Esta pesquisa foi apoiada em parte pelo NIH prêmios 5T32AI07506, 5R01AI033774, 5R37AI033142, 5R01AI052733.

Todo o trabalho animal foi feito de acordo com as normas e diretrizes do Instituto de Estudos de Animais no Albert Einstein College of Medicine. 1. Crescimento Condições de C. neoformans H99 (sorotipo A) <ol><li> Crescer indivíduo C. neoformans (Cn) colônias em placas de ágar Sabouraud. </li><li> Um dia antes do experimento, selecione uma colônia e inocular 10 ml de caldo Sabouraud. </li><li> Permitir que a cultura a crescer durante a noite a 37 ° C com agitação a uma velocidade de 250 rpm. </li></ol> 2 Isolamento e Preparação de primário macrófagos Osso de C57 / Bl6 Ratos <ol><li> Eutanásia ratos colocando animal numa câmara de CO 2 até não mais respiração. Como segunda medida, cervicalmente deslocar o pescoço antes de começar a dissecção. Esterilizar todo o corpo com álcool etílico a 70%. </li><li> Remova os dois fêmures e tíbia e os ossos no lugar estéril DMEM. </li><li> Retire o excesso de tecido e músculo usando delicate limpa até os ossos estão completamente limpos. </li><li> Coloque os ossos intactos apenas em uma placa de Petri contendo álcool etílico a 70% e incubar por 3 min para matar microorganismos associados. Retirar os ossos e lugar em placa de Petri com DMEM estéril. </li><li> Corte cuidadosamente extremidades dos ossos. Segurar osso sobre uma vazios tubo de 50 ml colocados em gelo e cuidadosamente descarga 10 ml de DMEM frio usando uma agulha G 25 através de cada osso. NOTA: Cada rato irá produzir quatro ossos (2 fêmures, duas tíbias). Portanto, um rato deve produzir aproximadamente 40 ml de suspensão de células. </li><li> Centrifugar a suspensão de células à temperatura ambiente durante 10 min a 650 x g. </li><li> Durante esta centrifugação, preparar e esterilizar filtro de macrófagos da medula óssea que consiste em meios de DMEM suplementado com 20% de L929, 10% de soro fetal de vitelo, 10% de NCTC-109, 1% de Pen-Strep, 1% de HEPES, 1% de L-Glutamina alimentação , 1% de aminoácidos não essenciais, 0,1% de 2-mercaptoetanol. </li><li> Ressuspender resultando pellet com 10 ml de alimentação de mídia. Passe suspens celularesião positivo através de um filtro de 70 um da célula para interromper aglomerações de células e remover os detritos grandes. </li><li> Placa 1 ml de suspensão de células para 10 ml de meio de alimentação em placas de Petri, que são especificadas para uso de cultura de tecidos (para um total de 10 placas). </li><li> Permitir que as células aderir a 37 ° C com 10% de CO 2. Adicionar 5 ml de meio de alimentação fresca no dia 3 No dia 7, substituir completamente a mídia de alimentação e macrófagos está pronto para ser usado. </li><li> O dia antes do experimento, retire macrófagos de placa removendo mídia alimentação e adicionar 5 ml de uma célula suave descascar reagente ao prato. </li><li> Incuba-se durante 5-10 minutos a 37 ° C e remover macrófagos com pipetagem suave. </li><li> Células de pelotas por centrifugação a 650 xg durante 10 min. </li><li> Ressuspender o sedimento em 1 ml de alimentação de mídia. Usando uma diluição de 1:20, o cálculo da concentração de macrófagos por pipetagem de 10 ul de suspensão de células para um hemocitómetro. </li><li> Usando 14 milímetros com fundo de vidro placas de Petri, placa Aconcentração de 1 x 10 5 macrófagos diretamente sobre o bem interior, que tem um máximo de 200 l, por isso tome cuidado para não exceder este volume como mostrado na Figura 4. </li><li> Permitir que as células adiram ao prato de Petri de vidro, colocando os pratos em uma incubadora a 37 ° C durante 1 hora. </li><li> Adicionar 1-2 ml de alimentação de meio suplementado com LPS a 1 mg / ml e IFNg em 500 u / ml, e permitir que as células a incubar durante a noite. </li></ol> 3 Co-incubação de macrófagos murino primário e C. neoformans <ol><li> No dia da experiência, remover um ml de células e de cultura de sedimento de levedura durante a noite inoculados por centrifugação durante 5 min a 420 x g. </li><li> Lave as células três vezes com PBS estéril para remover a mídia e produtos criptocócicas extracelulares como o glucuronoxylomannan polissacarídeo (GXM) na velocidade e tempo mencionado acima. </li><li> Ressuspender o sedimento de células em 1 ml de PBS estéril e fazer uma diluição a 1: 100. Pipeta de 10 mL de tele diluição para um hemocitómetro e contar as células. Adicionar células de levedura a uma MOI de 1: 5. Por exemplo, se houver um x 10 5 macrófagos / bem, faça uma suspensão de células de 5 x 10 6 / ml de Cn e adicione 100 ml desta solução para um montante total de 5 x 10 5 Cn. </li><li> Adicionar o volume calculado de suspensão de células criptocócica a 1 ml de meios de alimentação com o anticorpo mAb 18B7 a uma concentração de 10 ug / ml. Incubar suspensão de células à temperatura ambiente durante 5 min. </li><li> Remova a mídia de alimentação de vidro de fundo placa de Petri e lavar 1x com PBS estéril. Adicionar 100 ul de opsonizado Cn directamente a bem. </li><li> Permitir que as células a incubar durante 2 horas a 37 ° C com 10% de CO 2. </li><li> Depois de co-incubação, verifique com o microscópio que os macrófagos internalizaram Cn. Para ser considerado internalizada, as células criptocócicas deve ser visto claramente dentro dos limites do macrófago. </li><li> Lavar 3x prato de Petri com PBS estéril para remover todo e qualquer extracellular Cn. </li><li> Adicionar 1-2 ml de alimentação de mídia sem a adição de mAb 18B7 e configurar microscópio. </li></ol> Exocitose 4 Visualizando Non-lítica em tempo real Digital Utilizando Microscopia de Luz NOTA: Para realizar estes experimentos, um microscópio que vem com uma câmara de incubação anexo que inclui CO 2 entrega e uma unidade de aquecimento é necessário. <ol><li> Antes da experiência, pré-aquecer a câmara de incubação de, pelo menos, 30 minutos a 37 ° C. Certifique-se de que a unidade de CO 2 lê 5% no início da experiência. </li><li> Lugar com fundo de vidro placa de Petri na plataforma de microscópio, cubra com CO 2 tampa e feche todas as portas para garantir que nenhum calor escapa. </li><li> Usando a objetiva de 10X com o microscópio em contraste de fase, o foco em um campo claro de macrófagos infectados. Com base na experiência, determinar o objectivo usado por quantos macrófagos precisam de ser estudados. </li><li> Configurar o seu software microscópio para take uma imagem a cada 4 minutos, por um período de 24 horas. </li><li> Após 24 horas, o experimento terá atingido a conclusão e um total de 361 imagens terão sido recolhidas. Exportar estas imagens como .jpegs a uma compressão de 5%. Usando o software Image J, ver imagens como uma pilha Visual em 5 quadros por segundo. Se o filme tem que ser visto para publicação ou uma apresentação, salvá-lo como qualquer um avi ou mov arquivo dependendo do formato que funciona melhor para o telespectador. </li></ol>

<ol>
	<li>Voelz, K., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcal+Interactions+With+the+Host+Immune+System.">Cryptococcal Interactions With the Host Immune System.</a> Eukaryotic cell. 9 (6), 835-846 (2010).</li><li>Johnston, S. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcus+interactions+with+macrophages:+evasion+and+manipulation+of+the+phagosome+by+a+fungal+pathogen.">Cryptococcus interactions with macrophages: evasion and manipulation of the phagosome by a fungal pathogen.</a> Cellular microbiology. 15 (3), 403-411 (2012).</li><li>Sabiiti, W., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+infection+by+the+human+fungal+pathogen+Cryptococcus+neoformans.">Mechanisms of infection by the human fungal pathogen Cryptococcus neoformans.</a> Future microbiology. 7 (11), 1297-1313 (2012).</li><li>Bliska, J. B., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+pathogenic+bacteria+and+fungi+&#8212;+a+case+of+convergent+evolution.">Intracellular pathogenic bacteria and fungi &#8212; a case of convergent evolution.</a> Nature Reviews Microbiology. 7, 165-171 (2008).</li><li>Alvarez, M., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+spread+and+massive+vacuole+formation+after+Cryptococcus+neoformans+infection+of+murine+macrophages.">Cell-to-cell spread and massive vacuole formation after Cryptococcus neoformans infection of murine macrophages.</a> BMC immunology. 8 (1), 16 (2007).</li><li>Ma, H., Croudace, J. E., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+cell-to-cell+spread+of+a+pathogenic+yeast.">Direct cell-to-cell spread of a pathogenic yeast.</a> BMC immunology. 8, 15 (2007).</li><li>Alvarez, M., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosome+extrusion+and+host-cell+survival+after+Cryptococcus+neoformans+phagocytosis+by+macrophages.">Phagosome extrusion and host-cell survival after Cryptococcus neoformans phagocytosis by macrophages.</a> Curr Biol. 16 (21), 2161-2165 (2006).</li><li>Ma, H., Croudace, J. E., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expulsion+of+live+pathogenic+yeast+by+macrophages.">Expulsion of live pathogenic yeast by macrophages.</a> Curr Biol. 16 (21), 2156-2160 (2006).</li><li>Johnston, S. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+fungal+pathogen+Cryptococcus+neoformans+escapes+macrophages+by+a+phagosome+emptying+mechanism+that+is+inhibited+by+Arp2/3+complex-mediated+actin+polymerisation.">The human fungal pathogen Cryptococcus neoformans escapes macrophages by a phagosome emptying mechanism that is inhibited by Arp2/3 complex-mediated actin polymerisation.</a> PLoS pathogens. 6 (8), (2010).</li><li>Carnell, 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=Actin+polymerization+driven+by+WASH+causes+V-ATPase+retrieval+and+vesicle+neutralization+before+exocytosis.">Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis.</a> The Journal of cell biology. 193 (5), 831-839 (2011).</li><li>Voelz, K., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+signaling+regulates+the+outcome+of+intracellular+macrophage+parasitism+by+Cryptococcus+neoformans.">Cytokine signaling regulates the outcome of intracellular macrophage parasitism by Cryptococcus neoformans.</a> Infection and immunity. 77 (8), 3450-3457 (2009).</li><li>Bain, M. J., Lewis, E. L., Okai, B., Quinn, J., Gow, A. R. N., Erwig, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-lytic+expulsion/exocytosis+of+Candida+albicans+from+macrophages.">Non-lytic expulsion/exocytosis of Candida albicans from macrophages.</a> Fungal genetics and biology. 49 (9), 677-678 (2012).</li></ol>

Os autores declaram que não temos concorrentes interesses financeiros ou outros conflitos de interesse.

Aqui, descrevemos um método através do qual as interacções macrófagos-fúngicas podem ser registados e analisados ​​em tempo real, ao longo de um período de 24 horas. Nosso laboratório tem usado esse protocolo para estudar muitos dos aspectos temporais da exocitose não-lítica e continuará a usar microscopia em tempo real para determinar os componentes morfológicos que cercam este processo. Para esta técnica para produzir resultados ideais, deve ser dada especial atenção a d...

As fases de uma infecção fúngica entre células criptocócica e macrófagos são bem documentados 1-3. Após as células de levedura são ingeridos pelos macrófagos, uma grande variedade de interacções podem ocorrer: o macrófago pode lisar a libertar a sua carga de fungos para o espaço extracelular, ou pode controlar a infecção, mantendo as células de levedura dentro dos limites da sua membrana celular 4. No entanto, há vários anos, um novo resultado foi descrito de forma independente por dois grupos: exocitose não-lítico, um processo em que um macrófago expurgado algumas ou todas as células criptocócica para o ambiente circundante ou uma célula vizinha e tanto o hospedeiro e patógeno permanecer viáveis ​​5 -8. Vários estudos têm tentado compreender os mecanismos moleculares por trás dessa interação, mas que ainda não têm uma compreensão completa sobre o que impulsiona este fenômeno. A capacidade de capturar imagens em tempo real e posteriormente analisar infectar múltiplased macrófagos permite responder a muitas perguntas sobre as propriedades físicas que cercam exocitose não-lítica em relação ao tempo, a capacidade espacial, mudança na morfologia e até mesmo estresse de macrófagos durante uma infecção fúngica. Ao permitir que as células a serem alojados num ambiente que seja comparável ao de uma incubadora, podemos entrever a natureza dinâmica das interacções celulares de macrófagos-fúngica. Os pesquisadores usaram esta técnica para estudar vários componentes deste processo. O papel do fagossoma neste processo foi feito utilizando a coloração fluorescente aparente e actina agentes de bloqueio 9, enquanto que um outro grupo utilizado este método para mostrar que a exocitose não-lítico foi bloqueada em células que têm tido a LAVAGEM nucleação domínios proteicos suprimido 10. O efeito de sinalização de citoquinas também foi verificado por microscopia em tempo real 11. Este processo não está limitado a C. neoformans como foi também observado com Candida albicans, diferentr patógeno, que tem a capacidade de infectar macrófagos 12. Estes achados são exemplos de como este método pode fornecer uma riqueza de informações para uma área que sabemos tão pouco.

<table border="0" cellpadding="0" cellspacing="0" width="976">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sabouraud Dextrose Agar</td>
			<td style="width:183px;">BD</td>
			<td style="width:227px;">DF0109-17-1</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sabouraud Dextrose Broth</td>
			<td>BD</td>
			<td>DF0382-17-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dubelcco&#39;s Modified Eagle&#39;s Medium(DMEM)</td>
			<td>Corning Cellgro</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Calf Serum ( FCS)&#160;</td>
			<td>Atlanta Biologicals</td>
			<td>S12450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NCTC 109</td>
			<td>Invitrogen</td>
			<td>21340-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES Buffer</td>
			<td>Corning Cellgro</td>
			<td>25-060-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamax</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-essential Amino Acids</td>
			<td>Corning Cellgro</td>
			<td>25-025-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985-023</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3003 Tissue Culture Petri Dishes&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>08772E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellStripper</td>
			<td>Corning Cellgro</td>
			<td>25-056-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mat-tek Glass Bottom Culture Dishes</td>
			<td>MatTek</td>
			<td>P35GC-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LPS</td>
			<td>Sigma</td>
			<td>L3137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse IFN-g</td>
			<td>Roche</td>
			<td>NC 9222016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mAb 18B7</td>
			<td colspan="2">&#160;&#160;&#160;&#160;&#160;&#160; Non-commerical antibody produced in our lab</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axiovert 200M Microscope with Incubating Chamber</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing non-lytic exocytosis of cryptococcus neoformans from macrophages using digital light microscopy

Muitos aspectos da infecção de macrófagos por Cryptococcus neoformans têm sido extensivamente estudada e bem definido. No entanto, uma interação especial, que não é claramente compreendido é exocitose não-lítico. Neste processo, as células de levedura são libertadas para o espaço extracelular através de um mecanismo mal entendido que deixa ambos os macrófagos e Cn viável. Aqui, nós descrevemos a seguir como um grande número de macrófagos infectados individualmente por um período de 24 horas de infecção por microscopia de tempo decorrido. Macrófagos infectados estão alojados numa câmara de aquecimento, com uma atmosfera de CO 2 ligado a um microscópio que fornece as mesmas condições que uma incubadora de cultura de células. Microscopia digital ao vivo pode fornecer informações sobre as interações dinâmicas entre um hospedeiro e patógeno que não está disponível a partir de imagens estáticas. Ser capaz de visualizar cada célula infectada pode fornecer pistas de como macrófagos lidar com infecções fúngicas, e vice-versa. Esta técnica isa ferramenta poderosa para estudar as dinâmicas que estão por trás de um fenômeno complexo.

As imagens que esta técnica produz podem ser analisados ​​em uma variedade de maneiras de coletar informações em torno do que acontece depois que as células tenham fagocitado C. neoformans. Como pode ser visto na Figura 1, há aproximadamente 100 macrófagos que estão infectados ou não infectados. Cada um desses macrófagos dá ao espectador a oportunidade de estudar interações patógeno-hospedeiro em um nível muito microscópico. Como as Figuras 2 e 3</str...

Watch this Scientific Journal Video about Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy at JoVE.com

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Nós descrevemos como visualizar macrófago C. neoformans (Cn) interações em tempo real, com especial ênfase para o processo de exocitose não-lítica usando microscopia de luz digital. Usando esta técnica individualmente macrófagos infectados podem ser estudados para determinar os vários aspectos deste fenómeno.

A visualização não-lítica exocitose de<em&gt; Cryptococcus neoformans</em&gt; De macrófagos Usando Digital Microscopia de Luz

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

visualizing-non-lytic-exocytosis-cryptococcus-neoformans-from

Research

JoVE Journal

Immunology and Infection

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

11.7K visualizações.  Albert Einstein College of Medicine.  Nós descrevemos como visualizar macrófago C. neoformans (Cn) interações em tempo real, com especial ênfase para o processo de exocitose não-lítica usando microscopia de luz digital. Usando esta técnica individualmente macrófagos infectados podem ser estudados para determinar os vários aspectos deste fenómeno. 

Vídeo: Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

Visualizando transferência de células-célula de HIV usando Clones fluorescente de HIV e Microscopia Confocal ao vivo

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Imunologia e Infecção

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

Avaliar a atividade anti-fúngica isolada de macrófagos alveolares por microscopia confocal

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Macrófago Colesterol Exaustão e seu efeito sobre a fagocitose de<em&gt; Cryptococcus neoformans</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

Investigando Mastócito Secretory grânulos; de Biossíntese de Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

Transformação Biolistic de uma fluorescente Tagged Gene para o oportunista fúngica Pathogen<em&gt; Cryptococcus neoformans</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Medindo respostas fisiológicas de<em&gt; Drosophila</em&gt; Neurônios sensoriais para Lipid feromonas Usando Imagem de cálcio ao vivo

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Isolamento, caracterização e purificação dos macrófagos dos tecidos afetados pela inflamação obesidade

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

Visualizando o macrófago armadilhas extracelular usando microscopia Confocal

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

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. ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Visualizando intracelular SNARE tráfico pela vida de fluorescência, microscopia de imagem

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...