We thank Dr. Alexandre Benmerah (Institut Imagine Necker, Paris, France) for initial discussions on the experimental approach and Dr. Jamil Jubrail for reading the manuscript. Nad&#232;ge Kambou and Susanna Borreill are acknowledged for performing experiments with the method in our laboratory. This work was supported by grants from CNRS (ATIP Program), Ville de Paris and Agence Nationale de la Recherche (2011 BSV3 025 02), Fondation pour la Recherche M&#233;dicale (FRM INE20041102865, FRM DEQ20130326518 including a doctoral fellowship for FMA) to FN, and Agence Nationale de Recherche sur le SIDA et les h&#233;patites virales" (ANRS) including a doctoral fellowship for JM.

Nota: O plasmídeo utilizado Lifeact-mCherry é um dom tipo de Dr. Guillaume Montagnac, Institut Curie, Paris, gerada após 17. 1. Células e Transfecção Nota: RAW264.7 de macrófagos são crescidas até à sub-confluência em meio completo (meio RPMI (Roswell Instituto Parque Memorial) 1640, HEPES 10 mM, piruvato de sódio 1 mM, 50 ^ M β-mercaptoetanol, 2 mM de L-glutamina e 10% de FCS ( soro fetal de vitelo)) em uma placa de 100 mm. Eles são transf ectadas com plasmídeos que codificam para proteínas marcadas fluorescentemente por electroporação. Rotineiramente aproximadamente 5-6 x 10 6 células são transfectadas com 20 ug ou 10 ug de plasmídeo para cada transfecção e co-transfecção, respectivamente. Note-se que outros meios de transfecção à base de electroporação ou lipofecção podem ser utilizados como abordagens alternativas. <ol><li> Raspar as células com um levantador de células e ressuspender-los em 10 ml de meio de cultura por pipetagem para cima e para baixo várias vezes. </li><li> Centrifugar asuspensão de células 300 x g, 5 min a RT no rotor de ângulo oscilante. </li><li> Pré-aqueça 10 ml de meio completo suplementado com 10 ug / ml de gentamicina, a 37 ° C. </li><li> A seguir à centrifugação, desprezar o sobrenadante e ressuspender o sedimento em 3 ml de &quot;tampão de lavagem A&quot; a partir do kit de electroporação. </li><li> Centrifugar a suspensão de células a 300 xg, 5 min a temperatura ambiente no rotor de ângulo oscilante. </li><li> Enquanto isso preparar a mistura de ADN à temperatura ambiente: 120 ul de 2x tampão B, 20 ^ g de ADN plasmidico que codifica para a proteína de interesse, qsp 240 mL de H 2 O. </li><li> A seguir à centrifugação, eliminar todo o sobrenadante. </li><li> Ressuspender o sedimento de células na mistura de ADN e transferência em cuvetes de electroporação de 4 mm. </li><li> Incubar à temperatura ambiente durante 3 min. </li><li> Electroporate a 250 V, 900 uF. </li><li> Imediatamente ressuspender as células no pré-aquecido (37 ° C) de meio completo suplementado com gentamicina e a chapam em uma placa de 100 mm. </li><li> Incubam-se as células transfectadas durante a noite a 37 ° C, 5% de CO 2. </li><li> Na manhã seguinte, substituir o meio completo com 10 ml de meio de microscopia isento de soro (RPMI 1640 sem vermelho de fenol, 10 mM de HEPES, 1 mM de piruvato de sódio, 50 uM β-mercaptoetanol, 2 mM de L-glutamina). </li></ol> 2. Opsonização de glóbulos vermelhos Nota: Como um modelo de meta de partículas para os macrófagos, células do sangue ovelhas vermelhas (SRBCs) são utilizados. Normalmente, cerca de 7 x 10 6 SRBCs por 35 milímetros prato fundo de vidro é usado. <ol><li> Lavar os SRBCs com 100 ul de 1x PBS (fosfato salino tamponado) /0.1% de BSA (albumina de soro bovino) e centrifugação (600 xg, 4 min). </li><li> A seguir à centrifugação, desprezar o sobrenadante e ressuspender as SRBCs em 100 ul de 1x PBS / 0,1% BSA e centrifugação (600 xg, 4 min). </li><li> A seguir à centrifugação, desprezar o sobrenadante e ressuspender as SRBCs em 1X PBS / BSA a 0,1% com IgG de coelho anti-SRBCsna sub-aglutinar concentração. Utilizar 500 ul de solução contendo anticorpo / 5 ul de SRBCs. Nota: A concentração sub-aglutinação de anticorpos corresponde à concentração mais baixa que não induziu aglutinação detectada como a formação de uma rede. Em geral, a concentração sub-aglutinação é de 8,2 ug / ml. <ol><li> Determinar a concentração de anti-IgG de SRBCs necessários para opsonizar os SRBCs por um teste de neutralização. Serialmente dilua a anti-IgG de SRBCs (estoque a 13,1 mg / ml) entre 1/50 - 1 / 25.600 de uma microplaca. Adicionar 2 x 10 6 SRBCs em cada poço. Incubar a placa no escuro à temperatura ambiente durante várias horas. </li></ol></li><li> Incubar à temperatura ambiente durante 30 minutos com rotação lenta. </li><li> Após duas lavagens em 1X PBS / BSA a 0,1% como descrito acima, ressuspender os SRBCs-IgG (IgG-opsonizadas SRBCs) em meio isento de soro microscopia pré-aquecido (2 ml / prato). </li></ol> 3. Revestimento de poli-lisina de lamelas <ol><li> Enquanto isso, Tratar pratos 35 milímetros de vidro de fundo com 2 ml de 0,01% de poli-L-lisina durante 30 min à temperatura ambiente. </li><li> Lavar os pratos duas vezes com 2 ml de PBS 1x. </li></ol> 4. Fixação não-covalente de SRBCs em pratos com fundo de vidro <ol><li> Despeje 2 ml de SRBCs suspensão por 35 milímetros prato de vidro de fundo. </li><li> Centrifugar com um rotor girando a 500 xg durante 2 min para 35 milímetros de vidro pratos de fundo. </li><li> Remover o sobrenadante e lavar uma vez com 2 ml de PBS 1X / BSA a 10%. </li><li> Incubam-se as partículas durante 30 min com 2 ml de PBS 1X / BSA a 10% por placa de Petri. </li><li> Lavar os pratos três vezes com 2 ml de PBS 1x. </li><li> Substituir 1x PBS com 2 ml de meio pré-aquecido (37 ° C) sem soro microscopia. </li></ol> 5. A fagocitose visualizado por TIRFM <ol><li> Microscópio Nota: TIRFM foi realizada usando um microscópio equipado com uma objectiva de imersão em óleo (N 100X, NA1.49), uma câmara de aquecimento com CO 2, e duas cantele câmeras de detecção de fótons EMCCD (Electron Multiplicando Charge Coupled Device) acoplado com uma lente de 1.5X. <ol><li> O dia antes da sessão de microscópio TIRF, ligar a câmara de aquecimento a 37 ° C para permitir um aquecimento homogéneo da platina do microscópio. </li></ol></li><li> Determinação ângulo crítico Nota: software ImageJ Color Profiler é usado para processar fluxos de imagem TIRF. <ol><li> Coloque um 35 milímetros prato de vidro de fundo contendo SRBC opsonizado com meio de microscopia sem soro sob o microscópio. </li><li> Raspar as células e ressuspender-los na forma antes de os adicionar (100 - 500 ul) no prato. </li><li> Utilizar o software &quot;Aquisição vivo&quot; para controlar o microscópio e executar de excitação com um 491 nm e / ou um laser de 561 nm para identificar as células que expressam as proteínas marcadas fluorescentemente. </li><li> Identificar uma célula que expressa as proteínas marcadas fluorescentemente. </li><li> Coloque-o no meio do campo e adquirir 500 imas idades em um comprimento de onda de excitação, com ângulos diferentes a partir de 0º até 5º, com um incremento de 0.01º (Figura 2A). Os ângulos são automaticamente alterada pelo sistema de microscópio. </li><li> Determinar o ângulo crítico permitindo que a luz incidente seja totalmente reflectida na interface vidro / médio e gerar a onda evanescente. Usando o software ImageJ Color Profiler, abra a sequência de imagens, clicando em &quot;Arquivo&quot;, &quot;Abrir&quot; e selecione o arquivo. </li><li> Seleccionar uma região de interesse (ROI), com a ferramenta rectangular, na célula com uma fluorescência uniforme (Figura 2B amarelo 1). </li><li> Traçar o &quot;perfil do eixo Z&quot; significa intensidade de fluorescência medida no ROI com a função dos ângulos no eixo x, clicando na guia &quot;Imagem&quot;, &quot;Pilhas&quot; no menu suspenso e &quot;Plot-eixo Z perfil&quot; para baixo (Figura 2B vermelho 2). Nota: O ângulo crítico é o ângulo que conduz à mafluorescência ximum antes de uma queda acentuada na fluorescência. </li><li> Usar qualquer valor de ângulo sobre o eixo x superior ao ângulo crítico durante a sessão de microscopia para obter um sinal TIRF como exemplo 2.00 (Figura 2C). </li></ol></li><li> aquisições <ol><li> Usando o software vivo Aquisição, configurar os parâmetros de aquisição com o módulo &quot;Protocolo Editor&quot; (Figura 3A). <ol><li> Criar um protocolo com um &quot;loop&quot; compreendendo &quot;Canais Multi&quot; aquisição para adquirir sinal fluorescente a partir de proteínas de interesse na região de TIRF. Introduzir o ângulo TIRF (por exemplo 2,00), o tempo de exposição (por exemplo, 50 ms) e a intensidade do laser (por exemplo, 50%) (Figura 3B 1). </li><li> Introduzir um &quot;movimento Z&quot; da mm objectivo dos 3 acima da região TIRF para obter a aquisição de sinal na epifluorescência com o &quot;Canais Multi&quot; ferramenta. Insira um ângulo abaixo tele ângulo crítico (por exemplo 1,00), o tempo de exposição (50 ms) e a intensidade do laser (50%) (Figura 3B, 2 e 3). </li><li> Em seguida adicione um &quot;z move&quot; da mm objectiva 3 abaixo, para retornar na região de TIRF (Figura 3B 4). Adicionar um &quot;instantâneo&quot; da célula em LED (Light-Emitting Diode) (Figura 3B 5) luz brilhante. </li></ol></li><li> Encontrar uma célula de interesse, com um nível moderado de expressão de proteína marcada com fluorescência que inicia a fagocitose de SRBC por membrana plasmática que se estende em torno da partícula. </li><li> Coloque-o no meio do campo. </li><li> Iniciar a transmissão de aquisição de 500 - 1000 quadros. Na aba &quot;contagem de loop&quot;, digite &quot;750 frames&quot; (Figura 3B 1). Nota: software ImageJ Color Profiler é usado para processar fluxos de imagem TIRF. </li><li> Abra as imagens da sequência de programa Image J clicando&quot;Arquivo&quot;, &quot;Abrir&quot; e escolher o arquivo. </li><li> Separar os canais clicando na guia &quot;Imagem&quot;, &quot;Hyperstacks&quot; no menu suspenso e em &quot;Stack para hyperstack&quot; função. Conclua a janela apareceu: Ordem: xyctz; Channel (c): número de canais (ex: &quot;2&quot; se você tem dois fluorocromos); Slices (z): número de fatias em eixo z (ex: &quot;1&quot; se não há movimento no eixo Z); Frames (t): número de imagens divididas por número de canais; Modo de exibição: escala de cinza. Nota: Duas sequências de imagem separados são gerados, correspondendo aos dois canais diferentes. </li></ol></li></ol>

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The authors have nothing to disclose.

The experimental protocol described here proposes an unprecedented method to follow in real time and in living cells, with high-resolution, the formation of a phagosome and in particular its closure. Several technical aspects have to be discussed. Firstly, the assay is very sensitive to temperature. It is very important to check that the heating chamber is at 37 &#176;C and that all media, devices or cells are kept within the chamber to avoid temperature changes that could impair the efficiency of phagocytosis. We notice...

Fagocitose é uma função de célula grande que começa com o reconhecimento e ligação de material para a superfície receptores, que depois leva à internalização e degradação do material ingerido. Enquanto eucariotas unicelulares como o Dictyostelium discoideum molde e amebas usar fagocitose para a alimentação de bactérias, organismos superiores evoluíram com células profissionais. Os macrófagos ou células dendríticas são a primeira linha de defesa contra agentes patogénicos em vários tecidos e órgãos, e são cruciais para activar o sistema imunitário adaptativo através de apresentação de antigénio e produção de citocinas 1-4. Sob certas circunstâncias a fagocitose pode ser realizada por células fagocíticas não profissionais, por exemplo, células endoteliais e epiteliais. Este processo é importante para manter a homeostase durante o desenvolvimento e na vida adulta de renovação do tecido normal e de remodelação. fagócitos Finalmente, especializados, tais como células de Sertoli nos testículos ou da retinanas células epiteliais pigmentares são extremamente potentes fagócitos 5. A formação de um fagossoma onde a degradação de microrganismos ou fragmentos celulares ocorre começa com o agrupamento de receptores fagocíticos na superfície da célula fagocítica. eventos de sinalização a jusante seguintes agrupamento de receptores opsónicos, tais como receptores Fc (FcR) ou receptores de complemento (CRS) têm sido bem caracterizadas. No entanto, também existem numerosas receptores não-opsónicos incluindo receptores semelhantes a Toll (TLRs), lectinas, receptores de manose e receptores scavenger. Estes receptores reconhecem determinantes sobre a superfície das partículas, tais como resíduos de manose ou fucose, fosfatidilserina, e lipopolissacarídeos 1,6-9. Agente patogénico ou fragmentos de células de reconhecimento envolve a ligação ao agrupamento e de vários tipos de receptores de fagocitária, que, em seguida, conduzir a uma intensa e transiente remodelação da actina. Em exocitose paralelo, focal de compartmen intracelularTS contribui para a libertação da tensão da membrana e é importante para a fagocitose eficiente de grandes partículas. Os eventos de sinalização que levam à deformação de polimerização e membrana actina foram dissecados em modelos experimentais que desencadearam um único receptor fagocítica. Durante a fagocitose FcR-mediada, há intensa polimerização de actina, que é regulada por pequenas GTPases (Rac, Cdc42). Entre os seus efectores a jusante, a proteína síndroma de Wiskott-Aldrich (WASP) conduz à activação da proteína relacionada com a actina 2/3 complexo (Arp2 / 3) que nucleia 1,2,4,10 filamentos de actina. A produção local de fosfatidilinositol-4, 5-bifosfato (PI (4,5) P 2) é crucial para a polimerização de actina inicial que dirige a formação pseudópode. Sua conversão em PI (3,4,5) P 3 é necessário para a extensão pseudopod e fechamento fagossomo 11. Diversas vias de contribuir para o desaparecimento de PI (4,5) P 2. Em primeiro lugar destacamento de fosfatidilinositol kina fosfatoses (PIPKIs) do PI prisões fagosoma (4,5) P 2 síntese. Em segundo lugar, pode ser fosforilada e consumido por PI3K de classe I-quinases (PI3K) e convertido em PI (3,4,5) P 3 12. Um papel para fosfatases e fosfolipases também tem sido implicado em PI (4,5) P 2 hidrólise e remoção de F-actina durante a fagocitose em células de mamífero e em Dictyostelium 13,14. O δ fosfolipase C (PLC) hidrolisa PI (4,5) P 2 em diacilglicerol e fosfato de tris-inositol-1,4,5-. O OCRL PI (4,5) P 2 e PI (3,4,5) P 3 fosfatase (síndrome oculocerebrorrenal de Lowe) também tem sido implicado na formação do fagossoma. formação local preciso da F-actina e sua despolimerização é estreitamente regulada no espaço e no tempo e temos demonstrado que o recrutamento de compartimentos intracelulares é importante para entregar localmente a fosfatase OCRL, contribuindo assim para a despolimerização de actina locais na base do copo fagocitária <sup&gt; 13,15. Para isso, utilizou-se o conjunto experimental descrita aqui. Os jogadores mecanismo e moleculares necessários para o encerramento fagossomo e cisão da membrana permanecem pouco conhecidos devido às dificuldades em visualizar e monitorar o local de encerramento fagossomo. Até recentemente, a fagocitose foi observado em células fixas ou vivos que internalizam partículas na face dorsal ou em seus lados, tornando a visualização em tempo útil do local de fechamento fagossomo difícil. Além disso, métodos de fixação pode causar retração de membranas e enviesar os resultados na extensão pseudópodos e encerramento. Em contrapartida, o ensaio que criámos e descrever aqui nos permite visualizar a extensão pseudopod ea etapa de encerramento da fagocitose em células vivas 13, com base em microscopia interna total reflexão (TIRFM) 16. Esta técnica utiliza óptica uma onda evanescente para excitar fluoróforos numa zona fina na interface entre um modo transparentetampa (lamela) e um (meio de cultura de células) de líquido. A espessura da profundidade de excitação é de cerca de 100 nm a partir da superfície sólida, permitindo a visualização de eventos moleculares perto da membrana plasmática. TIRFM permite uma elevada relação de sinal-para-fundo, e limita a fluorescência para fora de foco recolhida e a citotoxicidade devido à iluminação de células. Tomando vantagem de TIRFM, foi desenvolvido o &quot;ensaio de encerramento fagossoma&quot; em que lamelas são activados com poli-lisina e, em seguida, revestida com as células vermelhas do sangue de IgG-IgG-opsonizados (SRBCs). Macrófagos que expressam proteínas transitoriamente fluorescente etiquetado de interesse são, então, autorizado a engolir as IgG SRBCs. Enquanto as células separar as partículas que são alvo não-ligado de forma covalente à superfície do vidro, as pontas dos pseudopods podem ser observadas e registadas no modo de TIRF. aquisições TIRF são combinados com aquisições no modo de epifluorescência, após o deslocamento da uM fase 3 acima, o que permite a visualization da base do copo fagocítica. A adição de drogas farmacológicas, tais como os da inibição da actina ou dinamina durante o processo também é possível, para dissecar ainda mais o processo, a nível molecular. O protocolo descrito em pormenor aqui é para uma linha celular de macrófagos de murídeo e RAW264.7 partículas opsonizadas, mas virtualmente, ele pode ser adaptado a qualquer outra célula fagocítica e com outros objectivos, tais como esferas. Este método permitirá uma melhor caracterização da regulação no tempo e no espaço dos jogadores moleculares envolvidos na extensão pseudopod e fechamento fagossoma durante vários processos fagocíticas.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-sheep red blood cells IgG</td>
			<td>MP Biomedicals</td>
			<td>55806</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin heat shock fraction, pH 7, &#8805;98%&#160;</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lifter</td>
			<td>Corning</td>
			<td>3008</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvettes 4mm</td>
			<td>Cell project</td>
			<td>EP104</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Thermo Fischer Scientific</td>
			<td>14190-094</td>
			<td>Room temperature</td>
		</tr>
		<tr>
			<td>Electrobuffer kit</td>
			<td>Cell project</td>
			<td>EB110</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm TC-Treated Cell Culture Dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Gene X-cell pulser</td>
			<td>Biorad</td>
			<td>165-2661</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin solution</td>
			<td>Sigma</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Dishes 35 mm uncoated 1.5</td>
			<td>MatTek corporation</td>
			<td>P35G-1.5-14-C Case</td>
			<td></td>
		</tr>
		<tr>
			<td>iMIC&#160;</td>
			<td>TILL Photonics</td>
			<td></td>
			<td>&#160;Oil-immersion objective&#160;(N 100x, NA1.49.),&#160; heating chamber with CO2, a camera single photon detection EMCCD ( Electron Multiplying Charge Coupled Device) and a 1.5X lens</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine Solution 0.1%</td>
			<td>Sigma</td>
			<td>P8920-100ml</td>
			<td>Dilution at 0.01% in water&#160;</td>
		</tr>
		<tr>
			<td>RPMI 1640 medium GLUTAMAX&#160; Supplement</td>
			<td>Life technologies</td>
			<td>61870-010</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium, no phenol red (10x500 ml)</td>
			<td>Life technologies</td>
			<td>11835-105</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Sheep red blood cells (SRBCs)</td>
			<td>Eurobio</td>
			<td>DSGMTN00-0Q</td>
			<td>Conserved in Alsever buffer at 4&#176;C before use</td>
		</tr>
	</tbody>
</table>

"phagosome closure assay" to visualize phagosome formation in three dimensions using total internal reflection fluorescent microscopy (tirfm)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

O sistema experimental descrito neste manuscrito está esquematicamente representada na Figura 1. Macrófagos RAW264.7 transfectadas que expressam as proteínas de interesse fundida com uma marcação fluorescente são colocados em contacto com glóbulos vermelhos de ovelha IgG-opsonizado (SRBCs) que eram não-covalentemente fixo sobre a lamela. Os macrófagos pode destacar o SRBC da lamela para afundá-lo. O microscópio utilizado TIRF permite a aquisição concomitante...

Watch this Scientific Journal Video about "Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM at JoVE.com

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

&quot;Fagossomo Encerramento Ensaio&quot; para Visualize Formação fagossomo em Três Dimensões Usando Total Internal Reflection Microscopia fluorescente (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

phagosome-closure-assay-to-visualize-phagosome-formation-three

Research

JoVE Journal

Immunology and Infection

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

6.4K visualizações.  Université Paris Descartes, Sorbonne Paris Cité. We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

Vídeo: "Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

Neutrófilos Traps Extracelular: Como Gerar e visualizá-los

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

Imunologia e Infecção

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

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

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

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

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

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

A observação direta de Fagocitose e NET-formação por neutrófilos em pulmões infectados usando dois fótons Microscopia

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Extração de proteínas e 2-D métodos Eletroforese em Gel de<em&gt; Burkholderia</em&gt; Espécies

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

Usando proteínas fluorescentes para visualizar e Quantificar<em&gt; Chlamydia</em&gt; Vacúolo Crescimento Dynamics em células vivas

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Estudo da fagolisossomo Biogenesis em macrófagos ao vivo

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Utilizando Proteínas Fluorescentes para monitorar glicossomo Dynamics do Trypanosoma Africano

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

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

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

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

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

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

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

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Medindo phagosome pH Raciometrico por microscopia de fluorescência

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

Imaging o neutrófilos fagossoma e citoplasma utilizando um indicador de pH Ratiometric

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

"Fagossomo Encerramento Ensaio" para Visualize Formação fagossomo em Três Dimensões Usando Total Internal Reflection Microscopia fluorescente (TIRFM)

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