<p class="jove_content"&gt; RN foi parcialmente financiado por uma Marie Curie Europeia concede Re-integração (ERG) do Sétimo Programa-Quadro da Comunidade Europeia, número do projeto 268324.</p&gt;

1. Isolamento dos PMNs a partir de sangue humano por centrifugação de densidade <ol><li> Desenhe 5 9 tubos ml de sangue venoso heparinizado. </li><li> Preparar gradientes de Ficoll dupla camada (4 gradientes para 5 tubos de sangue) do seguinte modo: 12 ml de Despeje a g / ml de solução de 1,119 densidade Ficoll num tubo de 50 ml e cuidadosamente camada de 10 ml de uma g / ml de solução de densidade Ficoll 1.077 no topo. </li><li> Dilui-se o sangue com um volume igual de PBS. </li><li> Camada do sangue diluído cuidadosamente sobre o gradiente de Ficoll de dupla camada; 20-25 ml por gradiente. </li><li> Centrifugar 20 min a 396 x g num rotor de balde oscilante, 22 ° C sem travagem. </li><li> Prepara RPMI frio contendo 0,05% de Albumina de Soro Humano (RPMI-HSA). Também preparar 9 ml estéril deionizada H 2 O. Pré-cool tanto o meio RPMI e H2O em gelo. </li><li> Aspirar a camada de Ficoll topo contendo plasma (de cor amarela) e PBMC, e a segunda camada de Ficoll (cor branca) com a utilização de uma bomba de vácuo (colocar uma ponta de pipeta esterilizada ema pipeta). </li><li> Recolha as PMNs em tubos de 50 ml, utilizando uma pequena pipeta de plástico (1 tubo para as frações PMN de cada 2 gradientes), e colocá-los no gelo. </li><li> Adicionar RPMI-HSA fria até um volume total de 50 ml e centrifugar durante 10 minutos a 249 xg, a 4 ° C. </li><li> Tira-se o sobrenadante com o uso de uma bomba de vácuo e do vortex suavemente o sedimento (eritrócitos e PMN). </li><li> Adicionar 9 ml deionizada estéril H 2 O e iniciar o temporizador. Parar o choque osmótico hiper após 30 seg exatamente, adicionando 1 ml 10 vezes concentrado PBS. Nota: a 30 segundos são muito críticas. </li><li> Adicionar RPMI-HSA fria até um volume total de 50 ml e centrifugar durante 10 minutos a 249 xg, a 4 ° C. </li><li> Tira-se o sobrenadante com o uso de uma bomba de vácuo e recolher o sedimento em 1 PMN tubo com um volume definido (1-2 ml) RPMI-HSA. </li><li> Determinar a quantidade de células e ajustar a concentração de 1,10 a 7 células / ml. Dependendo do dador, o rendimento isolado de PMN estarão entre nípt 5 x 10 6 e 3 x 10 7 PMNs de cada 9 ml tubo de sangue </li></ol> 2. Por citometria de fluxo para avaliação da mobilização de cálcio em PMNs humanos <ol><li> As células de carga (5 x 10 6 células / ml) com 2 uM Fluo-3-AM em RPMI-HSA e incubar durante 20 min à temperatura ambiente lentamente de balanço, utilizando, por exemplo, um agitador de plataforma oscilante, protegido da luz. </li><li> Entretanto preparar uma diluição em série (por exemplo, 3 vezes) do estímulo a 10x a concentração final. PSMα3 é usado neste protocolo, no entanto qualquer estímulo GPCR, que actua sobre os neutrófilos é adequado. </li><li> Prepara 10x concentrado inibidor do receptor em RPMI-HSA. Quando o inibidor actua no estímulo (eg HDL em PSMα3) pré-incubado 25 ul estímulo com um volume igual de inibidor, durante 10 min à temperatura ambiente. </li><li> Lave as células por adição de 10 ml de RPMI-HSA e centrifugar durante 249 xg à temperatura ambiente. Suspenda as células a 5 x10 6 células / ml. </li><li> Imediatamente antes do ensaio, diluir as células a 2x10 6 células / ml em RPMI-HSA e adicionar 200 ul por tubo de células FACS. PMNs diluído são muito frágeis. Mantê-los por muito tempo a esta concentração levará à auto-ativação, o mesmo vale para agitar vigorosa ou pipetagem. </li><li> Fixe o tubo com o citômetro de fluxo, espere 3 segundos e começar a aquisição. Depois de um período de tempo fixo (por exemplo, 8 sec), tire o tubo e adicionar rapidamente 50 mL de estímulo à amostra. Imediatamente voltar a ligar o tubo no suporte da amostra e continuar a aquisição. </li><li> Comece com o menor e terminam com a maior concentração de estímulo. Lava-se a agulha de citómetro de fluxo regularmente após cada ensaio com RPMI-HSA. </li><li> Analisar os dados com software de análise de citometria de fluxo. Comparar o sinal de fluorescência média, antes da adição do estímulo para que, após a adição do estímulo e usar os controlos positivos e negativos adequados para calcular a corrente alternadavação força medida para cada diluição. </li></ol> Alternativamente, quando o inibidor actua sobre os receptores pré-incubar as células com o inibidor. 3. A análise por microscopia de fluorescência de expressão de GFP bactérias após fagocitose por PMNs <ol><li> Crescer S. estirpes de Staphylococcus que contêm uma construção repórter de interesse durante a noite em caldo (com antibióticos quando necessário para manter o plasmídeo repórter). Neste caso MW2 estirpe contendo uma construção repórter da GFP é utilizado 10 PSMα, cultivadas em um tubo de plástico de 50 ml com 5 ml de meio de cultura LB. </li><li> Para remover toda a GFP a partir de O / N expressão, dilui-se as culturas a DO660 de 0,01 e crescer a DO660 de 0,1. Redilute esta cultura 1:30, e monitorar o crescimento até a cultura atingir uma DO660 de 0,1. Recolher as bactérias por centrifugação e lavar uma vez em DPBS. Ressuspender em 1:10 do volume original para se obter uma DO 660 de 1,0 ou roughly 5.10 8 UFC / ml. </li><li> Misturar as bactérias (1,10 7 / ml) com PMN isoladas de fresco (1,10 6 / ml) em RPMI-HSA (proporção de 10:1) num microtubo de 1,5 mL e adicionar soro humano a uma concentração final de 10%. Agitar numa plataforma de agitação durante 10 min a 37 ° C para estimular a fagocitose. Diluir as PMNs carregados com bactérias para 5,10 5 PMN / ml e pipeta de 250 mL em um poço de oito bem compartimentado lamínula. </li><li> Imagem dos PMNs com bactérias em um microscópio. Um microscópio invertido equipado com uma objectiva de NA 40X/0.85 funciona bem e deverá ser encerrada em um ambiente escuro da câmara para manter o ambiente de forma estável, a 37 ° C. Adquirir imagens para uma série de posições pré-definidas utilizando a câmara a cada 5-10 min em ambos os campos luminosos e do canal de GFP para acompanhar a produção da GFP no tempo. </li></ol>

<ol>
	<li>Rigby, K. M., DeLeo, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+in+innate+host+defense+against+Staphylococcus+aureus+infections.">Neutrophils in innate host defense against Staphylococcus aureus infections.</a> Semin. Immunopathol. 34, 237-259 (2012).</li><li>Voyich, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+Mechanisms+Used+by+Staphylococcus+aureus+to+Avoid+Destruction+by+Human+Neutrophils.">Insights into Mechanisms Used by Staphylococcus aureus to Avoid Destruction by Human Neutrophils.</a> The Journal of Immunology. 175, 3907-3919 (2005).</li><li>Kobayashi, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+neutrophil+destruction+following+phagocytosis+of+Staphylococcus+aureus.">Rapid neutrophil destruction following phagocytosis of Staphylococcus aureus.</a> J. Innate Immun. 2, 560-575 (2010).</li><li>DeLeo, F. R., Otto, M., Kreiswirth, B. N., Chambers, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Community-associated+meticillin-resistant+Staphylococcus+aureus.">Community-associated meticillin-resistant Staphylococcus aureus.</a> The Lancet. 375, 1557 (2010).</li><li>David, M. Z., Daum, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Community-Associated+Methicillin-Resistant+Staphylococcus+aureus:+Epidemiology+and+Clinical+Consequences+of+an+Emerging+Epidemic.">Community-Associated Methicillin-Resistant Staphylococcus aureus: Epidemiology and Clinical Consequences of an Emerging Epidemic.</a> Clinical Microbiology Reviews. 23, 616-687 (2010).</li><li>Kretschmer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Formyl+Peptide+Receptor+2+Senses+Highly+Pathogenic+Staphylococcus+aureus.">Human Formyl Peptide Receptor 2 Senses Highly Pathogenic Staphylococcus aureus.</a> Cell Host &amp; Microbe. 7, 463 (2010).</li><li>Prat, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+homolog+of+formyl+peptide+receptor-like+1+(FPRL1)+inhibitor+from+Staphylococcus+aureus+(FPRL1+inhibitory+protein)+that+inhibits+FPRL1+and+FPR.">A homolog of formyl peptide receptor-like 1 (FPRL1) inhibitor from Staphylococcus aureus (FPRL1 inhibitory protein) that inhibits FPRL1 and FPR.</a> J. Immunol. 183, 6569-6578 (2009).</li><li>Prat, C., Bestebroer, J., de Haas, C. J. C., van Strijp, J. A. G., van Kessel, K. P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Staphylococcal+Anti-Inflammatory+Protein+That+Antagonizes+the+Formyl+Peptide+Receptor-Like+1.">A New Staphylococcal Anti-Inflammatory Protein That Antagonizes the Formyl Peptide Receptor-Like 1.</a> The Journal of Immunology. 177, 8017-8026 (2006).</li><li>de Haas, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+inhibitory+protein+of+Staphylococcus+aureus,+a+bacterial+antiinflammatory+agent.">Chemotaxis inhibitory protein of Staphylococcus aureus, a bacterial antiinflammatory agent.</a> The Journal of Experimental Medicine. 199, 687-695 (2004).</li><li>Surewaard, B. G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+Staphylococcal+Phenol+Soluble+Modulins+by+Serum+Lipoprotein+Particles.">Inactivation of Staphylococcal Phenol Soluble Modulins by Serum Lipoprotein Particles.</a> PLoS Pathog. 8, e1002606 (2012).</li><li>Kubica, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Potential+New+Pathway+for+Staphylococcus+aureus+Dissemination:+The+Silent+Survival+of+S.+aureus+Phagocytosed+by+Human+Monocyte-Derived+Macrophages.">A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages.</a> PLoS ONE. 3, e1409 (2008).</li><li>Surewaard, B. G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcal+alpha-phenol+soluble+modulins+contribute+to+neutrophil+lysis+after+phagocytosis.">Staphylococcal alpha-phenol soluble modulins contribute to neutrophil lysis after phagocytosis.</a> Cellular Microbiology. , (2013).</li><li>Queck, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAIII-Independent+Target+Gene+Control+by+the+agr+Quorum-Sensing+System:+Insight+into+the+Evolution+of+Virulence+Regulation+in+Staphylococcus+aureus.">RNAIII-Independent Target Gene Control by the agr Quorum-Sensing System: Insight into the Evolution of Virulence Regulation in Staphylococcus aureus.</a> Molecular Cell. 32, 150 (2008).</li><li>Baban, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+Bacterial+Imaging+In+Vivo.">Bioluminescent Bacterial Imaging In Vivo.</a> J. Vis. Exp. (69), e4318 (2012).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

Nos métodos aqui descritos alguns passos são muito crítico. Vamos destacar estes aqui. Para o isolamento dos neutrófilos, por centrifugação em gradiente de densidade é importante para não perturbar as camadas, durante ou após os passos de centrifugação. Quando a aspiração dos neutrófilos utilizando uma pipeta de plástico, certifique-se de não apertar o balão, enquanto na camada de células, tal como ejectar líquido irá perturbar as camadas. Além disso, inspecione visualme...

Os neutrófilos (PMN) são fagócitos profissionais que desempenham um papel chave na resposta imune inata contra Staphylococcus aureus 1. A constante batalha entre o anfitrião eo micróbio levou a uma corrida armamentista de ambos. Recentemente, comunidade-associado (CA) das cepas de S. meticilina resistente aureus (MRSA) surgiram que parecem ser muito eficiente na neutralização de neutrófilos assassinato 2,3. Modulin produção excessiva solúvel fenol (PSMs) de MRSA-CA foi associada a uma maior virulência 4,5. Os neutrófilos humanos pode reconhecer estas PSMs através FPR2 que levam à activação deste acoplado à proteína G do receptor 6. Um dos primeiros eventos é a mobilização das reservas intracelulares de cálcio (Ca2 +). Ca 2 + actua como um mensageiro secundário para uma variedade de funções efectoras incluindo desgranulação de PMN e fagocitose 7. Portanto Ca2 + é um indicador muito sensível dacapacidade funcional dos PSMs para ativar PMNs. Para estudar os efeitos dos PSMs para os neutrófilos, os neutrófilos são isolados frescos e carregado com um corante que fluoresce na mobilização do cálcio. Numa experiência de citometria de fluxo, este rápido mobilização pode ser medido. Usando este método, é possível estudar os efeitos directos de componentes tóxicos e de outros para os neutrófilos, e determinar a concentração mais baixa à qual estes estão activas. Para nós, é uma ferramenta muito útil para estudar o efeito de muitas proteínas produzidas por S. aureus envolvidos na evasão imune, como FPR2 proteína inibidora (FLIPR) 8, FLIPR-like 7 e Quimiotaxia proteína inibidora de Staphylococcus aureus (CHIPS) 9. Todas estas proteínas foram mostradas para inibir a mobilização de cálcio no número de neutrófilos por ligação ao receptor que reconhece o agonista. Recentemente, o nosso grupo descrito que os PSMs estão funcionalmente inibida por lipoproteínas séricas 10 &lt;/ Sup&gt;. Estas lipoproteínas estão abundantemente presentes no sangue e no tecido humano, o que indica que os PSMs exercer a sua função, principalmente no meio intracelular. A disponibilidade do ensaio de mobilização do cálcio nos permitiu medir com precisão o efeito de lipoproteínas do soro sobre a activação dos neutrófilos por os PSMs, indicado pela inibição considerável por concentrações muito baixas de soro. Uma vez que os PSMs estão funcionalmente inibida por soro, a hipótese de que existe uma função importante para os PSMs como toxinas intracelulares. Por isso, procuraram determinar o papel de PSMs após a fagocitose. Para investigar a expressão dos PSMs no espaço intercelular, construímos fusões repórter do promotor do operão psmα para GFP. Quando essas cepas repórter de S. aureus foram fagocitados pelos neutrófilos, indução de expressão foi observado por microscopia de fluorescência de 10. Obviamente, esta technique permite o estudo da expressão de um grande número de genes em S. aureus ou outros agentes patogénicos depois de fagocitose. Uma vez que para S. aureus sobreviver no nicho intercelular é muito importante para superar o sistema imune inato 11 10 12, estudando o papel dos genes ativados neste nicho é altamente relevante para a compreensão de sua virulência.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Fluo-3, AM</td>
			<td>Molecular Probes / Life Technologies</td>
			<td>F-1241</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ficoll-Paque</td>
			<td>GE Healthcare</td>
			<td>17-5442-03</td>
			<td>density 1.077 g/ml</td>
		</tr>
		<tr>
			<td>Histopaque</td>
			<td>Sigma</td>
			<td>11191</td>
			<td>density 1.119 g/ml</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco, Life Technologies</td>
			<td>52400-025</td>
			<td>contains 25 mM HEPES and L-glutamine</td>
		</tr>
		<tr>
			<td>Leica TCS SP5 microscope</td>
			<td>Leica Microsystems, The Netherlands</td>
			<td>TCS SP5</td>
			<td>objective: HCX PL APO 40X/0.85</td>
		</tr>
		<tr>
			<td>FACSCalibur</td>
			<td>BD Biosciences</td>
			<td>FACSCalibur</td>
			<td>Very important that the tube can be removed and replaced during the measurement process</td>
		</tr>
	</tbody>
</table>

studying interactions of staphylococcus aureus with neutrophils by flow cytometry and time lapse microscopy

Nós apresentamos métodos para estudar o efeito de modulins solúveis fenol (PSMs) e outras toxinas produzidas e secretadas por Staphylococcus aureus, nos neutrófilos. Para estudar os efeitos dos PSMs de neutrófilos nos isolar neutrófilos frescos utilizando centrifugação em gradiente de densidade. Estes neutrófilos são carregadas com um corante que fluoresce na mobilização do cálcio. A activação dos neutrófilos por PSMs inicia um aumento rápido e transitório na concentração de cálcio intracelular livre. Numa experiência de citometria de fluxo, este rápido mobilização pode ser medida por monitorização da fluorescência de um corante pré-carregada, que reage com o aumento da concentração de Ca2 + livre. Usando esse método, podemos determinar a concentração PSM necessário para ativar os neutrófilos, e medir os efeitos de inibidores específicos e gerais da ativação de neutrófilos. Para investigar a expressão dos PSMs no espaço intracelular, we foram construídas fusões repórter do promotor do operão PSMα para GFP. Quando essas cepas repórter de S. aureus são fagocitados pelos neutrófilos, a indução da expressão pode ser observada por meio de microscopia de fluorescência.

Citometria de fluxo para análise da mobilização de cálcio em PMNs humanos A incubação de neutrófilos com uma série de concentração de PSMα3 sintéticos, resultando em activação rápida medida pelo fluxo de cálcio, o que é mostrado por um aumento no sinal em FL-1. Pré-incubação de PSMα3 sintético com 0,01%, 0,1% ou 1% de soro humano inibiu significativamente a capacidade de provocar fluxos de cálcio (Figura 1). Análise da expressã...

Watch this Scientific Journal Video about Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy at JoVE.com

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

Nós apresentamos métodos para estudar o efeito de PSMs e outras toxinas segregadas por<em&gt; Staphylococcus aureus</em&gt; Em neutrófilos utilizando citometria de fluxo e microscopia de fluorescência.

Estudo das interacções de<em&gt; Staphylococcus aureus</em&gt; Com neutrófilos por citometria de fluxo e Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

studying-interactions-staphylococcus-aureus-with-neutrophils-flow

Research

JoVE Journal

Immunology and Infection

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

13.0K visualizações.  University Medical Center Utrecht. Nós apresentamos métodos para estudar o efeito de PSMs e outras toxinas segregadas por Staphylococcus aureus Em neutrófilos utilizando citometria de fluxo e microscopia de fluorescência.

Vídeo: Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

O uso do fluxo de gotejamento e Reatores de disco rotativo para Staphylococcus aureus Análise de biofilme

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Imunologia e Infecção

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

Infecção subcutânea de Staphylococcus aureus resistente (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

Staphylococcus aureus usando hemoglobina humana como uma fonte de ferro

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

Um método simples e eficiente para detectar ativação do fator nuclear em neutrófilos humanos por citometria de fluxo

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Detecção de nanopartículas fluorescentes Interações com Primário imunitário subpopulações de células por citometria de fluxo

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imagiologia neutrófilos e monócitos em Mesenteric Veins por Microscopia intravital em ratos anestesiados em Tempo Real

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

genotipagem de<em&gt; Staphylococcus aureus</em&gt; por Ribossômico Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Metodologia para o Estudo da transferência horizontal de genes em<em&gt; Staphylococcus aureus</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

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 ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Um modelo do rato para avaliar a resposta imune inata à infecção de Staphylococcus aureus

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...