The authors thank Ian Clarke and P. Scott Hefty for the pGFP-SW2 and pASK-GFP-mKate2-L2 plasmids, respectively. We thank Paul Miller for technical assistance and Richard Stephens for other resources. This work was funded by NIH grant AI095603 (KH).

1. Geração de linhas celulares hospedeiras fluorescente <ol><li> Dia 1. Placa células 293T (ou outra linha de empacotamento retroviral) em placas de 6 poços a 2 x 10 6 células / poço, para ser ~ 75% confluentes no dia seguinte. Se desejado, placa de poços duplicados para cada um retrovírus para ser utilizado. </li><li> Dia 2. células Aspirar e adicionar 2 ml de meio de crescimento fresco (DMEM + 10% de FBS + 2 mM de L-glutamina). Transfectar células com 5-8 ug de cada vector retroviral (contendo GFP) e vector de embalagem (por exemplo., PVSVg) usando Lipofectamine 2000 ou reagente semelhante, e seguindo as instruções do fabricante. </li><li> Incubar as células com ADN de 4-6 h num banho a 37 ° C incubadora de CO 2, e subsequentemente substituir meio com 1 ml de meio de crescimento. </li><li> Incubar as células durante 48 h em 37 ° C incubadora de CO 2. </li><li> Dia 3. Placa células alvo (por exemplo., HeLa) em placas de 6 poços a uma densidade aproximada de 5 a 10 células / poço, para ser ~ 50% confluentes apróximo dia. Verifique transfecção positiva em células 293T por monitorização de fluorescência de GFP em um microscópio invertido. </li><li> Dia 4. Recolher as células sobrenadantes 293T usando uma pipeta e filtrar o sobrenadante através de um filtro de celulose de 0,45 um seringa de etilo contendo retrovírus. </li><li> Remova o meio a partir de células HeLa e adicionar 0,5 ml de meio de crescimento contendo 16 ug / ml de polibreno. Adicionar ~ 0,5 ml de retrovírus filtrado às células gota a gota, e incuba-se as células num banho a 37 ° C incubadora de CO 2 durante 24 horas. Armazenar quaisquer retrovírus restantes (isto é, a partir do duplicar poço) a 4 ° C. </li><li> Dia 5. Se poços duplicados foram feitas para gerar retrovírus extra, repita o passo 1.7 para infectar em série com os restantes partículas retrovirais. </li><li> Dia 6. Passagem transfectadas células HeLa 1: 1 num prato contendo selecção de antibiótico 10 cm (por exemplo, 500 ug / ml de neomicina). </li><li> Aguarde tempo suficiente para que as células transduzidas para voltar a crescer na presença de selecção antibiotlc, após o qual as células podem ser propagadas por técnicas padrão com uma concentração mais baixa de antibiótico de selecção (por exemplo., 200 ug / ml de neomicina). Nota: As células podem também ser seleccionadas por clonagem de brilho ideal neste momento, se necessário. </li></ol> 2. Geração de-expressando GFP Chlamydia trachomatis <ol><li> Prepare 2x tampão de CaCl2 (20 mM de Tris pH 7,4, 100 mM CaCl2) e tampão 4SP (0,4 M de sacarose, 16 mM de Na 2 HPO 4). </li><li> Dia 1. Descongele congelados C. estoque trachomatis no gelo e diluir imediatamente 10 bactérias ul em 50 ul 2x CaCl2 buffer. Adiciona-se 3 ug de ADN de plasmídeo (por exemplo., PASK-GFP-mKate2-L2), misturar bem e incubar durante 30 min a 25 ° C. </li><li> Durante esta etapa, preparar células hospedeiras por trypsinizing um frasco T-75 de células McCoy em um tubo de centrífuga. Centrifugar a suspensão de células a 50 xg durante 5 min e desprezar o sobrenadante. Resuspend sedimento de células num volume apropriado de tampão 1x CaCl 2 para dar uma concentração final de 4 x 10 7 células / ml. </li><li> Após a incubação de 30 minutos (a partir do passo 2.2), adicionar 100 ul de células McCoy preparadas para transformação tubo de mistura (volume total de 200 uL) e incubar 20 min a 25 ° C. Adicionar 100 ul desta mistura de células transformadas para um único poço de uma placa de 6 poços e sobreposição com 2 ml de meio de crescimento. Incubar a 37 ° C em uma incubadora de CO 2 durante 2 dias. </li><li> Dia 3. lise de células infectadas com C. McCoy trachomatis, substituindo meio com 1 ml de dH2O e raspar as células com uma inclinação de 1.000 ul pipeta de ponta. Adicione 1 ml de tampão 4SP e passar a suspensão através de uma agulha de 27,5 G ~ 5 vezes para a lise celular eficiente e liberação de C. trachomatis. </li><li> Transferir 2 ml do lisado celular contendo C. trachomatis para um frasco T-75 contendo uma monocamada confluente de recém-banhado McCcélulas oy; adicionar 8 mL de DMEM (sem FBS) e incubar durante 2 horas a 25 ° C para permitir C. trachomatis para aderir às células. </li><li> Aspirar as células e adicionar meio de crescimento fresco, suplementado com 10 U / ml de penicilina G e 1 ug / ml de ciclo-heximida. Incubar o balão durante 48 h numa incubadora a 37 ° C, CO 2. </li><li> Dia 4. Verifique por microscopia de luz que as células são infectadas e inclusões contêm Chlamydia aberrante. Identificar inclusões como vacúolos brilhantes em um microscópio de luz padrão e organismos Chlamydia aberrantes como objetos em forma de anel dentro inclusões que são maiores que 2 mm de diâmetro. </li><li> Dia 5. Lise de cultura por meio de substituição com 2 ml de dH2O e raspar as células numa suspensão. Adicionar 2 ml de tampão 4SP e passar a suspensão através de uma agulha de 27,5 G ~ 5 vezes. </li><li> Utilizar 2 ml de lisado celular para infectar uma monocamada fresca de células McCoy em um único poço de uma placa de 6 poços. Incubar as células com lisado durante 2 horas a 25 ° C, aspirado e adicionar crescimento frescomeio contendo penicilina G e ciclo-heximida. </li><li> Incubar as células em uma incubadora a 37 ° C de CO2 durante 3-5 dias, dependendo do estado de saúde geral das células. </li><li> Repita os passos 2.9-2.10 uma ou mais vezes, conforme necessário, culturas Passaging em poços frescas em uma placa de 6 poços até inclusões normais que procuram (desprovidos de corpos aberrantes) têm surgido. Neste momento usar um microscópio de fluorescência invertido para verificar se C. trachomatis expressar mKate2 (vermelho). </li><li> Intensificar as culturas infectadas para geração de stocks por clamídia título elevado, por exemplo colhendo transformantes Chlamydia partir de células de McCoy ou HeLa infectadas cultivadas em frascos T-150. </li></ol> 3. infectar as células com Chlamydia <ol><li> Placa células GFP-HeLa no recipiente de cultura desejada, por exemplo pratos com fundo de vidro ou lâminas de câmara para a imagem latente de alta resolução, ou placas de 24 poços para determinação IFU e triagem com vários poços. </li><li> Descongele frozen tubo contendo C. trachomatis, C. muridarum, ou C. pneumoniae em gelo e diluído em HBSS a desejada multiplicidade de infecção (MOI), por exemplo MOI = 1. </li><li> Realizar infecções estáticos ou auxiliado por centrifugação, com base na estirpe de Chlamydia em particular que irá ser utilizado. <ol><li> Para as infecções com C. trachomatis serovar L2 LGV ou C. muridarum, lavar as células GFP-HeLa com HBSS e incubar com bactérias diluídas durante 2 horas a 25 ° C. Aspirar e lavar as células com HBSS, e incuba-se as células em meio de crescimento com 37 ° C incubadora de CO 2. </li><li> Para as infecções com C. trachomatis serovar D ou C. pneumoniae, lavar as células GFP-HeLa com HBSS e adicionar bactérias diluídas para as células. Coloque placa com múltiplas cavidades ou prato fundo de vidro (fixo por fita em uma placa da tampa com vários poços) em um suporte de placa com múltiplas cavidades de um anexo de rotor de caçamba móvel em uma centrífuga de bancada. </li><li> Células centrifugar a 900 xg a 25 ° C durante 1 h. </li&gt; <li> Remover os recipientes de cultura a partir de centrifugação e colocar numa incubadora a 37 ° C de CO2 durante 1 hora. </li><li> Aspirar células em uma cabine de segurança biológica, lave as células duas vezes com HBSS, e adicionar meio de crescimento fresco. Células lugar em um C incubadora de CO 2 37 °. </li></ol></li></ol> 4. Ao vivo Visualization celular de clamídia Inclusões <ol><li> Remover Chlamydia infectadas células GFP-HeLa de incubadora de CO 2 e substituir meio com RPMI sem vermelho de fenol + 5% FBS. </li><li> Células montar em um microscópio de fluorescência invertido (Nikon Eclipse Ti-E ou similar) usando uma objectiva 20X ou superior petróleo. </li><li> Usando o software de imagem (Volocity 6 ou similar), o foco em identificar e Chlamydia infectados células GFP-HeLa: verde células que contêm grandes buracos negros (inclusões de clamídia). </li><li> Marque os locais xyz de regiões que contêm células infectados por clamídia de interesse usando software de imagem (Volocity 6 ou semelhante). Realizar isso manualmente &quot;Adicionando Points &#39;enquanto no modo de exibição XY Stage. Deste modo, as coordenadas XYZ são guardadas para cada local marcado. </li><li> Na caixa de diálogo Configuração de Aquisição, configurar o software para adquirir verde (GFP, HeLa citosol) e vermelho (mKate2, C. trachomatis) canais, e em uma taxa de novos quadros por canal a cada 5 min. </li><li> Adquirir a sequência de imagens utilizando a aquisição protocolo estabelecido acima, clicando no botão &quot;Capture / Record&quot;. </li></ol> 5. A quantificação da inclusão parâmetros de crescimento em células vivas <ol><li> Às vezes desejadas pós-infecção (ex., 24, 48 hpi) Chlamydia remover células infectadas GFP-HeLa da incubadora e montar em um microscópio de fluorescência invertido. Nota: Um dos objectivos 20X ou 40X ar com uma NA elevada é melhor adequada para placas de 24 poços, e uma objectiva de 40X óleo é recomendado para lâminas de câmara. Cultivar células em qualquer substrato para este burroay. </li><li> Concentre-se em midplanes de células, onde inclusões estão em seu diâmetro máximo e produzir bordas nítidas. </li><li> Aquisição de imagens para muitos campos em cada poço (pelo menos 10 por poço); poços normalmente representam repetições ou variáveis ​​experimentais. </li><li> Enumerar o número de inclusões manualmente, por olho (inclusões são compartimentos negros em células HeLa-GFP) ou usar algoritmos de software de imagem para identificar e contar automaticamente inclusões. <ol><li> Encontre células GFP-HeLa por intensidade de fluorescência (&#39;encontrar&#39; Objetos de comando) e ajustar a intensidade limiar de fluorescência baseada em intensidades relativas de células. </li><li> Filtro de objetos selecionados usando diretrizes de tamanho de manter somente aqueles superior a 5 m 2. Esta é a população 1 &#39;. </li><li> Aplicar &#39;tapar buracos em objetos&#39; de comando usando &quot;população 1 &#39;como a entrada. Esta etapa gera &#39;população 2&#39;. </li><li> Subtrair &#39;população 1&#39; de &#39;pOPULAÇÃO 2 &#39;para produzir marcado positivamente inclusões de clamídia, designadas por &quot;população 3&#39;. </li><li> Aplicar filtro de tamanho de &#39;população 3&#39; (&#39;Filtro População&#39; comando), por exemplo, tratar de objectos maiores do que 25 um para 2 inclusões nas células infectadas durante 24 horas ou mais. Para tempos de início da infecção, tais como antes de 18 horas, conjunto de filtros de tamanho para manter objectos maiores do que 4 mm 2, uma vez que estas inclusões são muito menores. Tamanho de filtragem resulta numa população de objectos designados como inclusões &#39;&#39;. </li><li> Calcular dados quantitativas de imagens, por exemplo, o número de inclusão, inclusão tamanho e circunferência inclusão, usando software de imagem. </li></ol></li></ol>

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	<li>Stamm, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydia+trachomatis+infections:+progress+and+problems.">Chlamydia trachomatis infections: progress and problems.</a> The Journal of Infectious Diseases. 179, S380-S383 (1999).</li><li>Schachter, J., Stephens, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+and+disease+epidemiology.">Infection and disease epidemiology.</a> Chlamydia: Intracellular Biology, Pathogenesis, and Immunity. , 139-169 (1999).</li><li>Campbell, L. A., Kuo, C. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology.">Cell biology.</a> Chlamydia: Intracellular Biology, Pathogenesis, and Immunity. , 101-138 (1999).</li><li>Wang, Y., Kahane, S., Cutcliffe, L. T., Skilton, R. J., Lambden, P. R., Clarke, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+transformation+system+for+Chlamydia+trachomatis:+restoration+of+glycogen+biosynthesis+by+acquisition+of+a+plasmid+shuttle+vector).">Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector).</a> PLoS Pathogens. 7 (9), e1002258 (2011).</li><li>Hackstadt, T., Scidmore, M. A., Rockey, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+metabolism+in+Chlamydia+trachomatis-infected+cells:+directed+trafficking+of+Golgi-derived+sphingolipids+to+the+chlamydial+inclusion.">Lipid metabolism in Chlamydia trachomatis-infected cells: directed trafficking of Golgi-derived sphingolipids to the chlamydial inclusion.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (11), 4877-4881 (1995).</li><li>Boleti, H., Ojcius, D. M., Dautry-Varsat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+labelling+of+intracellular+bacteria+in+living+host+cells.">Fluorescent labelling of intracellular bacteria in living host cells.</a> Journal of Microbiological Methods. 40 (3), 265-274 (2000).</li><li>Hybiske, K., Stephens, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+host+cell+exit+by+the+intracellular+bacterium+Chlamydia.">Mechanisms of host cell exit by the intracellular bacterium Chlamydia.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (27), 11430-11435 (2007).</li><li>Heinzen, R. A., Hackstadt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Chlamydia+trachomatis+parasitophorous+vacuolar+membrane+is+not+passively+permeable+to+low-molecular-weight+compounds.">The Chlamydia trachomatis parasitophorous vacuolar membrane is not passively permeable to low-molecular-weight compounds.</a> Infection and Immunity. 65 (3), 1088-1094 (1997).</li><li>Wickstrum, J., Sammons, L. R., Restivo, K. N., Hefty, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+expression+in+Chlamydia+trachomatis+using+the+tet+system.">Conditional gene expression in Chlamydia trachomatis using the tet system.</a> PLoS One. 8 (10), 10-1371 (2013).</li><li>Chin, E., Kirker, K., Zuck, M., James, G., Hybiske, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+recruitment+to+the+Chlamydia+inclusion+is+spatiotemporally+regulated+by+a+mechanism+that+requires+host+and+bacterial+factors.">Actin recruitment to the Chlamydia inclusion is spatiotemporally regulated by a mechanism that requires host and bacterial factors.</a> PLoS One. 7 (10), e46949 (2012).</li><li>Agaisse, H., Derr&#233;, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+C.+trachomatis+cloning+vector+and+the+generation+of+C.+trachomatis+strains+expressing+fluorescent+proteins+under+the+control+of+a+C.+trachomatis+promoter.">A C. trachomatis cloning vector and the generation of C. trachomatis strains expressing fluorescent proteins under the control of a C. trachomatis promoter.</a> PLoS One. 8 (2), e57090 (2013).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

Aqui nós descrevemos a estratégia experimental para a geração de células hospedeiras fluorescentes para visualização em tempo real e análise de inclusões de clamídia. Esta abordagem visualização vacúolo confere a poderosa capacidade de iluminar, controlar e medir quantitativamente as propriedades dinâmicas de inclusões por clamídia entre as populações de células ou em células isoladas ao longo do tempo. Inclusões de clamídia em células proteína marcada fluorescentes são notavel...

Doenças infecciosas causadas por espécies da bactéria intracelular Chlamydia provocar um grande fardo para a saúde global, incluindo doença sexualmente transmissível, doença inflamatória pélvica, cegueira, pneumonia e possivelmente aterosclerose 1-4. A capacidade de Chlamydia para interagir com a célula hospedeira, a partir de dentro de um vacúolo (denominado a inclusão), constitui um factor determinante para a sua infecção bem sucedida de células e o hospedeiro. A inclusão de um compartimento é patogénico romance que permite o crescimento e clamídia é dinamicamente modificada ao longo de todo o ciclo de desenvolvimento de 2-3 dias de Chlamydia 5. A natureza intracelular obrigatório de clamídias apresenta inúmeros desafios para a comunidade de pesquisa, em especial para estudar diretamente a biologia única da inclusão. A grande desvantagem tem sido a incapacidade de visualizar de forma eficiente, quer intracelular Chlamydia ou a sua inclusão pela abordagem fluorescentees em células vivas. Uma descoberta recente revelou finalmente os meios de produção de GFP expressando C. trachomatis 6; no entanto, esta constatação não conduziu ainda a rotulagem específica da inclusão. Algumas técnicas têm sido descritas para a rotulagem de bactérias e inclusões de 7,8, mas eles sofrem de deficiências, como a não-especificidade, transitoriedade e susceptibilidade à fotodegradação. Uma descoberta chave do nosso grupo estabeleceu uma nova estratégia para iluminar a inclusão usando GFP expressando células hospedeiras 9. Esta estratégia explora a impermeabilidade racionalmente intrínseca da membrana inclusão a moléculas maiores do que 520 Da 10. Quando as células são manipuladas para expressar estavelmente uma proteína fluorescente cytosolic particular (por exemplo, ou GFP mCherry), inclusões de clamídia são visíveis com uma nitidez notável por sua exclusão completa de fluorescência. Esta estratégia de imagem inversa permite a visualização imediata das inclusões para todos Chlespécies amydia e pode ser facilmente adaptado para a maioria das células hospedeiras de interesse. Como uma demonstração da sua utilidade, este método foi utilizado anteriormente para revelar e definir as vias celulares de saída para Chlamydia spp 9. Aqui, nós ainda demonstrar como esse método é realizado, e pode ser explorado para obter dados quantitativos chave sobre dinâmica de crescimento inclusão. Além disso, pode substituir de forma eficaz para a enumeração métodos baseados em anticorpos dispendiosos e pode ser usado em conjunto com outros marcadores fluorescentes, tais como mKate2-11 expressando Chlamydia. Esta poderosa combinação de ferramentas permite a exploração das propriedades físicas da membrana de clamídia inclusão dentro das células hospedeiras de estar.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-Mem</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#181;m filters</td>
			<td>Fisher</td>
			<td>09-719D</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Invitrogen</td>
			<td>10131</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Invitrogen</td>
			<td>14025</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11995</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Invitrogen</td>
			<td>11875</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 w/o phenol red</td>
			<td>Cellgro</td>
			<td>17-105-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G</td>
			<td>Sigma</td>
			<td>13752</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>C7698</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Chamber slides, Lab-Tek II</td>
			<td>Nunc</td>
			<td>154526, 154534</td>
			<td></td>
		</tr>
	</tbody>
</table>

using fluorescent proteins to visualize and quantitate chlamydia vacuole growth dynamics in living cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

As células de mamífero que expressam as proteínas citosólicas fluorescentes (por exemplo, GFP) pode ser modificado para permitir a iluminação de inclusões de clamídia em células infectadas, culturas vivas. Após a infecção com clamídia, as inclusões são facilmente visíveis como pontos negros nas células hospedeiras (Figura 1). A clareza das inclusões-falta de fluorescência pode ser explorada para a identificação automática de inclusões em numerosos campos...

Watch this Scientific Journal Video about Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells at JoVE.com

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

Usando proteínas fluorescentes para visualizar e Quantificar<em&gt; Chlamydia</em&gt; Vacúolo Crescimento Dynamics em células vivas

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

using-fluorescent-proteins-to-visualize-quantitate-chlamydia-vacuole

Research

JoVE Journal

Immunology and Infection

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

7.5K visualizações.  University of Washington. A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

Vídeo: Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Medir a atividade das calpaínas em células fixas e Vivendo por citometria de fluxo

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Imunologia e Infecção

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Sete Passos para Stellate Células

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Seguindo o destino celular, em<em&gt; E. coli</em&gt; Após a infecção por um fago Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. 
We generated model xenograft tumors in mice by subcutaneous injection of a human ovarian cancer cell line, OVCAR-8 (NCI DCTD Tumor Repository, Frederick, MD). 
We transformed attenuated strains of S. typhimurium bacteria (ELH430:SL1344 phoPQ- 1) with a constitutively expressed luciferase (luxCDABE) plasmid for visualization2. These strains specifically colonize tumors while remaining essentially non-virulent to the mouse1. 
Once measurable tumors were established, bacteria were injected intravenously via the tail vein with varying dosage. Tumor-localized, bacterial gene expression was monitored in real time over the course of 60 hours using an in vivo imaging system (IVIS). At each time point, tumors were excised, homogenized, and plated to quantitate bacterial colonies for correlation with gene expression data. 
Together, this data yields a quantitative measure of the in vivo growth and gene expression dynamics of bacteria growing inside tumors.

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting.

Medindo o Crescimento e Expressão Gênica Dynamics do Tumor-Targeted<em&gt; S. Typhimurium</em&gt; Bactérias

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium ...

Forward Genetic Approaches in Chlamydia trachomatis

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis&#160;despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype&#8211;phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.

Forward Genetic Approaches in Chlamydia trachomatis

We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.

Abordagens para a frente genéticos em<em&gt; Chlamydia trachomatis</em

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Acompanhamento da evolução das Membrane Polaridade, integridade de membrana e intracelulares Ion concentrações no<em&gt; Streptococcus pneumoniae</em&gt; Usando corantes fluorescentes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Utilizando Proteínas Fluorescentes para monitorar glicossomo Dynamics do Trypanosoma Africano

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

&quot;Phagosome Closure Assay&quot; to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

&quot;Fagossomo Encerramento Ensaio&quot; para Visualize Formação fagossomo em Três Dimensões Usando Total Internal Reflection Microscopia fluorescente (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Rato pulmão Slices para Visualize células vivas pulmonar dendríticas precisão de corte

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.