Nós gostaríamos de agradecer Mathias S. Dick, Roland F. Dreier e Sebastian Rühl para discussão. Este trabalho foi apoiado por uma SNSF Professorship PP00P3_139120 / 1 e uma Universidade de Basel concessão projeto ID2153162 para PB

1. Digitonin Ensaio com mCherry expressando Salmonella (Análise baseada em FACS) <ol><li> Preparando bactérias Nota: Salmonella de tipo selvagem SL1344 (resistente à estreptomicina) expressando mCherry partir de um plasmídeo (pFPV mCherry que codifica sob o promotor rpsM) é usado neste ensaio. Fagocitose bacteriana e proliferação onde não alterada pela expressão constitutiva de mCherry (dados não mostrados) 16. <ol><li> Inocular a estirpe 1 dia antes da infecção em 3 ml de meio LB suplementado com estreptomicina (90 ug / ml) e ampicilina (100 ug / ml, mCherry vector de expressão) num agitador a 37 ° CO / N. </li></ol></li><li> Chapeamento de células para a infecção. <ol><li> Semente de tipo selvagem BMDMs em uma placa de 24 poços a uma densidade de 1,25 x 10 5 células / poço em 1 ml de meio de macrófagos primários (DMEM suplementado com 10% de Soro Fetal de Vitela (FCS, inactivado pelo calor, a 56 ° C, 30 min ), 1% de HEPES, 1% de aminoácidos não-essenciais (NEAA), 1% de L-glutamina e 10% L929 sobrenadante (meio condicionado a partir de macrófagos factor estimulador de colónias (M-CSF), produzindo células L929). Poços de sementes de acordo com a Tabela 1 para ter em conta as condições individuais. Adicionar lamelas aos poços utilizados como controlos (Tabela 1). </li><li> Incubar as células durante a noite numa incubadora a 37 ° C e 5% de CO 2. </li></ol></li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td></td><td></td><td colspan="2"> Permeabilização </td><td colspan="3"> Coloração </td></tr><tr><td></td><td> Lamela de vidro </td><td> Salmonella </td><td> Digitonin </td><td> Saponin </td><td> anti-Salmonella </td><td> anti-Calnexina </td><td> anti-PDI </td></tr><tr><td> A1 (amostra) </td><td> - / + * </td><td> + </td><td> + </td><td></td><td> + </td><td></td><td></td></tr><tr><td> A2 (amostra) </td><td> - / + * </td><td> + </td&gt; <td> + </td><td></td><td> + </td><td></td><td></td></tr><tr><td> A3 (amostra) </td><td> - / + * </td><td> + </td><td> + </td><td></td><td> + </td><td></td><td></td></tr><tr><td> A4 (sem controle de permeabilização) </td><td> - / + * </td><td> + </td><td></td><td></td><td> + </td><td></td><td></td></tr><tr><td> A5 (controle completo permeabilização) </td><td> - / + * </td><td> + </td><td></td><td> + </td><td> + </td><td></td><td></td></tr><tr><td> B1 (coradas para calnexina) </td><td> + </td><td></td><td></td><td></td><td></td><td> + </td><td></td></tr><tr><td> B2 (coradas para calnexina) </td><td> + </td><td></td><td> + </td><td></td><td></td><td> + </td><td></td></tr><tr><td> B3 (coradas para calnexina) </td><td> + </td><td></td><td></td><td> + </td><td></td><td> + </td><td></td></tr><tr><td> C1 (manchadopara PDI) </td><td> + </td><td></td><td></td><td></td><td></td><td></td><td> + </td></tr><tr><td> C2 (coradas para PDI) </td><td> + </td><td></td><td> + </td><td></td><td></td><td></td><td> + </td></tr><tr><td> C3 (coradas para PDI) </td><td> + </td><td></td><td></td><td> + </td><td></td><td></td><td> + </td></tr></tbody></table> Tabela 1: Regime de chapeamento de medula óssea macrófagos derivados (BMDMs) num formato de 24 poços para a infecção subsequente com Salmonella, permeabilização e coloração * Dependendo se um protocolo ou protocolo 2 é feito.. <ol start="3"><li> Preparando as bactérias para a infecção. <ol><li> No dia seguinte, determinar a concentração de Salmonella em cultura durante a noite por diluição da cultura a 1:10 em PBS e medindo a OD 600 de encontro a um único controlo PBS. Isto assegura que a OD 600 é medida na lgama inear do espectrofotômetro. </li><li> Calcular a concentração de salmonelas por ml. Uma OD600 de 1 corresponde a 1 x 10 9 bactérias. </li><li> Dilui-se as bactérias em forma de macrófagos para atingir uma concentração de Salmonella de 1,25 x 10 6 células / ml. </li></ol></li><li> A infecção de células com Salmonella. <ol><li> Remover o meio de BMDMs. Adicionar 1 ml de meio aos poços de macrófagos de controlo não infectados (B1-B3, C1-C3). Adicionar 1 ml de suspensão de Salmonella poços A1 - A5 a chegar a uma multiplicidade de infecção (moi) de 10 bactérias por célula. </li><li> Centrifugar a placa durante 15 minutos a 300 xg a 37 ° C para sincronizar a infecção. Transferir a placa numa incubadora a 37 ° C e 5% de CO 2. </li></ol></li><li> Matar Salmonella extracelular com gentamicina. <ol><li> A 1 h pós-infecção, remova a placa da incubadora e adicionar 0,1 ml de meio de macrófagos contendo 0,1 mg / ml de gentamicinapara matar as bactérias extracelulares. Adicionar gentamicina a todas as cavidades, mesmo com os controlos não infectados, para assegurar que todas as células são tratadas da mesma maneira. Transferir a placa numa incubadora a 37 ° C e 5% de CO 2. </li></ol></li><li> Lavar as células. <ol><li> Às 2 h após a infecção, remover placa da incubadora e lavar os poços 2x com 1 ml de DMEM simples (pré-aquecido a 37 ° C) e substituí-lo por meio de macrófagos (pré-aquecido a 37 ° C) contendo 10 ug / ml &#160; Gentamicina para prevenir o crescimento de todas as bactérias extracelulares restantes. Transferir a placa numa incubadora a 37 ° C e 5% de CO 2. </li></ol></li><li> Preparando padrões frescos com detergentes e anticorpos. <ol><li> Imediatamente antes do ponto de tempo de análise desejado, preparar uma solução de reserva fresca de digitonina a 1 mg / ml em tampão de KHM (acetato de potsio 110 mM, HEPES 20 mM, 2 mM de MgCl2, pH 7,3). Filtrar o estoque e diluir em KHM tampão para uma concentração de trabalhode 50 ug / ml de digitonina. Além disso, preparar uma solução de saponina a 0,1% em tampão de KHM, anticorpo anti- Salmonella acoplado a FITC (CSA-1-FITC) a 1/500, anti-calnexina a 1/100 ou anti-PDI a 1/100 em KHM tampão suplementado com 3% de BSA. </li></ol></li><li> Permeabilização celular. <ol><li> Retire a placa da incubadora e lavar os poços 3x com 0,5 ml de tampão de KHM (pré-aquecido a 37 ° C). Remover KHM tampão e adicionar 0,25 ml planície KHM buffer para poços A4 e B1 / C1, KHM tampão com digitonina para poços A1 - A3 e B2 / C2 ou KHM tampão com saponina para poços A5 e B3 / C3 (de acordo com a Tabela 1). Incubar durante exactamente 1 min à temperatura ambiente e imediatamente lavar os poços 3x com 0,5 ml de tampão de KHM (pré-aquecido a 37 ° C). </li></ol></li><li> Coloração de anticorpo primário. <ol><li> Remover o tampão de KHM e adicionar as soluções de anticorpos primários (anticorpo de cabra anti -FITC Salmonella, 250 ul / poço, 0,1 ug / ml) aos poços apropriados (Figura 1 </strong&gt;, Tabela 1). Incubar a placa durante 15 minutos num incubador a 37 ° C e 5% de CO 2. </li><li> Lavam-se as células com 1 ml de 1x PBS. </li></ol></li><li> As células infectadas (poços A1 - A5): análise FACS <ol><li> Lavar as células 3x com PBS e adicionar 0,5 ml de PBS gelado contendo 0,1% de Triton. Transferir para um tubo de 5 ml com tampão FACS coador de células para romper os agregados de células. Manter as amostras em gelo. </li><li> Analisar as amostras de um FACS. Usando os controles totalmente permeabilizadas, defina a porta para as bactérias totais com base na dispersão frontal e lateral (FSC / SSC) em primeiro lugar, eo sinal mCherry (610 nm ± 20 nm filtro). </li><li> Em seguida, analisar o sinal FITC na população bacteriana total usando os controles permeabilizadas totalmente permeabilizadas e não para definir as portas para as bactérias citossólicos (FITC +, mCherry +) e bactérias vacuolares (FITC, mCherry +) (Figura 3). </li><li> Com os portões definido, analisar as amostras e determinar a percentagem de vs citosólicavacuolares bactérias que utilizam software FlowJo de acordo com o protocolo do fabricante. </li></ol></li><li> Permeabilização controles não infectados (poços B1 - B3, C1 - C3): microscopia <ol><li> Fixação <ol><li> Remover PBS e adicionar 250 ul de PFA a 4% em PBS. Incubar durante 10 min a 37 ° C. </li><li> Lavar os poços 2x com 1 ml de PBS e adicionar 250 ul de 0,1 M de glicina em PBS durante 10 minutos para extinguir o fixador. Lavar os poços 2x com 1 ml de PBS. </li></ol></li><li> Coloração com anticorpo secundário. <ol><li> Controlos permeabilização da mancha por 1 hora à TA com anticorpos secundários acoplados com fluoróforos adequados em PBS suplementado com 0,1% de saponina e BSA a 3% para permeabilizar células completamente. </li><li> Lavar 3x lamelas com 1 ml de PBS e montar as lamelas para análise no meio de montagem com DAPI (1,5 ug / mL). </li></ol></li><li> Análise de microscopia. <ol><li> Analisar as lamelas com os controles em um aumento de 40 vezes 63X ou usando um fluo confocalmicroscópio rescência. Se a permeabilização tem trabalhado, não deve haver nenhuma coloração para células não-permeabilizadas, coloração Calnexina para ambas as células digitonin- e Saponina-permeabilizadas e PDI coloração apenas para células permeabilizadas-saponina (Figura 4). </li></ol></li></ol></li></ol> 2. Digitonin Ensaio com Unlabeled Salmonella (Análise baseada em Microscopia) <ol><li> Preparando bactérias para o / n culturas. <ol><li> Inocular não marcado Salmonella de tipo selvagem SL1344 (resistente à estreptomicina) 1 dia antes da infecção em 3 ml de meio LB suplementado com estreptomicina (90 ug / ml) num agitador a 37 ° CO / N. </li><li> Chapeamento de células para a infecção. <ol><li> BMDMs semente em uma placa de 24 poços contendo lamelas de vidro esterilizadas a uma densidade de 1,25 x 10 5 células / poço em 1 ml de meio de macrófagos (DMEM suplementado com 10% de Soro Fetal de Vitela (FCS, complementado-de, 56 ° C,30 min), 1% de HEPES, 1% de aminoácidos L-aminoácidos não-essenciais (NEAA), 1% de L-glutamina e 10% de sobrenadante L929). Poços de sementes de acordo com a Tabela 1 para ter em conta as condições individuais. </li><li> Incubar as células S / N em uma incubadora a 37 ° C e 5% de CO 2. </li></ol></li><li> Preparando as bactérias para a infecção. <ol><li> No dia seguinte, determinar a concentração de Salmonella na cultura D / N por diluição da cultura a 1:10 em PBS e medindo a OD 600 de encontro a um único controlo PBS. Isto assegura que a OD 600 é medida na gama linear do espectros otómetro. </li><li> Determinar a concentração de Salmonella por mililitro. Uma OD600 de 1 corresponde a 1 x 10 9 bactérias. </li><li> Dilui-se as bactérias em forma de macrófagos para atingir uma concentração de Salmonella de 1,25 x 10 6 células / ml. </li></ol></li><li> A infecção de células com Salmonella. <ol&gt; <li> Remover o meio de todos os poços. Adicionar 1 ml de meio de macrófagos claro para poços de controlo não infectados (B1 - B3, C1 - C3). Adicionar 1 ml de suspensão de Salmonella poços A1 - A5 a chegar a uma multiplicidade de infecção (moi) de 10 bactérias por célula. </li><li> Centrifugar a placa durante 15 minutos a 300 xg a 37 ° C para sincronizar a infecção. Transferir a placa numa incubadora a 37 ° C e 5% de CO 2. </li></ol></li><li> Matar Salmonella extracelular com gentamicina. <ol><li> A 1 h pós-infecção, remova a placa da incubadora e adicionar 0,1 ml de meio contendo macrófagos 1 mg / ml de gentamicina para matar bactérias extracelulares para todos os poços. Transferir a placa numa incubadora a 37 ° C e 5% de CO 2. </li></ol></li><li> Lavar as células. <ol><li> Às 2 h pós-infecção, remova a placa da incubadora e cada poço lavar 3x com 1 ml de meio fresco contendo macrófagos 10 ng / mL Gentamicina para evitaro crescimento de todas as bactérias extracelulares restantes. Transferir a placa numa incubadora a 37 ° C e 5% de CO 2. </li></ol></li><li> Preparando padrões frescos com detergentes e anticorpos. <ol><li> Imediatamente antes do ponto de tempo de análise desejado, preparar uma solução de reserva fresca de digitonina a 1 mg / ml em tampão de KHM (acetato de potsio 110 mM, HEPES 20 mM, 2 mM de MgCl2, pH 7,3). Filtra-se o estoque e diluir em tampão de KHM para uma concentração de trabalho de 50 ug / ml de digitonina. </li><li> Além disso, preparar uma solução de saponina a 0,1% em tampão de KHM, anti- Salmonella -FITC (CSA-1, 0,1 ug / ml), anti-calnexina a 1/100 ou anti-PDI a 1/100 em tampão de KHM suplementado com BSA a 3% (ver tabela materiais para a concentração de anticorpo). </li></ol></li><li> Permeabilização celular. <ol><li> Retire a placa da incubadora e lavar os poços 3x com 0,5 ml de tampão de KHM (pré-aquecido a 37 ° C). Remover KHM tampão e adicionar 0,25 ml planície KHM bufferpara poços A4 e B1 / C1, KHM tampão com digitonina para poços A1 - A3 e B2 / C2 ou KHM tampão com saponina para poços A5 e B3 / C3 (de acordo com a Tabela 1). Incubar durante exactamente 1 min à temperatura ambiente e imediatamente lavar os poços 3x com 0,5 ml de tampão de KHM (pré-aquecido a 37 ° C). </li></ol></li><li> Coloração de anticorpo primário. <ol><li> Remover o tampão de KHM e adicionar as soluções de anticorpos primários (anticorpo de cabra anti-FITC Salmonella AST1, 250 ul / poço, 0,1 ug / ml) aos poços apropriados (Figura 2, Tabela 1). Incubar a placa durante 15 minutos num incubador a 37 ° C e 5% de CO 2. Lavar 3x com 1 ml de PBS. </li></ol></li><li> Fixação. <ol><li> Remover PBS e adicionar 250 ul de PFA a 4% em PBS. Incubar durante 10 min a 37 ° C. </li><li> Lavar os poços 2x com 1 ml de PBS e adicionar 250 ul de 0,1 M de glicina em PBS durante 10 minutos para extinguir o fixador. Lavar os poços 2x com 1 ml de PBS. </li></ol></li><li> TotalSalmonella coloração de amostras infectadas. <ol><li> Lavar lamelas 3x com PBS com 0,1% de saponina / BSA a 3% e incubar as amostras com um anticorpo anti-AST1 (de cabra anti-AST1, 0,1 ug / ml, de ratinho anti-LPS funciona bem, 0,1 ug / ml) durante 1 hora à TA em PBS com 0,1% de saponina / BSA a 3%. </li><li> Lavar 3x em PBS com 0,1% de saponina / BSA a 3%. Incubar as amostras com um anticorpo secundário anti-cabra acoplado a escolha do fluoróforo em PBS com 0,1% de saponina / 3% de BSA durante 30 min à temperatura ambiente no escuro. </li><li> Lavar 3x lamelas com 1 ml de PBS e montar as lamelas para análise no meio de montagem com DAPI (1,5 ug / mL). </li></ol></li><li> Análise de microscopia. <ol><li> Aquisição de dados com um microscópio confocal por contagem de Salmonella citosólica (anti- Salmonella -FITC positivo) e Salmonella total (duplo positivo) (Figura 5). Controles permeabilização deve mostrar: nenhuma coloração para células não permeabilizadas, Calnexina stenção para células permeabilizadas-digitonina e coloração PDI para células permeabilizadas-saponina (Figura 4). Wells A4 e A5 vai servir como controles internos para a coloração de Salmonella. </li></ol></li></ol></li></ol>

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We have nothing to disclose.

Diferencial de permeabilização é um método fácil e robusto para analisar e quantificar a distribuição subcelular de agentes patogénicos bacterianos entre compartimentos vacuolares e o citosol. O mesmo ensaio foi usado com sucesso com bactérias tais como Francisella novicida 2,4 e 12 Shigella flexneri. No entanto, uma vez que muitos patógeno intracelular alterar ou modificar as estruturas de acolhimento de Endomembrane, a robustez da permeabilização digitonina terá de ...

Agentes patogénicos bacterianos intracelulares replicar quer directamente no citosol da célula hospedeira, ou em compartimentos vacuolares especializados 1. Durante a fase inicial da infecção a maioria dos patógenos são internalizadas por fagocitose ou por células especializadas (tais como macrófagos) ou por promover activamente a sua própria absorção em células não fagocíticas. Em células fagocíticas, o fagossoma funde normalmente com lisossomas para formar um compartimento de degradação, em que as partículas são digeridas fagocitados. Bactérias citosólicas especializados, tais como Francisella novicida ou Shigella flexneri, escapar degradação phagosomal por induzir a ruptura do fagossoma e subsequentemente escapar para o citosol 2,3. Isso requer associada a virulência mecanismo como a ilha de patogenicidade Francisella (FPI) ou a Shigella flexneri T3SS, que injeta proteínas efetoras que promovem a ruptura vacuolar 2-4. As características citosol da célula hospedeiraum número de receptores de reconhecimento de padrões conservados que normalmente reconhecem a presença de patógenos e induzem imune inata sinalização 5. Além disso, xenophagy, uma forma anti-microbiana de autofagia pode degradar bactérias que entram no citosol. Citossólicos bactérias são normalmente bem adaptado para diminuir ou burlar estas respostas usando estratégias diferentes. Por exemplo, Francisella modifica suas LPS para evitar o reconhecimento de acolhimento e Shigella impede recrutamento autofagia utilizando proteínas efetoras secretadas 6,7. Outra estratégia para escapar degradação lisossomal é empregado pelo modelo vacuolar patógeno Salmonella enterica sorotipo Typhimurium, a seguir referido como Salmonella typhimurium. Salmonella usa um T3SS para injetar effectors que remodelar o phagosome inicial em seu nicho intracelular, a Salmonella -contendo vacúolo (SCV) 8,9. Manipulação contínua de vias de acolhimento énecessária para manter este vacúolo recrutando lípidos e outros nutrientes para o vacúolo. De facto, um mutante de Salmonella sifa falha para manter a estabilidade vacuolar, e entra no citosol das células hospedeiras em questão de horas após a infecção, o que resulta na activação de vias imunes inatas e recrutamento autofagia 8. Fuga vacuolar de Salmonella varia dependendo do tipo de célula que é estudos, e vários relatórios demonstraram que em células epiteliais mesmo WT Salmonella pode escapar do VCE e hyperreplicate no citosol 10. Relatórios recentes mostram que a detecção de agentes patogénicos imune inata vacuolares também depende da capacidade do hospedeiro em reconhecer e desestabilizar vacúolos contendo agentes patogénicos PCVs (11-14). Dado que tanto o hospedeiro ou bactérias genótipo pode afectar a distribuição de bactérias intracelulares entre o vacuolar e compartimento citosólico, é necessário para quantificar o número de vacuolar vs.bactérias citosólicas. Uma vez que, em configurações experimentais mais células hospedeiras são infectados com mais do que uma bactéria e, subsequentemente, pode abrigar várias bactérias em diferentes compartimentos subcelulares, uma técnica é necessário que permite a quantificação do nível de bactérias individuais. Permeabilização diferencial (também conhecido como teste de protecção phagosomal) fornece essa resolução 2,4,10,12. O ensaio é baseado na permeabilização selectiva da membrana plasmática da célula hospedeira com o detergente digitonina, o qual deixa intacta membranas vacuolares e, assim, permite que a coloração selectiva de bactérias citosólicas com anticorpos. Aqui nós fornecemos dois protocolos para a quantificação de cytosolic e vacuolar Salmonella typhimurium usando diferencial permeabilização. O princípio deste método é descrita na Figura 1: macrófagos da medula óssea derivadas (BMDMs) são infectadas com fase estacionária mCherry-expressando Salmonella. Fase estacionária Salmonella precisa tO ser utilizados porque regular negativamente a expressão do SPI-1 T3SS, cuja actividade de outro modo seriam reconhecidos pelo inflammasome NLRC4 e induzir a morte celular rápida (pyroptosis) do BMDMs 15. Após a infecção as células são lavadas e tratadas com 50 ug / ml de digitonina para 1 minuto. As células são lavadas mais uma vez e imediatamente incubadas com anticorpos anti- Salmonella acoplado a FITC para marcar as bactérias com o acesso ao citosol. Após um passo de lavagem adicional células são lisadas e a percentagem das bactérias mCherry + (vacuolar) e FITC + / + mCherry (citosólica) é determinada por FACS. Nós também relatam uma adaptação deste método que pode ser utilizado se não há estirpes que expressam proteínas fluorescentes estão disponíveis (Figura 2). Os passos adicionais são introduzidas após a marcação com FITC, em que as células são fixadas e permeabilizadas completamente. Posteriormente, todas as bactérias são coradas com anticorpos anti-Salmonella e anticorpos secundários correspondentes. Detec ç ã o é em seguida feito por microscopia, em vez de análise de FACS.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Digitonin</td>
			<td>Sigma</td>
			<td>D5628</td>
			<td>50ug/mL</td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>mpbio</td>
			<td>219998380</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Life Technologies</td>
			<td>15630</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Acetate</td>
			<td>Sigma</td>
			<td>791733</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Salmonella CSA-1-FITC</td>
			<td>KPL</td>
			<td>01-91-99-MG</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>anti-Salmonella CSA-1</td>
			<td>KPL</td>
			<td>02-91-99-MG</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>anti-Calnexin</td>
			<td>Enzo Lifesciences</td>
			<td>SPA-860D</td>
			<td>1/100</td>
		</tr>
		<tr>
			<td>anti-PDI</td>
			<td>Enzo Lifesciences</td>
			<td>SPA-890</td>
			<td>1/100</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vectorlabs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti Rabbit antibody-488</td>
			<td>Molecular Probes</td>
			<td>A-11070</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Life Technologies</td>
			<td>15710-49</td>
			<td>100ug/mL and 10ug/mL</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Promega</td>
			<td>H5141</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>20012-019</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Amimed</td>
			<td>5-13K00-H</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>imaging done at 63x</td>
		</tr>
		<tr>
			<td>FACS Fortessa</td>
			<td>BD technologies</td>
			<td></td>
			<td>detection mCherry 610 nm</td>
		</tr>
		<tr>
			<td>FACS Fortessa</td>
			<td>BD technologies</td>
			<td></td>
			<td>detection FITC 530 nm</td>
		</tr>
	</tbody>
</table>

quantification of cytosolic vs. vacuolar salmonella in primary macrophages by differential permeabilization

Agentes patogénicos bacterianos intracelulares podem replicar no citosol ou no vacúolo contendo agentes patogénicos especializados (PCVs). Para atingir o citossol, bactérias como Shigella flexneri e Francisella novicida precisa para induzir a ruptura do fagossoma. Em contraste, Salmonella typhimurium replica num compartimento vacuolar, conhecido como Salmonella vacúolo molecular contendo (SCV). Todavia, certos mutantes de Salmonella não conseguem manter a integridade do VCE e são, assim, libertado para o citosol. A percentagem de bactérias citosólica vs vacuolares sobre o nível de bactérias individuais podem ser medidos por permeabilização diferencial, também conhecido como ensaio de protecção fagossoma. A abordagem faz uso da propriedade de detergente digitonina para se ligar seletivamente o colesterol. Uma vez que a membrana de plasma contém mais colesterol do que outras membranas celulares, digitonina pode ser usado selectivamente para permeabilizar a membrana plasmática, enquanto deixando as membranas intracelularesintacta. Em resumo a seguir à infecção, com o agente patogénico expressando uma proteína marcador fluorescente (por exemplo mCherry entre outros), a membrana plasmática de células hospedeiras é permeabilizadas com uma curta incubação em tampão contendo digitonina. As células são então lavadas e incubadas com um anticorpo primário (acoplado a um fluoróforo de escolha) dirigido contra a bactéria de escolha (por exemplo, anti- -FITC de Salmonella) e lavou-se novamente. Se forem utilizadas as bactérias não marcados, um passo adicional pode ser feito, em que todas as membranas são permeabilizadas e todas as bactérias coradas com um anticorpo correspondente. Após a coloração, a percentagem de vacuolares e citosólicos bactérias podem ser quantificados por microscopia de FACS ou por contagem de eventos simples ou duplos positivos. Aqui nós fornecemos detalhes experimentais para a utilização desta técnica com a bactéria Salmonella typhimurium. A vantagem deste teste é que, em contraste com o outro ensaio, que fornece uma quantificação do nível de único bactericidaRia, e se analisou por microscopia fornece o número exacto de citosólica e bactérias vacuolares numa dada célula.

Figura 1 e Figura 2 mostra o esquema do ensaio digitonina descritos no protocolo 1 e 2 protocolo ilustra as etapas críticas do protocolo e os resultados obtidos. A Figura 3 mostra os resultados típicos de FACS. Os controlos positivos e negativos são usados ​​para definir as portas para mCherry + / bactérias FITC (vacuolar Salmonella) e mCherry + / FITC + bactérias (cytosolic Salmonella). Com base nestes portões a percentagem de bactérias citosólicas e vacuolare...

Watch this Scientific Journal Video about Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization at JoVE.com

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Quantificação de citosólico vs vacuolar<em&gt; Salmonella</em&gt; Em macrófagos primários por Permeabilização Diferencial

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

quantification-cytosolic-vs-vacuolar-salmonella-primary-macrophages

Research

JoVE Journal

Immunology and Infection

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

10.8K visualizações.  University of Basel. We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Vídeo: Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

Medição do Ca citosólico 2 + Em Isolated linfáticos contráteis

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

Imunologia e Infecção

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 ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

De alta capacidade: Ensaio para Fenótipo<em&gt; Salmonella enterica</em&gt; Typhimurium Association, invasão e replicação em macrófagos

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

A transfecção altamente eficiente de THP-1 por macrófagos humanos Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

Proteômica Profiling de macrófagos por eletroforese 2D

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

Migração fagossomo e velocidade medida no Live primárias macrófagos humanos infectados com o HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Caracterização fenotípica dos macrófagos a partir de rim de rato por Citometria de Fluxo

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento de Salmonella typhimurium-contendo Phagosomes de macrófagos

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Time-Lapse imagem em 3D da fagocitose por macrófagos de rato

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

Quantificação de realce dependente de anticorpo do vírus nas células humanas primárias Zika

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...