</tr>
		<tr>
			<td>FITC-goat anti-mouse IgM</td>
			<td>Zymed</td>
			<td>62-6811</td>
			<td>Fluorescein isotiocyanate (FITC)-labelled goat anti-mouse secondary&#160; antibody</td>
		</tr>
		<tr>
			<td>TRITC- goat anti-rabbit IgG (H+L)</td>
			<td>Zymed</td>
			<td>816114</td>
			<td>Tetramethyl-rhodamine isothiocyanate (TRITC)-labelled&#160; goat anti-rabbit IgG&#160; secondary antibody.</td>
		</tr>
		<tr>
			<td>STX2 Electrode</td>
			<td>World Precision Instrument&#160;</td>
			<td>102711</td>
			<td>Consists of a fixed pair of double electrodes, 4 mm wide and 1 mm thick. Each stick of the electrode pair contains a silver/silver-chloride pellet for measuring voltage and a silver electrode for passing current. For use with EVOM</td>
		</tr>
		<tr>
			<td>EVOM epithelial voltohmmeter</td>
			<td>World Precision Instrument&#160;</td>
			<td>12111</td>
			<td>Use in resistance mode and maintain unplugged during TER measurements</td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>MEARIENFELD</td>
			<td>610610</td>
			<td>Hemocytometer&#160;</td>
		</tr>
		<tr>
			<td>Leica TCS_SP5_MO</td>
			<td>Leica</td>
			<td>Without/number</td>
			<td>Laser confocal microscopy with Leica microsystems CMS Gmbh/leica Las af Lite/BIN software</td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories, Inc.</td>
			<td>H-1000</td>
			<td>Mounting medium for fluorescence</td>
		</tr>
		<tr>
			<td>4&#180;, 6-diamino-2-phenylindole (Dapi)</td>
			<td>SIGMA</td>
			<td>D-9542</td>
			<td>0.05 mM final concentration</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>US Biological</td>
			<td>A-1310</td>
			<td>0.5%&#160; final concentration for blocking solution</td>
		</tr>
	</tbody>
</table>

analysis of the epithelial damage produced by entamoeba histolytica infection

Entamoeba histolytica é o agente causador da amebíase humana, uma das principais causas de diarréia e abscesso hepático em países tropicais. A infecção inicia-se por interacção do agente patogénico com células epiteliais intestinais. Essa interação leva à ruptura das estruturas intercelulares, como junções apertadas (TJ). TJ garantir a vedação da camada epitelial para separar tecido hospedeiro do lúmen intestinal. Estudos recentes fornecem evidências de que a interrupção do TJ pelo EhCPADH112 proteína parasitária é um pré-requisito para E. invasão histolytica que é acompanhada por disfunção da barreira epitelial. Assim, a análise dos mecanismos moleculares envolvidos na TJ desmontagem durante E. invasão histolytica é de suma importância para melhorar a nossa compreensão da amebíase patogênese. Este artigo apresenta um modelo simples que permite a avaliação das interações iniciais patógeno-hospedeiro eo potencial de invasão do parasita. Os parâmetros a serem analisados ​​incluem tresistência eléctrica ransepithelial, interacção de EhCPADH112 com receptores da superfície do epitélio, as alterações na expressão e localização de marcadores epiteliais juncionais e localização das moléculas do parasita no interior das células epiteliais.

Para uma E. sucedida cultura histolytica, duas condições importantes devem ser cumpridas: o crescimento em condições axênicas e colheita em fase logarítmica. Anteriormente, culturas de E. histolytica foram prontamente estabelecido em associação com certas espécies de bactérias ou tripanossomatídeos 22. No entanto, hoje em dia é comum ter cultivo axênico deste parasita que significa uma subcultura indefinido de amebas em um ambiente livre de bactérias que metabolizam, f...

Watch this Scientific Journal Video about Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection at JoVE.com

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

A infecção humana por Entamoeba histolytica leva a amebíase, uma das principais causas de diarréia em países tropicais. A infecção é iniciada pelo agente patogénico com as células epiteliais do intestino, provocando a abertura dos contactos célula-célula e, consequentemente, a diarreia, por vezes, seguido por uma infecção do fígado. Este artigo fornece um modelo para avaliar as interações patógeno-hospedeiro iniciais para melhorar a nossa compreensão da amebíase patogênese.

Análise do dano epitelial Produzido pela<em&gt; Entamoeba histolytica</em&gt; Infecção

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

analysis-epithelial-damage-produced-entamoeba-histolytica

Research

JoVE Journal

Immunology and Infection

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

18.6K visualizações.  Center for Research and Advanced Studies of the National Polytechnic Institute.  A infecção humana por Entamoeba histolytica leva a amebíase, uma das principais causas de diarréia em países tropicais. A infecção é iniciada pelo agente patogénico com as células epiteliais do intestino, provocando a abertura dos contactos célula-célula e, consequentemente, a diarreia, por vezes, seguido por uma infecção do fígado. Este artigo fornece um modelo para avaliar as interações patógeno-hospedeiro iniciais para melhorar a nossa compr...

Vídeo: Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

Ensaio in vitro de metilação tRNA com a Entamoeba histolytica DNA e Dnmt2 tRNA metiltransferase (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

Imunologia e Infecção

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Desenvolvimento de um marcador negativo selecionáveis ​​para Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

Aplicação de um rato ligado patch Peyer Assay alça intestinal para avaliar absorção pelas células bacterianas M

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Isolamento de citometria de fluxo de células primárias Murino Tipo II epiteliais alveolares de Estudos funcionais e Molecular

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

Análise da acessibilidade de solvente de resíduos de cisteína em<em&gt; Milho rayado fino vírus</em&gt; Partículas semelhantes a vírus produzidas em<em&gt; Nicotiana benthamiana</em&gt; Plantas e cross-linking de peptídeos para VLP

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Capsular sorotipagem de<em&gt; Streptococcus pneumoniae</em&gt; Por aglutinação em látex

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Rápida análise de aberrações cromossômicas em linfócitos B mouse por PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

Uso da técnica de sobreposição de Soft-agar para triagem de compostos inibidores produzida por bactérias

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

Imagem de Ca2 + respostas durante Shigella infecção das células epiteliais

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Caracterização de subconjuntos de monócitos humanos por sangue total Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...