This work was supported by Inserm. Marion Bouvet is a fellow of the French Ministry for Research and Technology. Annie Turkieh is a fellow granted by European Union FP7 HOMAGE (305507).

O protocolo segue as diretrizes da nossa humana comitê de ética em pesquisa das instituições. Buffy coat de dadores humanos saudáveis ​​foram obtidos a partir do Centro Regional de Sangue Transfusion (Lille, França). As amostras obtidas a partir dos casacos de Buffy são declaradas como uma coleção Inserm (n ° DC2010-1209). 1. Material e Meios de Cultura Preparação <ol><li> Diluir 10x de Tampão Fosfato Salino (PBS) em água destilada estéril para obter 1x PBS. </li><li> Adicione meio RPMI 1640 suplementado com gentamicina (40 ug / ml) e L-glutamina (2 mM), com e sem 10% de soro humano reunido. </li></ol> 2. As culturas primárias de macrófagos derivados de monócitos (MDM) <ol><li> Diluir buffycoat (25 ml) com 1x PBS. Encher cuidadosamente num gradiente de Ficoll tubo de centrífuga e / Leucosep a 1.600 xg durante 20 min, à temperatura ambiente. </li><li> Recolhe monócitos na interface para um novo tubo. Lavar 3x com 10 ml de 1x PBS contendo 0,1% etilenodiaminotetraácido acético (EDTA) por centrifugações sucessivas a 1.000 xg, 370 xg e 160 xg durante 10 min cada, e depois uma vez em 1x PBS sozinho a 160 xg durante 10 min. </li><li> Ressuspender o sedimento celular em 5 ml de meio RPMI-1640 sem soro e semear as células em pratos de 35 mm a uma densidade de 1 x 10 6 células por prato. </li><li> Após sedimentação durante 90 minutos na incubadora, desprezar o sobrenadante contendo as células não aderentes. Lavam-se as células aderentes, que consiste de monócitos, 3x com 1 ml de PBS; em seguida adicionar 1 ml de meio fresco contendo 10% (v / v) de soro humano para as células livres de soro anteriormente. </li><li> Após 6 dias de cultura, para se obter os macrófagos diferenciados alternativos (M2), adicionar recombinante humana IL-4 (15 ng / ml) e manter durante 6 dias. Em seguida, o tratamento de macrófagos diferenciados com lipopolissacárido (100 ng / mL) no dia 12 durante 4 horas para se obter os macrófagos M1. </li></ol> 3. Extração de macrófagos M1 e M2 Proteínas para 2D Eletroforese <ol><li> Wash macrophidades três vezes com Tris 25 mM, pH 7,4, e resíduos em tampão contendo 30 mM Tris pH 8, 4% 3 - [(3-colamidopropil) dimetilamónio] -1-propanossulfonato (CHAPS), 2 M tioureia e 7 M de ureia. </li><li> Lisar células utilizando um misturador adequado para tubos de microcentrífuga de 1,5 ml para 5 min em gelo e armazenar a -20 ° C. </li><li> Determinar a concentração de proteína usando um reagente de Bradford comercial. Armazenar aliquotas de 100 ul das proteínas à temperatura de -20 ° C até à sua utilização. </li></ol> 4. Isoelectrofocusing NOTA: Realizar todos os procedimentos de rotulagem no escuro. <ol><li> Reduzir a 5 ug de cada amostra (M1 e M2 macrófagos) ajustado para 9 mL com tampão de lise com Tris 2 mM (2-carboxietil) fosfina (TCEP) durante 1 hora a 37 ° C. </li><li> Adicionar cianina 3 (Cy3) de extracto de M1 e M2 extracto corante sulfidrilo reactivo, a uma concentração de 0,8 nM de proteína / ug. Adicionar cianina 5 (Cy5) a M1 e M2 extrai a uma concentração de 0,8 nM / mg de proteína e incubardurante 30 min a 37 ° C. </li><li> Pare a reacção com a adição de um volume igual de tampão de amostra contendo 7 M de ureia, 2 M tioureia, 4% CHAPS, 130 mM de ditiotreitol (DTT) e 2% pharmalytes. </li><li> Mix Cy3 marcado amostras M1 com amostras M2 Cy5-rotulados em uma mão e Cy3 marcado amostras M2 com amostras M1 Cy5-rotulados em outro lado. </li><li> Reidratar um gradiente de pH imobilizado (IPG) faixa (240 milímetros, pH 3-10 gradiente linear) com 450 mL de amostras misturadas marcados em tampão contendo 7 M uréia, 2 M de tiouréia e 4% CHAPS em uma focalização isoelétrica (IEF) sistema de célula para 24 horas, sem aplicar qualquer corrente. </li><li> Realizar concentrando a 300 volts (V) durante 3 horas, e, em seguida, em um gradiente a 1.000 V durante 6 h, em um gradiente a 8000 V durante 3 horas e, finalmente a 8000 V durante 3 horas. </li></ol> 5. Segunda Dimensão NOTA: Execute todos os procedimentos de eletroforese no escuro. <ol><li> Incubar as tiras de IPG em tampão de equilíbrio contering 0,1 mM Tris-HCl (pH 8), 6 M de ureia, 2% (w / v) de dodecil sulfato de sódio (SDS) e 30% (v / v) de glicerol durante 10 min. </li><li> Transferem-se as tiras de IPG equilibradas para a segunda dimensão (electroforese em gel de SDS-poliacrilamida (PAGE)) em 12,5% PAGE e selar com baixo ponto de fusão de agarose. </li><li> Levar a cabo a electroforese a 20 ° C utilizando um sistema Ettan-Daltsix a uma voltagem constante de 70 V durante a noite seguido de 300 V até o azul de bromofenol a frente atinge o fundo do gel. </li></ol> 6. Aquisição de Imagem e Análise Bioinformatic <ol><li> Geles de digitalização expressos entre as duas placas de vidro de baixa fluorescência com um scanner DIGE Imager em comprimentos de onda de excitação / emissão de 532/580 nm para Cy3 e Cy5 para 633/670 nm para se obter imagens com um tamanho de pixel de 100 um. </li><li> Realizar análise de imagem com software comercial, como anteriormente descrito 12. </li><li> Calcule e normalizar volumes pontuais em cada imagem. Atribuir um volume local normalizada como um suporteortion do valor total de cada mancha detectada no gel. </li><li> Analise as diferenças de volumes pontuais de proteína para cada tipo de macrófagos por comparação do valor de volume normalizado local entre os dois grupos (M1 e M2). Considere a diferença entre os volumes pontuais para ser significativa se a mudança é de 1,5 vezes (p &lt;0,05, de um modo análise ANOVA). </li></ol>

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There are no declared conflict of interest.

O protocolo aqui descrito detalha um método para analisar o impacto de vários estímulos dos dois subtipos de macrófagos, M1 (pró-inflamatória) e M2 (anti-inflamatório). As culturas primárias de M1 e M2 macrófagos foram obtidos a partir da diferenciação de monócitos como publicado anteriormente 7. O procedimento de eletroforese em gel 2D DIGE requer materiais e equipamentos especializados, como o IEF celular para placas isoelectrofocusing, baixa fluorescência para a SD...

Os macrófagos são células heterogéneas e de plástico que são capazes de adquirir fenótipos funcionais distintos. In vivo, estas células respondem a uma grande variedade de micro signalssuch ambiental como produtos microbianos, citoquinas, etc. 1. In vitro, o fenótipo pró-inflamatória (M1) de macrófagos pode ser induzida por lipopolissacarídeo (LPS) e o fenótipo anti-inflamatória (M2) por algumas citocinas tais como a interleucina-4 (IL-4). Além disso, os macrófagos podem alternar de um M1 M2 activado para fenótipo, e, inversamente, mediante sinais específicos 2. Dependendo do fenótipo, os macrófagos possuem diferentes funções. M1 macrófagos são células que produzem citocinas pró-inflamatórias, tais como o factor de necrose tumoral α (TNF-α), para matar os microorganismos ou células tumorais 3. Em contraste, os macrófagos M2 evitar estes resposta inflamatória como na cicatrização de feridas e fibrose através da produção de anti-inflammatory factores, tais como TGF-β 3,4. As células mononucleares do sangue periférico humano a partir de dadores saudáveis ​​foram isoladas por centrifugação em gradiente de densidade de Ficoll como previamente descrito 5 utilizando uma técnica adaptada de Boyum 6. Os macrófagos em cultura podem ser diferenciadas em M1 ou M2 fenótipo após 6 dias de cultura primária 7. Análise da expressão de proteínas ou mudanças de proteína entre os dois subtipos de macrófagos sob vários estímulos controlados, tais como patogenicidade hospedeiro ou toxinas microbianas, será útil para decifrar a funcionalidade do pro- macrófagos e anti-inflamatórios. Proteômica são ferramentas exclusivas para o monitoramento direto de proteínas que são especificamente para cima ou para baixo-regulado em macrófagos cultivados humanos sob vários estímulos. Corantes fluorescentes resolveu algumas das limitações de eletroforese em gel 2D, como a baixa sensibilidade e análise de imagem de 8 &lt;<html> / Sup&gt;. Os corantes que reagem com resíduos de cisteína aumentou a sensibilidade de detecção em comparação com aqueles reagir com 9 resíduos de lisina. Em um estudo anterior, demonstramos a utilidade de rotulagem saturação DIGE para a análise de amostras de escassos 10 em comparação com o 2D eletroforese manchada de prata clássica 11. Esta tecnologia é útil para analisar rapidamente modificações de proteína entre os dois subtipos de macrófagos ou entre os macrófagos não tratados e tratados do mesmo subtipo. As vantagens desta técnica são proteómica ter acesso às informações do tamanho da proteína e modificações pós-tradução através da análise do gel 2D 12. Deve ser tomado em consideração que esta não é uma técnica de alto rendimento, o que limita o número de amostras que podem ser analisadas. O desenvolvimento de ensaios de alto rendimento com base em espectrometria de massa como revisto recentemente 13 pode melhorar esta. _content &quot;&gt; Aqui apresentamos como realizar análise 2D DIGE da extração de proteínas de macrófagos em cultura através dos processos de eletroforese, isoelectrofocusing e SDS-PAGE, bem como informações sobre a utilidade do software 2D adequada.

<table border="0" cellpadding="0" cellspacing="0" width="472">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:208px;"><a name="RANGE!A1:C43">Name</a></td>
			<td style="width:155px;">Company</td>
			<td style="width:111px;">Catalog number</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RPMI 1640</td>
			<td>Invitrogen</td>
			<td>31870-074</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PBX 10 X</td>
			<td>Invitrogen</td>
			<td>14200-083</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">L-glutamine-200mM-100X</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">gentamycin 10 mg/mL</td>
			<td>Invitrogen</td>
			<td>15710-049</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">human serum</td>
			<td>Invitrogen</td>
			<td>34005100</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ficoll d=1,077</td>
			<td>ATGC</td>
			<td>L6115</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Leucosep</td>
			<td>Dutscher</td>
			<td>16760</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#160;6-wells plate PRIMARIA&#160;</td>
			<td>Becton Dickinson</td>
			<td>353846</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IL-4</td>
			<td>Promocell</td>
			<td>B-61410</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">lipopolysaccharide</td>
			<td>Sigma-Aldrich</td>
			<td>L-2654</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Giemsa</td>
			<td>Fluka</td>
			<td>48900</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">EDTA MM372,2</td>
			<td>Research Organics&#160;</td>
			<td>3.00E+01</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Filter 0,22&#181;m</td>
			<td>Millipore</td>
			<td>SCGPTORE</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">100 mL cylinder</td>
			<td>Corning</td>
			<td>430182</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TCEP</td>
			<td>Interchim</td>
			<td>UP242214</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bradford reagent</td>
			<td>Bio-Rad</td>
			<td>5000006</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cy3+Cy5-reactive dye</td>
			<td>GE Healthcare</td>
			<td>25-8009-83</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IPG strip 3-10 24cm</td>
			<td>GE Healthcare</td>
			<td>17-6002-44</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Protean IEF cell</td>
			<td>Bio-Rad</td>
			<td>165-4000</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Low-melting agarose</td>
			<td>Invitrogen</td>
			<td>15517-014</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ettan-Daltsix system</td>
			<td>GE Healthcare</td>
			<td>80-6485-08</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ettan DIGE Imager scanner</td>
			<td>GE Healthcare</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Progenesis Samespot</td>
			<td>Non linear dynamics</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">50 ml tubes</td>
			<td>any supplier</td>
			<td>n/a</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">15 ml tubes</td>
			<td>any supplier</td>
			<td>n/a</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">CHAPS</td>
			<td>Sigma-Aldrich</td>
			<td>C5070</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ur&#233;e</td>
			<td>Merk</td>
			<td>108484-500</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Thiour&#233;e</td>
			<td>Sigma-Aldrich</td>
			<td>T7875</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DTT</td>
			<td>Bio-Rad</td>
			<td>1610611</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">APS</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TEMED</td>
			<td>Sigma-Aldrich</td>
			<td>T9281</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tris Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tris HCl</td>
			<td>Sigma-Aldrich</td>
			<td>T3253</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pharmalytes 3-10</td>
			<td>GE Healthcare</td>
			<td>17-0456-01</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">SDS</td>
			<td>Sigma-Aldrich</td>
			<td>L3773</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>114391</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G6279</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Acrylamide 40%</td>
			<td>Bio-Rad</td>
			<td>161-0148</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">2D clean Up</td>
			<td>GE Healthcare</td>
			<td>80-6454-51</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dim&#233;thylformamide</td>
			<td>Sigma-Aldrich</td>
			<td>22705-6</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">electrode wicks</td>
			<td>Bio-Rad</td>
			<td>165-4071</td>
		</tr>
	</tbody>
</table>

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.

Para realizar a análise proteômica diferencial adequado, o processamento de amostras a serem analisadas devem ser verificadas. No exemplo apresentado, a qualidade de cultura de células de macrófagos é necessário para os aspectos morfológicos e moleculares, tal como publicado anteriormente 5. A diferenciação de monócitos em macrófagos e a homogeneidade da cultura foi seguido por microscopia de fase. A Figura 1 mostrou um exemplo de culturas primárias de...

Watch this Scientific Journal Video about Proteomic Profiling of Macrophages by 2D Electrophoresis at JoVE.com

Proteomic Profiling of Macrophages by 2D Electrophoresis

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 ...

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JoVE Journal

Immunology and Infection

12.5K visualizações.  University Lille Nord de France. 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.

Vídeo: Proteomic Profiling of Macrophages by 2D Electrophoresis

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Depleção e reconstituição de macrófagos em Ratos

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Imunologia e Infecção

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Isolamento de monócitos humanos por duas vezes centrifugação em gradiente e sua diferenciação de macrófagos em revestidos de teflon Cultura de Células Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Utilização de<em&gt; Shigella flexneri</em&gt; Para estudar interações Autophagy-Cytoskeleton

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

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 ...

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. ...

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

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

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

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

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

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

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 ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.

SILAC Based proteômica Caracterização de exossomas de HIV-1 células infectadas

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

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. ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Análise de Microglia e Macropagos derivados de Monócitos do Sistema Nervoso Central por Citometria de Fluxo

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...