Name: assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy
Uploaded: 2014-07-09T21:30:00
Description: Watch this Scientific Journal Video about Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy at JoVE.com

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 etiqueta...

<ol>
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F., 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=Differential+polarization+of+alveolar+macrophages+and+bone+marrow-derived+monocytes+following+chemically+and+pathogen-induced+chronic+lung+inflammation.">Differential polarization of alveolar macrophages and bone marrow-derived monocytes following chemically and pathogen-induced chronic lung inflammation.</a> J Leukoc Biol. 88, 159-168 (2010).</li><li>Schaffner, A., Douglas, H., Braude, A. I., Davis, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killing+of+Aspergillus+spores+depends+on+the+anatomical+source+of+the+macrophage.">Killing of Aspergillus spores depends on the anatomical source of the macrophage.</a> Infect Immun. 42, 1109-1115 (1983).</li><li>Goodridge, H. 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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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			<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>
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		<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>
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			<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>
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			<td height="21" style="height:21px;width:216px;">insulin syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">6 ml syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<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>
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			<td height="21" style="height:21px;width:216px;">suture thread</td>
			<td style="width:71px;"></td>
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			<td height="21" style="height:21px;width:216px;">50 ml conical centrifuge tubes</td>
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			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">gauze sponges (4 inch square)</td>
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			<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>
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		<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>
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		<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>
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			<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>
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			<td height="63" style="height:63px;width:216px;">Aspergillus fumigatous strain expressing green fluorescent protein</td>
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			<td style="width:119px;"></td>
			<td>provided by Dr Margo Moore, Simon Fraser University, Burnaby, BC, Canada</td>
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			<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>
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			<td height="21" style="height:21px;width:216px;">scissors</td>
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			<td height="21" style="height:21px;width:216px;">forceps</td>
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			<td height="42" style="height:42px;width:216px;">Biological Safety Cabinet Class II</td>
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			<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>
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			<td height="21" style="height:21px;width:216px;">Leica DM1000 light microscope</td>
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			<td height="21" style="height:21px;width:216px;">Hemocytometer</td>
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			<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>
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			<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>
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		<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>
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		<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>
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			<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>
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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

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

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

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JoVE Journal

Immunology and Infection

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Microondas Assistida Diagnóstico Rápido de Doenças vírus de planta por Microscopia Eletrônica de Transmissão

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Imunologia e Infecção

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

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. ...

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

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

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 ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

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

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

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

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

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

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 ...