Este trabalho foi financiado pelo Instituto Nacional de Alergia e Doenças Infecciosas Grant R01AI079253 (a BHS) e pelo Instituto Nacional do Câncer Cancer Center Suporte Grant CA016056 para Roswell Park Cancer Institute.

Todos os procedimentos realizados em animais neste estudo foram aprovados pelo Comitê Animal Care e Use em Roswell Park Cancer Institute e cumpriu todo o estado, federal, e os Institutos Nacionais de Saúde regulamentos. 1. In vivo macrófago Labeling Nota: PHK26 é um corante lipofílico que serão absorvidos pelas células fagocíticas, tanto na circulação e nos tecidos quando administrados por via intravenosa (iv). PKH26 foi utilizado para a etiquetagem in vivo para minimizar a quantidade de manipulação pós-colheita do macrófago. PKH-26, tem a vantagem de estavelmente acumulação nos macrófagos alveolares ao longo de períodos prolongados. Esperar 10 dias após a administração permite a limpeza de todas as células marcadas de circulação, deixando apenas alveolar macrófagos estável rotulados com PKH26 8, 9. PKH26 é um fluorocromo vermelho que tem de excitação (551 nm) e emissão (567 nm) características compatíveiscom os sistemas de detecção de rodamina ou ficoeritrina. Qualquer procedimento rotulagem pode introduzir artefato, incluindo perda de viabilidade. No entanto, uma vez que usamos a mesma abordagem para a rotulagem de macrófagos em diferentes genótipos de mouse (por exemplo, WT vs NADPH oxidase com deficiência), os efeitos desses artefatos seria uniforme. <ol><li> Diluir solução estoque de PKH26 (1 x 10 -3 M em etanol) 1:05 em diluente C fornecido pelo fabricante. </li><li> Carga de 100 ul de PKH26 diluída para uma seringa de 1/2 ml de insulina (29 L x 1/2 &quot;) para cada rato. </li><li> Administrar aos ratos por injeção iv (ou veia da cauda ou retro orbital) pelo menos 10 dias antes da colheita. </li></ol> 2. Colheita de macrófagos alveolares de Lavagem Broncoalveolar (BAL) O número de ratos para usar para uma experiência pode variar. Um rendimento típico de um ratinho não estimulado é na gama de 3-5 x 10 5 macrófagos alveolares, no entanto, este número pode variar muito based em fatores como o tamanho do mouse, a experiência da pessoa que realiza a lavagem, eo número de injeções de fluido de lavagem eo volume retirado dos pulmões. Neste protocolo de lavagem pulmonar foi realizada com uma cavidade torácica fechada, que, juntamente com a repetidas lavagens de 1 ml ajuda a aumentar o rendimento das células. <ol><li> Eutanásia rato por administração de uma dose letal de anestésico avertina (12,5 mg) por via intraperitoneal (ip) usando condições assépticas para manter a solução estéril. </li><li> Spray de rato cuidadosamente com etanol a 70%. </li><li> Remover a pele a partir da extremidade craniana animal expondo o tórax (Figura 1A). <ol><li> Faça um pequeno corte na pele logo abaixo do diafragma. </li><li> Inserir o dedo dentro do corte e puxe a pele para longe da extremidade craniana do animal (Figura 1A). </li><li> Empurre-se ligeiramente sobre a cabeça, estendendo-se o lado ventral do pescoço (Figura 1A). </li></ol></li><li> Corte um pequeno buraco através da caixa torácicano lado direito do tórax para esvaziar os pulmões e também permitir a visualização dos pulmões durante as etapas seguintes. </li><li> Localize a traquéia e dissecar cuidadosamente o tecido circundante. <ol><li> Retire as glândulas salivares, músculos esterno hioideo e laringe, etc com uma pinça curva. </li><li> Retire cuidadosamente com uma pinça curva e cortar com uma tesoura o tecido fino que cobre a traquéia (adventícia) revelando a estrutura da cartilagem rodeado subjacente. </li></ol></li><li> Inserção do cateter na traqueia. <ol><li> Usando pinças curvas, colocar uma sutura de trás (dorsal) da traqueia e puxar uma 10 cm de comprimento através da abertura. </li><li> Começando acima da cricóide (o anel engrossado da cartilagem) guiar cuidadosamente um cateter 22 G até a traquéia parar onde a traquéia desaparece por trás do esterno. </li><li> Firmemente amarrar sutura em torno de traquéia e cateter para um ajuste confortável. Remova a agulha do cateter. </li></ol></li><li> Monte 4-way torneira. <ol><li> Preencherum 6 ml seringa com DPBS, 1% de FBS e anexar a 4-way torneira, como indicado na Figura 1B. </li><li> Anexar um vazio 6 ml de seringa na posição indicada na torneira de 4 vias (Figura 1B). </li><li> Anexar porta aberta no 4-way torneira de cateter (Figura 1B). Tenha cuidado para não desalojar cateter de traquéia. </li></ol></li><li> Recolha do fluido de lavagem dos pulmões. Nota: Os investigadores podem optar por utilizar menores volumes de fluido de lavagem para minimizar a possibilidade de danos para o tecido. É importante que a mesma abordagem pode ser aplicada de forma consistente em todos os grupos de ratos. <ol><li> Gire a alça da torneira para que a seringa vazia é fechado e injecte lentamente DPBS ~ 1 ml, 1% FBS para inflar os pulmões. Observe a inflação do pulmão através do buraco cortado no passo 2.4. </li><li> Tenha cuidado para não overinflate os pulmões. </li><li> Gire a alça da torneira para que a seringa cheia está fechado, e retire cuidadosamente a gripe<html> ID a partir dos pulmões. </li><li> Observe os pulmões esvaziar através do buraco cortado no passo 1.4. O cateter pode precisar ser movido ligeiramente para trás ou para frente para conseguir um bom empate. Tomar precauções especiais para manter o cateter no interior da traqueia. </li><li> Repita os passos 2.8.1-2.8.4 5-6x, até todo o DPBS, 1% de FBS foi injectado e retirado dos pulmões. Nota: recuperação do líquido do pulmão não será 100% do líquido injetado. </li></ol></li><li> Seringa vazia com lavado em um tubo cônico de 50 ml. Manter as células à temperatura ambiente. </li><li> Sedimentar as células por centrifugação a 300 xg durante 5 min à temperatura ambiente e deitar fora o sobrenadante. Isto pode ser guardado para análise de citocina, se desejado. </li><li> Ressuspender as células em 1-5 mL de meio RPMI 1640 com FBS a 10% e contagem num hemocitómetro. No rato não estimuladas,&gt; 95% de células colhidas por BAL são esperados ser os macrófagos, o que pode ser confirmado por citologia. </li></ol> 3. Cultivo e colheita Fungo &quot;&gt; O Aspergillus fumigatus utilizado neste protocolo expressa GFP ao longo de todas as fases do seu ciclo de vida:. Esporos, germlings e hifas fúngicas Esta estirpe foi fornecido pelo Dr. Margo Moore, Simon Fraser University, Burnaby, BC, Canadá. <ol><li> Placa de conídios de uma estirpe de Aspergillus fumigatus expressando GFP em Sabouraud Brain Heart Infusion (BHI) ágar slants com cloranfenicol (0,05 g / L) e gentamicina (0,5 g / L). </li><li> Incubar no escuro vagamente tampado por 7 a 10 dias em temperatura ambiente. </li><li> Colheita conídios por inclinação lavagem com 10 ml de 0,01% de Tween 20 em DPBS. Levemente raspar o fungo com a stripette a afrouxar. Lavar a inclinação várias vezes para aumentar o rendimento. </li><li> Passar a suspensão de conídios por meio de um filtro de células de 100 um em uma cónico de 50 ml. </li><li> Pellet conídios por centrifugação a 400 xg durante 10 min. </li><li> Conídios Ressuspender em 50 ml DPBS e contar com um hemocitômetro. </li><li> Lavar duas vezes com DPBS e diluir a concentração desejadatração. </li></ol> 4. Fúngica Desafio e microscopia confocal Para investigar o papel dos macrófagos da NADPH-oxidase na defesa do hospedeiro, a capacidade dos macrófagos alveolares isoladas a partir de ratinhos de três genótipos para controlar o crescimento de esporos de Aspergillus fagocitose foi avaliada. Os genótipos utilizados neste estudo incluem; 1) ratinhos de tipo selvagem, 2) NCF1 * / * Mn-ratinhos com uma mutação de ocorrência natural NCF1 (NCF1 codifica para phox p47, um componente necessário do fagócito NADPH-oxidase), deixando-os de NADPH-oxidase deficiente, e 3) NCF1 * / * Mn + ratinhos transgénicos (MN indica o transgene abrigando NCF1 tipo selvagem sob o controlo do promotor de CD68 humano), onde a actividade da NADPH-oxidase foi reconstituído na população de monócitos / macrófagos 10, 11. A microscopia confocal é o mais adequado para este ensaio, devido à presençae crescimento de conídios não fagocitados nas lâminas. O microscópio confocal irá permitir a visualização de planos individuais dentro do eixo Z, e permitir a diferenciação entre o fungo cresce em torno, em vez de no interior de macrófagos. Todas as imagens confocal são adquiridos por meio de uma lente de imersão em óleo 63X. Z-stacks são adquiridos com um fator de zoom eletrônico de 2,5. A espessura de uma pilha de-Z é determinada utilizando DAPI e as imagens de campo brilhante para localizar a parte superior e inferior do campo. Z-stacks variou 14-24 mM com fatias individuais ~ 1 mm de espessura. Para a quantificação dos macrófagos, os exames individuais de zoom eletrônico 1X foram realizados em 10 campos por genótipo. Macrófagos intactas foram identificados com base na PKH26 e coloração DAPI da membrana e do núcleo, respectivamente. Os conídios foram identificados com base na imagem de campo claro expressão e FITC. <ol><li> Prepare lamelas e placas estéreis <ol><li> Debaixo de uma cabine de segurança biológica, mergulhe 22 x 22 mm lamínulas de vidro em 70% etanol por 5-10 min. </li><li> Lavar lamínulas em PBS estéril. </li><li> Utilizando fórceps estéreis, colocar suavemente uma única lamela no fundo de cada poço de uma estéril embalado individualmente placa de 6 poços. </li></ol></li><li> Semente de ~ 1 x 10 6 colhidas PKH26 marcado macrófagos alveolares em suspensão em 300 ul de RPMI 1640, FBS a 10% para cada lamela. O número exacto pode variar, dependendo do rendimento da colheita. </li><li> Permitir macrófagos para aderir por incubação a 37 ° C com 5% de CO 2 durante a noite. </li><li> Na manhã seguinte, adicionar 1 x 10 ~ 7-expressando GFP A. fumigatus conidia suspensa em 100 ul de pré-aquecido (37 ° C), meio RPMI 1640, 10% FBS. </li><li> Retornar placa a 37 ° C incubadora com 5% de CO 2. </li><li> As células devem ser fixados para um mínimo de 2 horas à temperatura ambiente ou durante a noite a 4 ° C. No 3, 7, e 14 hr fixar as células por adição de um volume igual de 2% de formaldeído a cada cavidade (concentração final de 1% de formaldeído). Coloque placas para aOs pontos de tempo iniciais, a 4 ° C durante a noite. Para o ponto de tempo final (14 horas), fixar as células à temperatura ambiente durante 2 horas. </li><li> Após a fixação, remover suavemente lamínulas usando uma pinça e enxaguar em DPBS. </li><li> Remover o excesso de líquido, colocando a extremidade da lamela em um Kimwipe dobrado. </li><li> Aplicar 6 mL de fluorescência meio de montagem com DAPI para uma lâmina de vidro. </li><li> Colocar uma extremidade da lamela sobre a lâmina e cair suavemente com o lado das células para baixo para o meio de montagem. Meio de montagem vai se espalhar e formar uma camada uniforme por baixo da lamela. Não há necessidade de pressionar na lamela. </li><li> Selar bordas da lamínula com clara unha polonês e deixar secar ao ar. </li><li> Loja preparados slides de uma caixa corrediça (protegido da luz) à temperatura ambiente até estar pronta para análise por microscopia confocal. </li></ol>

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Utilizando esta abordagem de análise ex vivo da actividade antifúngica de macrófagos in vivo juntamente com o desafio de fungos, que anteriormente demonstrou que a NADPH oxidase nos macrófagos desempenha um papel importante na defesa contra A. fumigatus 4. A utilização de macrófagos alveolares isolados nos permite focar a sua actividade anti-fúngica, na ausência de outras células do sistema imune (por exemplo, recrutamento de neutrófilos) que protegem contra o <e...

Macrófagos alveolares são as células fagocíticas de primeira linha que encontram micróbios inalados. Os macrófagos reconhecem motivos Aspergillus por receptores de reconhecimento de patógenos, ingerir e limitar o crescimento de esporos inalados (conídios), e iniciar as respostas inflamatórias. A NADPH oxidase de fagócitos converte oxigênio molecular ao ânion superóxido e intermediários reativas de oxigênio a jusante (ROIs). A doença granulomatosa crônica (CGD) é uma desordem hereditária do sistema NADPH oxidase caracterizada por infecções bacterianas e fúngicas graves e por respostas inflamatórias excessivas. Apesar de NADPH-oxidase é crítica para a defesa do hospedeiro mediada pelos neutrófilos 1-3, a importância da NADPH-oxidase em macrófagos não está bem definido. Estudos anteriores mostraram que os macrófagos alveolares ingerir e matar os esporos de Aspergillus, enquanto que os neutrófilos como alvo principalmente o estágio de hifas 5. No entanto, tem havido resul conflitantests quanto ao papel da NADPH-oxidase em macrófagos para controlar o crescimento de A. fumigatus esporos 6, 7. O objetivo deste método foi delinear o papel específico da NADPH oxidase em macrófagos na mediação de defesa do hospedeiro contra A. esporos fumigatus. Descobrimos que a NADPH oxidase de macrófagos alveolares controla o crescimento de A. fagocitado fumigatus esporos 4. Para modelar mais de perto a resposta do hospedeiro a fungos inalados, usamos macrófagos alveolares colhidas por lavagem broncoalveolar de camundongos não estimuladas imediatamente após o sacrifício. O uso de macrófagos alveolares isolados nos permitiu concentrar em sua atividade antifúngica, na ausência de outras células do sistema imunológico (por exemplo, neutrófilos recrutados) que defendem contra Aspergillus in vivo. Neste método, os macrófagos alveolares foram coradas in vivo antes da colheita de modo a minimizar a quantidade de manipulação de pós-colheita. &lt;/ P&gt; Outra vantagem do presente protocolo é a utilização de um expressando GFP-A. fumigatus tensão. Este fungo expressa GFP a partir da fase de conídios através da fase de hifas e não tem necessidade de uma maior coloração. Para obter imagens com maior detalhe, escolhemos microscopia confocal que permite a reconstrução de estruturas tridimensionais a partir de imagens obtidas. A reconstrução tridimensional dá a capacidade de diferenciar entre hifas em torno, em vez de em ou através de um macrófago. Os conídios também pode ser justamente distinguido como intracelular contra extracelular.

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	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">PKH26GL-1KT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2,2,2 Tribromoethanol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T48402</td>
			<td>Used to make AvertinAnesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-methyl-2-butanol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A-1685</td>
			<td>Used to make AvertinAnesthetic</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dulbecco&#39;s Phosphate Buffered Saline (without calcium and magnesium)</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NH4CL</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A0171</td>
			<td>Used to make ACK red cell lysis buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">KHCO3</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P91444</td>
			<td>Used to make ACK red cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">E6758</td>
			<td>Used to make ACK red cell lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">22 gauge X 1.00 inch i.v. catheter</td>
			<td style="width:71px;">BD&#160;</td>
			<td align="right" style="width:119px;">381523</td>
			<td>Insyte Autoguard Winged</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">insulin syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 ml syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4-way stopcock</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">50-700-077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">suture thread</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 ml conical centrifuge tubes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gauze sponges (4 inch square)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 with L-glutamine</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">10-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Hyclone</td>
			<td style="width:119px;">SH30396.03</td>
			<td>heat inactivated</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hema 3 Stain Set</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">22-122-911</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cytoseal 60</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">8310-4</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sabouraud Brain Heart Infusion Agar with Chloramphenicol and Gentamicin Slants</td>
			<td style="width:71px;">BD&#160;</td>
			<td align="right" style="width:119px;">297252</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Aspergillus fumigatous strain expressing green fluorescent protein</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>provided by Dr Margo Moore, Simon Fraser University, Burnaby, BC, Canada</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100 micron Cell Strainers</td>
			<td style="width:71px;">BD Falcon</td>
			<td style="width:119px;">64753-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">scissors</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">forceps</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biological Safety Cabinet Class II</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sorval ST40 Centrifuge with swing bucket rotor</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leica DM1000 light microscope</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemocytometer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cytospin 2 centrifuge</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Cell Culture Incubator</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>37 &#176;C, 5%&#160; CO2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">22X22 mm micro cover glass</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">48366-227</td>
			<td>glass coverslips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 well Cell Culture Plates</td>
			<td style="width:71px;">Corning</td>
			<td align="right" style="width:119px;">3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10% Neutral Buffered Formalin</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BDH0502-1LP</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Vectashield Mounting medium with DAPI</td>
			<td style="width:71px;">Vector Laboratories</td>
			<td style="width:119px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Leica TCS SP2 system with a laser point scanner</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Mounted on DMIRE2 fluorescence microscope</td>
		</tr>
	</tbody>
</table>

assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy

O pulmão é uma interface em que as células hospedeiras são rotineiramente expostas a microorganismos e produtos microbianos. Macrófagos alveolares são células fagocíticas de primeira linha que encontram fungos inalados e outros micróbios. Os macrófagos e outras células do sistema imunológico reconhecem motivos Aspergillus por receptores de reconhecimento de patógenos e iniciar respostas inflamatórias a jusante. A NADPH oxidase de fagócitos gera intermediários reativas de oxigênio (ROIs) e é fundamental para a defesa do hospedeiro. Apesar de NADPH-oxidase é crítica para a defesa do hospedeiro mediada pelos neutrófilos 1-3, a importância da NADPH-oxidase em macrófagos não está bem definido. O objetivo deste estudo foi delinear o papel específico da NADPH oxidase em macrófagos na mediação de defesa do hospedeiro contra A. fumigatus. Descobrimos que a NADPH oxidase de macrófagos alveolares controla o crescimento de A. fagocitado fumigatus esporos 4. Aqui, descrevemos um método para avaliar a capacidade de um ratomacrófagos lveolar (AMS) para controlar o crescimento de esporos de Aspergillus fagocitados (conídios). Macrófagos alveolares são coradas in vivo e dez dias mais tarde isolado a partir de murganhos por lavagem broncoalveolar (BAL). Os macrófagos são semeadas em lamínulas de vidro, então semeado com a proteína verde fluorescente (GFP) expressando A. esporos fumigatus. Em momentos específicos, as células são fixadas e o número de macrófagos intactas com esporos fagocitados é avaliada por microscopia confocal.

<html> A recolha dos macrófagos alveolares pelo LBA de ratinhos não estimulados irá resultar numa população pura de&gt; 95% com base na citologia. A 3 e 7 horas (Figura 2) pontos de tempo preceder a fase de hifas de crescimento fúngico e permitir uma comparação da eficiência fagocítica de conídios entre genótipos. O ponto de tempo de 14 horas é onde genótipos pode ser comparado no que respeita à capacidade para inibir a passagem de conídios fagocitado para a fase de hifas tecido invasiva ...

Watch this Scientific Journal Video about Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy at JoVE.com

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

Um método para avaliar a capacidade dos isolados macrófagos alveolares do rato para controlar o crescimento de esporos de Aspergillus fagocitados por microscopia confocal.

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 ...

assessing-anti-fungal-activity-isolated-alveolar-macrophages-confocal

Research

JoVE Journal

Immunology and Infection

11.4K visualizações.  Roswell Park Cancer Institute.  Um método para avaliar a capacidade dos isolados macrófagos alveolares do rato para controlar o crescimento de esporos de Aspergillus fagocitados por microscopia confocal. 

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

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

Microscopia intravital do Baço: Análise Quantitativa da Mobilidade Parasite e do fluxo sanguíneo

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Imunologia e Infecção

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Imagens em tempo real de células mielóides em Dynamics<em&gt; Apc<sup&gt; Min / +</sup</em&gt; Intestinal Tumores girando Disk microscopia confocal

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

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 ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Geração e identificação de GM-CSF macrófagos derivados e dendríticas células alveolares do tipo de ratinho de Medula Óssea

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

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, 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 and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolamento e cultura In Vitro de macrófagos alveolares murino e humanos

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

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 ...

Methods Article

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