This work was supported by the Deutsche Forschungsgemeinschaft by grant Z within Sonderforschungsbereich 944 &#8216;Physiology and Dynamics of Cellular Microcompartments&#8217; and HE1964/18 within priority program 1580.

1. Síntese de 10 nm Nanopartículas de Ouro (Gold PN) 15 <ol><li> Preparar a solução A: adicionar 2 ml 1% de cloreto de ouro aquosa para 160 ml Milli-Q, ou bidestilada, água. </li><li> Preparar a solução B: adicionar 8 mL de 1% de tri-citrato de sódio x 2 H2O e ácido tânico 160 ul de 1% em 32 ml de Milli-Q, ou duas vezes destilada, água. </li><li> Aquecer a solução A e B a 60 ° C e misturá-los com agitação. Observe a cor azul escuro imediatamente. Observe cor vermelha depois de cerca de 15 min. Em seguida, aquece-se a 95 ° C, mantém-5 min e arrefecer a solução para a TA. </li><li> Adicionar uma gota de suspensão de NP para uma grelha revestida de carbono e deixa-se secar ao ar. Verifique o tamanho e morfologia de NPs por microscopia eletrônica de transmissão. </li></ol> 2. Revestimento de Ouro NPs com BSA e Marcando com Rhodamine N-hidroxisuccinimidilo Ester (NHS) 16 <ol><li> Adicionar 900 ul NPs de ouro para um tubo de Eppendorf de 1,5 ml, de centrifugação a 15.000 xg durante 30 min. Opnalmente, preparar vários tubos podem ao mesmo tempo. </li><li> Rejeitar o sobrenadante, re-suspender o sedimento em 900 ul esterilizadas água Milli-Q e adicionar 100 ul de 2 mg / ml de BSA / PBS, mistura-se num Vortex a 800 rpm durante 30 min. </li><li> Para remover o excesso de BSA, centrifugar a preparação a 15.000 xg durante 60 min. </li><li> Desprezar o sobrenadante e re-suspender as NPs de ouro-BSA em 125 mL PBS, adicionar 12,5 mL de 1 M de bicarbonato. </li><li> Imediatamente antes da utilização, preparar uma solução 10 mg / ml de rodamina NHS / DMSO, adicionar 30 ul de solução de rodamina / NHS DMSO a 1 ml da suspensão de ouro-BSA PN. Incubar a reacção durante 2 horas (ou O / N) à temperatura ambiente durante 800 rpm, mistura, evitando a exposição à luz. </li><li> Purificar as NPs de ouro BSA-rodamina através de diálise contra PBS a 4 ºC com 5 alterações no buffer durante o período de 36 - 48 h. </li><li> Para estabilizar NPs, adicionar 10 ul de 200 mg / ml de BSA / PBS em cada 1 ml de NPs de ouro-BSA-rodamina. Para remover a centrífuga livre ou liberado BSA-rodamina, aos 15,000 xg durante 60 min. Ressuspender o sedimento em 2 mg / ml de BSA / PBS, medir a OD 520, e armazenar a 4 ° C, evitando a exposição à luz. </li><li> Diluir os PN 10 vezes com Milli-Q ou água bidestilada, adicionar uma gota sobre uma grelha revestida de carbono, e deixar secar ao ar. Verifique o tamanho e morfologia de NPs por microscopia eletrônica de transmissão (TEM). Nota: Todos os materiais utilizados para a preparação de NP foram esterilizadas e a operação foi conduzida em uma capa de cultura celular ou em um banco ao lado de uma chama. </li></ol> 3. Cultura de células HeLa <ol><li> Células HeLa que expressam permanentemente LAMP1-GFP são cultivadas em DMEM com 10% de soro fetal de vitelo (FCS) e cultivadas a 37 ° C numa atmosfera contendo 5% de CO 2. </li><li> Semear as células a uma densidade de 50.000 por cavidade em uma lâmina de câmara de 8 poços (Ibidi) e incubar S / N. </li></ol> 4. Cultura de Bactérias <ol><li> Use Salmonella enterica sorotipo Typhimurium NCTC 12023 wild-type (WT) tensão. Para efeito de comparação, use cepas mutantes Δ ssaV defeituosos no SPI2-T3SS e Δ sifa falta efetoras chave sifa para SIF. Use cepas abrigando plasmídeo pFPV25.1 de expressão constitutiva de GFP reforçada. NOTA: As estirpes bacterianas são rotineiramente cultivadas em caldo de Luria-Bertani (LB) com adição de 50 ug / ml de carbenicilina (WT) e LB com adição de 50 ug / ml de carbenicilina e / ou 50 ug / ml de canamicina (Δ ssaV, Δ sifa ) do necessário para manter os plasmídeos. </li><li> Inocular uma única colônia de bactérias em 3 ml de LB com antibióticos apropriados e crescer O / N a 37 ºC em condições que agitam para aeração, então diluir 1:31 em fresco LB e continuar o crescimento para 3,5 horas. Neste ponto de tempo, as culturas atingem a fase de log tardia e bactérias são altamente invasivo. A &#39;drum rolo&#39; é conveniente para incubar culturas tubo de ensaio com arejamento. </li></ol> 5. A infecção das células HeLa pelaSalmonella e Gold-BSA-Rodamina NPs Pulso perseguição de rotulagem (ver Figura 1 para o esquema) <ol><li> Medida OD600 das bactérias sub-cultivada e dilui-se a OD 600 = 0,2 em 1 ml de PBS (~ 3 x 10 8 ufc / mL), adicionar quantidades apropriadas de bactérias às células HeLa em lâminas de câmaras de 8 poços para atingir uma multiplicidade de infecção (MOI) de 100. </li><li> Incubar durante 30 minutos na incubadora de células, lava-se 3 vezes com PBS para remover as bactérias não-internalizadas (este ponto de tempo foi definido como 0 horas após a infecção, ou 0 h pi). Adicionar 300 ul de meio de cultura fresco contendo 100 ug / ml de gentamicina e manter durante 1 hr. Em seguida substituir o meio com meio fresco contendo 10 ug / ml de gentamicina durante o resto do tempo de incubação. </li><li> Depois de incubação com meio de cultura contendo 100 ug / ml de gentamicina, durante 1 hora, o meio é substituído por meio de imagiologia (Eagles MEM sem FCS, L-glutamina, bicarbonato de vermelho de fenol e de sódio, com HEPES 30 mM, pH 7,4) containing 10 ug / ml de gentamicina. NPs de ouro-BSA-rodamina são adicionadas a células HeLa para obter uma concentração final de OD 520 = 0,1. NOTA: NPs de ouro-BSA-rodamina também podem ser adicionados a células antes da infecção, ou em vários pontos de tempo pi </li><li> Após 1 hora de incubação, remover o meio, lavar 3 vezes com PBS, e adicionar 300 ul de meio fresco contendo imagiologia FCS a 10% e 10 ug / ml de gentamicina durante o resto do tempo de incubação. NOTA: A duração da incubação pode variar em função da concentração de PN e linhas celulares utilizadas. Para RAW264.7 de macrófagos, observou-se que 30 minutos de incubação com PN a uma concentração de OD 520 = 0,05 permitido interiorização suficiente. </li></ol> 6. Criação de Imagens <ol><li> Usar um sistema de imagiologia confocal, como um laser de varredura confocal (CLSM) ou de disco rotativo (SD) microscópio com uma câmara de ambiente humidificado para imagem de alta resolução a diferentes pontos de tempo. </li><li> Ligue a temperatura control sistema e esperar até que ele é estável. Otimizar as configurações de imagem, como a ampliação, velocidade de digitalização, a resolução, Z-stack etc. Use as configurações de excitação / emissão adequadas para GFP e NPs ouro-BSA-rodamina. Para este protocolo, use uma velocidade de digitalização de 400 Hz, resolução de 512 x 512 pixels e tamanho Z-passo de 0,25 m. Excitar GFP e ouro-BSA-rodamina usando um laser de Ar (488 nm) e um laser de HeNe (543 nm), respectivamente. Incluir uma imagem de campo brilhante (BF) de canal para observar a forma das células. Para outros sistemas de microscopia e condições de infecção, a definição tem que ser ajustado em conformidade. NOTA: Use o mesmo laser Ar como fonte de luz de canal e sinal BF foi detectado com um detector multiplicador foto (PMT). </li><li> No indicada pontos temporais pi, montar as lâminas de câmara de 8 poços contendo células infectadas no palco microscópio e gravar imagens. </li></ol> 7. Análise de Imagens <ol><li> Use microscopia software de análise de imagem (veja <strong&gt; Materiais e Tabela de Equipamentos), para analisar a reforma do sistema de endo-lysosomal por Salmonella intracelular. Abra os dados clicando em &#39;Open&#39; e escolher o arquivo. NOTA: Como alternativa, os pacotes de software de código aberto, como ImageJ ou FIJI pode ser utilizado para a imagem de análises quantitativas. </li><li> Na barra de ferramentas objetos do clique Ultrapasse vista sobre o ícone <img alt="Ícone" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon1.jpg" /> para adicionar um novo item de superfície. Clique em <img alt="Ícone" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> (Next). </li><li> Para analisar uma região de interesse (ROI), selecione um segmento de ROI. Em uma área de visualização, uma seção delimitada-retângulo sobreposto na imagem está representando o ROI. Digite os valores no x- correspondente, y, e os campos Z-, ou clique diretamente nas setas no retângulo de visualização para modificar o tamanho ea posição do ROI. Em seguida, clique <img alt="Ícone" fo:content-width=".1in" src<html> = &quot;/ Files / ftp_upload / 52058 / 52058icon2.jpg&quot; /&gt; (Next). </li><li> Como um canal de origem, selecione o canal 3 (sinal for Gold-BSA-Rhodamine NPS). Verifique a opção suave para configurar a lisura da superfície resultante. Defina um valor manualmente ou aceite o valor gerado automaticamente. Para Threshold, selecione a opção de intensidade absoluta. </li><li> Para o ajuste de limiar, selecione a opção manual e definir um valor. Na área de visualização, uma prévia limiar superfície é exibido em cinza. </li><li> Na guia Classificar Surfaces a superfície resultante pode ser filtrado por vários critérios. Um filtro padrão é &#39;número de voxels&gt; 10&#39;, e outros filtros também podem ser incluídos, clicando em &quot;adicionar&quot;. Se a filtragem não é necessário, em seguida, elimine todos os filtros, clicando no botão excluir. Clique <img alt="Ícone" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> (Next). </li><li> Para concluir a criação do Surface, clique em/files/ftp_upload/52058/52058icon3.jpg &quot;/&gt; (Finish). Na lista de objetos, agora desmarque a caixa para o Volume item e nova superfície criado é exibido na área de visualização. </li><li> Exportar as estatísticas. Agora, na vista Ultrapassar a cor dos indivíduos, variar de púrpura para vermelho de acordo com a sua área. E a mesa &#39;Números Plot Area&#39; mostra a informação de estatísticas (por exemplo, número de identificação e área de objetos). Exportar todas as estatísticas de superfície 1 para um arquivo do Excel através de um clique <img alt="Ícone" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon4.jpg" /> (Save). </li></ol>

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No conflicts of interest declared.

O sistema de endo-lisossômico de células de mamíferos controla processos fisiológicos importantes, incluindo a absorção de nutrientes, transdução de sinal mediadas por hormônios masculinos, vigilância imunológica, e apresentação de antígenos 17. Até agora, uma grande variedade de marcadores foram utilizados para a rotulagem das vias de endocitose e estudos de rastreio. Por exemplo, as sondas Lysotracker são sondas fluorescentes acidotropic desenvolvidos pela Molecular Probes (Life Technologies...

Nanopartículas fluorescentes (PN), incluindo NPs metálicas, pontos quânticos, o NPS de polímero, o NPS de sílica, pontos de carbono, etc., têm atraído uma atenção considerável nas últimas décadas 1,2. Em comparação com os corantes orgânicos tradicionais, NPs fluorescentes mostram químicas, propriedades ópticas e mecânicas desejáveis, tais como a potência do sinal forte, a resistência à fotodegradação e elevada biocompatibilidade 3,4. Essas vantagens tornam o método de escolha para a detecção intracelular e imagens de células vivas. Além disso, uma variedade de NPs elétron-densas são visíveis por microscopia eletrônica (ME), facilitando a sua utilização para análise microscópica correlacionada, que permite combinação de células vivas de rastreamento com microscopia de luz (ML) e maior resolução em nível ultra-estrutural com EM 5. Por exemplo, NPs de ouro têm sido desde há muito tempo utilizado de forma eficiente em células como biossensores para o diagnóstico sensível vivo, bem como no domínio da imuno-6 de rotulagem. S recentestudies indicam que nanopartículas de ouro com diferentes tamanhos e formas podem ser facilmente captação por uma grande variedade de linhas de células e transporte de rotina, através da via endossomal, por conseguinte, ter grande potencial de ser aplicada para o transporte intracelular de seguimento da vesícula e do sistema de rotulagem de endo-7,8 lisossomal . Os agentes patogénicos microbianos, tais como Salmonella enterica, Shigella flexneri e histeria monocytogenes, têm desenvolvido diversos mecanismos para invadir as células hospedeiras não fagocíticas 9. Depois de ser internalizado, os agentes patogénicos, quer localizadas no citosol ou sequestrados nos compartimentos ligados à membrana, interage bastante com os seus ambientes de hospedeiro e modulam estes favorecem a sua própria sobrevivência 10. Por exemplo, Salmonella enterica reside e replica dentro de um compartimento phagosomal intracelular denominada -contendo vacúolo Salmonella (SCV) por infecção 11. O SCV amadurecimentotrafega em direção ao aparelho de Golgi, passando por interações contínuas com a via endocítica, e induz a formação de estruturas tubulares extensas, tais como Salmonella filamentos induzidas (SIF), seleção túbulos nexin, Salmonella transportadora secretora induzida por proteínas de membrana (3) SCAMP3 túbulos, etc . 12-14. Estudar como estas bactérias patogênicas manipular vias de células do hospedeiro é essencial para a compreensão de doenças infecciosas. Aqui, NPs de ouro-BSA-rodamina foram utilizados como marcadores de fluido para rotular o sistema celular hospedeiro endo-lisossomal, e o agente patogénico humano gastrointestinal Salmonella enterica serovar Typhimurium (Salmonella) foi utilizado como um modelo de bactéria para estudar as interacções entre o agente patogénico com o hospedar via endocítica. NPs de ouro-BSA-rodamina intracelulares em células não infectadas e células infectadas com WT ou estirpes mutantes de Salmonella foram fotografadas por um microscópio de varrimento a laser confocal (CLSM).Então Imaris software foi utilizado para quantificar a distribuição de NPs, indicando que a infecção por Salmonella induzida extremo rearranjo dos endossomas / lisossomas. Seguindo a descrição do presente método, as experiências análogas pode ser concebido para controlar destino a longo prazo das NPs internalizadas e para investigar a influência de várias substâncias exógenas ou factores endógenos na via endocítica de células eucarióticas.

<table border="0" cellpadding="0" cellspacing="0" width="684">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:193px;">Name of the Material/Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:149px;">Catalog Number</td>
			<td style="width:176px;">Comments/ Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gold chloride</td>
			<td>Sigma-Aldrich</td>
			<td>520918</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tannic acid</td>
			<td>Sigma-Aldrich</td>
			<td>403040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tri-sodium citrate</td>
			<td>Sigma</td>
			<td>C8532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NHS-Rhodamine</td>
			<td>Pierce</td>
			<td>46406</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO&#160;</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin</td>
			<td>Applichem</td>
			<td>A1492</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamcyin</td>
			<td>Roth</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbenicillin</td>
			<td>Roth</td>
			<td>6344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">8-well chamber slides</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td>tissue culture treated, sterile</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaris Software</td>
			<td>Bitplane</td>
			<td>version 7.6</td>
			<td>various configurations available</td>
		</tr>
	</tbody>
</table>

application of fluorescent nanoparticles to study remodeling of the endo-lysosomal system by intracellular bacteria

Nanopartículas fluorescentes (PN), com desejável propriedades ópticas e mecânicas química, são ferramentas promissoras para rotular organelas intracelulares. Aqui, apresentamos um método que utiliza ouro-BSA-rodamina NPs para rotular o sistema de endo-lisossômico de células eucarióticas e monitorar manipulações de acolhimento vias celulares pelo patógeno intracelular Salmonella enterica. As NPs foram prontamente internalizado pelas células HeLa e localizadas em endossomos tardios / lisossomos. Infecção por Salmonella induzida rearranjo das vesículas e acúmulo de NPs em estruturas de membrana Salmonella- induzidas. Implantamos o pacote de software Imaris para análises quantitativas de imagens de microscopia confocal. O número de objetos e sua distribuição de tamanho em células não infectadas eram distintas das que estão em células infectados por Salmonella, indicando extremamente remodelação do sistema de endo-lysosomal por WT Salmonella.

NPs de ouro foram gerados por meio de um método bem estabelecido através da redução de ácido cloroáurico por citrato e ácido tânico. Como mostrado na Figura 2A, as NPs de ouro foram sintetizados quase-esféricas com um tamanho de aproximadamente 10 nm. BSA-revestimento e rotulagem rhodamine não influenciou sua morfologia ou tamanho (Figura 2B). Tem sido relatado que NPs de ouro pode ser prontamente retirado pelas várias células de mamífero e term...

Watch this Scientific Journal Video about Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria at JoVE.com

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Este artigo descreve métodos para a síntese e marcação fluorescente de nanopartículas (PN). As NPs foram aplicados em experiências de pulse-chase para rotular o sistema de endo-lisossomal de células eucarióticas. A manipulação do sistema de endo-lisossomal por actividades do agente patogénico intracelular, Salmonella enterica foram seguidas de imagem de células vivas e quantificada.

Aplicação de nanopartículas fluorescentes para estudar Reformas do Sistema Endo-lysosomal por bactérias intracelulares

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

application-fluorescent-nanoparticles-to-study-remodeling-endo

Research

JoVE Journal

Immunology and Infection

9.9K visualizações.  Universität Osnabrück.  Este artigo descreve métodos para a síntese e marcação fluorescente de nanopartículas (PN). As NPs foram aplicados em experiências de pulse-chase para rotular o sistema de endo-lisossomal de células eucarióticas. A manipulação do sistema de endo-lisossomal por actividades do agente patogénico intracelular, Salmonella enterica foram seguidas de imagem de células vivas e quantificada. 

Vídeo: Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Detecção de multiplex de bactérias em amostras clínicas e Complexo Ambiental utilizando Oligonucleotídeos-coupled microesferas fluorescentes

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Imunologia e Infecção

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Purificação e visualização de lipopolissacarídeo de bactérias Gram-negativas por extracção aquosa de fenol-Hot

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Medidas de ruptura vacuolar única célula causadas por patógenos intracelulares

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Estabelecimento de um<em&gt; In vitro</em&gt; Sistema para estudar o comportamento intracelular de<em&gt; Candida glabrata</em&gt; Em humanos THP-1 macrófagos

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

Aplicando um sistema de expressão induzível para estudar interferência de fatores de virulência bacteriana com Sinalização Intracelular

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

Desenvolvimento de um sistema de entrega de DNA Economical por &quot;Acufection&quot; e sua aplicação à Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Sistema de Eficácia e citotoxicidade Rastreio de inibidores Segmentação intracelular<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Um novo sistema alimentador-livre para produção em massa de murino Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

Coloração imuno-histoquímica fluorescente multiplexada, imagem e análise de amostras histológicas de linfoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

Modelos de colonização vaginal murina por bactérias cultivadas anaerobicamente

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...