Agradecemos Wendy Salmon e Nicki Watson da instalação Keck no Instituto Whitehead, do MIT para a imagem latente.

1. Preparação de DC2.4 e RAW Lines 264,7 celulares NOTA: A linha de células semelhantes a macrófagos RAW 264.7 e o DC2.4 linha de células dendríticas são tanto de origem murina, e as seguintes condições foram usadas para crescer as células. <ol><li> Cultivar células RAW 264.7 em DMEM (meio de Eagle mínimo de Dulbecco) suplementado com 10% de soro bovino fetal inactivado (FBS) a 37 ° C numa incubadora humidificada com 5% de CO 2. </li><li> Cultivar células DC2.4 em condições semelhantes com o de células RAW 264.7, excepto meio de utilização de RPMI (Roswell Park Memorial Institute) suplementado com L-glutamina em vez de DMEM. </li></ol> 2. Preparação de fluorescentes conjugados Partículas: Beads C. albicans, zimosano, IgG-revestidas <ol><li> Crescer Candida albicans <ol><li> Inocular C. albicans estirpe SC5314 a partir de um stock de glicerol congelado em 25 ml de YPD suplementado com Uri (2% de Bacto-peptona, 1%extracto de levedura, 2% de glucose e 80 ug / ml de uridina) durante a noite a 30 ° C. NOTA: Evite a crescer a cultura Candida mais do que uma OD 600 de 12. </li><li> Tomar 1 ml da C. albicans e girá-lo para baixo a 2.000 xg durante 5 min à temperatura ambiente. </li><li> Aspirar o sobrenadante e ressuspender o pellet com 1 ml de PBS. </li><li> Recolhe-se o sedimento por centrifugação a 2000 xg durante 5 min à temperatura ambiente. </li><li> Repita o passo 2.1.4. </li><li> Calcula-se a multiplicidade de infecção (MOI) de C. albicans em relação ao número de fagócitos e prosseguir com o ensaio de fagocitose, como descrito na secção 3. um OD 600 de 1 é equivalente a 2,5 x 10 7 Candida albicans por 1 ml. NOTA: Geração de Candida-BFP é descrito na referência 13. </li></ol></li><li> Preparação de Zymosan e grânulos de IgG-revestidas NOTA: Zymosan é um contendo β-glucana-preparação de hidratos de carbono em partículas fúngica que derivada a partir de Saccharomyces cerevisiae. Zymosan é conhecido por provocar sinais inflamatórios em macrófagos e células dendríticas, e tem sido amplamente utilizado em estudos de fagocitose 13, 14, 15. <ol><li> Resumidamente vortex do tubo que contém as partículas, e aliquotar numa quantidade suficiente para um experimento em um tubo de 1,5 ml. NOTA: Normalmente usamos 1:10 (para uma célula, adicionar 10 partículas de zimosan ou contas de IgG-revestidas). </li><li> Adicionar 1 ml de PBS gelado, e recolher as partículas por centrifugação durante 1 min a 10.000 x g. </li><li> Aspirar o sobrenadante e ressuspender o pellet em PBS 1 ml de gelo-frio. </li><li> Repita os passos 2.2.2-2.2.3 NOTA: Vá para a etapa 2.2.5 ou guarde o tubo a 4 ° C até o uso. </li><li> Sonicar as partículas durante 10 s em sonicador de banho de ultra-sons (120 V, 50-60 kHz), a fim de evitar umaNY agregação das partículas. </li></ol></li></ol> 3. A fagocitose NOTA: A fagocitose é um processo complexo que começa com a ligação das partículas na superfície celular dos fagócitos através da interacção de PRRs com ligandos sobre a superfície da partícula. A ligação é seguida pela montagem de actina e das suas proteínas associadas na local de contacto, e a formação de um copo fagocitária. A desmontagem actina subsequente ocorre no fagossoma e resulta na imersão completa do particulado. Abaixo descrevemos as diferentes fases de fagocitose. <ol><li> Ensaio de Ligação NOTA: O objectivo desta experiência é monitorar o primeiro passo de fagocitose que envolve a interacção entre os ligandos alvo nas partículas e os receptores nos fagócitos. <ol><li> Semente de cerca de 5 x 10 4 células em uma lâmina de câmara de modo que eles são de 60-70% confluentes no momento da experiência. Alterntivamente, células da placa em lamelas (24 ou em formato de 12 poços) ou placas de 96 poços com fundo de vidro. Incubar as células durante a noite a 37 ° C incubadora humidificada com 5% de CO 2. NOTA: As células podem ser contadas usando hemocitômetro ou uma técnica semelhante. </li><li> Colocar a lâmina da câmara (ou placa) a 10 ° C durante 10 min. </li><li> Remova cuidadosamente o meio de cada poço com uma pipeta ou aspirador. ATENÇÃO: Execute esta etapa com cuidado como cada poço de um sistema lamela câmaras é pequeno; é fácil de perturbar as células com a ponta de uma pipeta ou um forte fluxo de ar de um aspirador. </li><li> Misturar as partículas marcadas com fluorescência com um meio de cultura de células (preparado como descrito acima). Adicionar -BFP Cândida (a uma MOI de 10), fluorescentemente grânulos-coelho conjugado com zimosano ou látex de IgG-FITC (10 partículas por célula) das células. Utilizar um volume final de 200 ul de meios de comunicação uma câmara bem para garantir que todas as células são cobertas. </li><li> Gire para baixo o slide câmara a 277xg durante 3 min a 10 ° C. NOTA: Este passo de centrifugação ajuda a aumentar o contato entre as partículas e as células, e sincroniza o processo de ligação. </li><li> Imediatamente Transferir as lâminas para uma câmara incubadora humidificada a 37 ° C com 5% de CO 2 durante 5 min. </li><li> Remover o slide câmara da incubadora e aspirar os meios de comunicação. </li><li> Lavam-se as células 3x com PBS gelado. NOTA: Verifique a presença de partículas livres, com um microscópio de luz e lavar mais vezes com PBS, se necessário. É essencial para remover as partículas não ligadas a partir do bem para minimizar o ruído de fundo durante a análise. </li><li> Fixar as células por adição de 500 ul de paraformaldeido a 4% (PFA) diluída em PBS, por incubação durante 30 min à temperatura ambiente. CUIDADO: Este passo requer o uso de PFA a 4%, que é altamente tóxico, corrosivo e potencial cancerígeno. cuidado apropriado deve ser tomado ao usar este líquido. Além disso, proteger as amostras da luz. </li><li&gt; Lavam-se as células por adição de 500 ul de PBS para dentro da câmara e aspirar a solução suavemente. </li><li> Repita 3.1.9 etapa duas vezes. NOTA: Nesta etapa, encher cada frasco imediatamente após a remoção do PBS anterior. </li><li> Pare a fixação por incubação com 500 ul de 50 mM de NH 4 Cl em PBS, durante 10 min. </li><li> Lavar as células duas vezes com PBS como descrito no passo 3.1.10. </li><li> Permeabilizar as células por adição de 250 uL de tampão de ligação (0,1% de saponina, 0,2% de BSA em PBS) durante 30 min à temperatura ambiente. </li><li> Mancha F-actina por adição de faloidina conjugada fluorescentemente (diluído 1: 500 em tampão de ligação). Incubar as células durante 1 h à temperatura ambiente. </li><li> Lave as células três vezes com PBS como descrito no passo 3.1.10. </li><li> Capturar imagens usando um microscópio confocal (objetiva 63X e 402 nm e 488 nm do laser) e analisar as imagens usando ImageJ 16. NOTA: A ligação Uma partícula de sucesso é caracterizada pela presença de um cu fagocitáriap no local onde o contacto da célula fagocítica partículas (Figura 1). Veja o passo 3.2.5. </li></ol></li><li> Fagocitose de Captação de Ensaio NOTA: Este experimento é projetado para monitorar a internalização completa das partículas marcadas fluorescentes pelos fagócitos meio de microscopia confocal (Figura 2). <ol><li> Semente de cerca de 5 x 10 4 células em uma lâmina de câmara de modo que eles são de 60-70% confluentes no momento da experiência. Alternativamente, células da placa em lamelas (24 ou em formato de 12 poços) ou placas de 96 poços com fundo de vidro. Incubar as células durante a noite a 37 ° C incubadora humidificada com 5% de CO 2. NOTA: As células podem ser contadas usando hemocitômetro ou uma técnica semelhante. </li><li> Manter as lâminas de câmara (ou placa) a 10 ° C durante 10 min. NOTA: Arrefecimento é necessário, a fim de bloquear a endocitose, bem como a fagocitose indesejados, e, subsequentemente, ter uma fagocitose sincronizado. </li><li> perfpassos ORM de 3.1.3 a 3.1.5 </li><li> Transferir as lâminas para uma câmara de 37 ° C incubadora humidificada com 5% de CO 2, e incubar por diferentes pontos de tempo. Fix células após a infecção 30, 60 e 90 min post. Observe C. albicans começam a se formar hifas na infecção pós 60 min; fagócitos começam a mostrar a morte celular (pyroptosis) após 90 min 8. NOTA: Este é uma recomendação, e optimização é essencial, dependendo do tipo de célula e as partículas usadas. </li><li> Execute os passos a partir 3.1.7 a 3.1.15. Analisar imagens usando ImageJ 16. NOTA: uma implantação bem sucedida é caracterizado por uma internalização completa de um material em partículas dentro do citoplasma de um fagócito (Figura 2). A coloração dos fagócitos com marcador da superfície celular, tais como faloidina ajuda a delinear o contorno das células. É também essencial para realizar z pilhas a fim de determinar se as partículas são obrigadas a superfície celular ou completely internalizado. </li></ol></li><li> Imagens ao vivo da fagocitose NOTA: Neste protocolo, descrevem a utilização de microscopia confocal para capturar as diferentes fases de fagocitose usando zimosan conjugado fluorescente. Usamos a linha de células dendríticas DC2.4 que expressa estavelmente mCherry marcada com F-actina 8, um biossensor que mostra a distribuição de actina filamentosa em células vivas. Desde a fagocitose é um processo orientado a actina a imagens ao vivo nesta célula não só nos permite captar o movimento ea dinâmica da rede de F-actina, mas também para visualizar os diferentes eventos fagocíticas no interior das células vivas (Figura 3, Movie 1). <ol><li> Semente de cerca de 5 x 10 4 células em uma lâmina de câmara com uma concentração de modo que eles são de 60-70% confluentes no momento da experiência. Incubar as células durante a noite a 37 ° C incubadora humidificada com 5% de CO 2. NOTA: As células podem ser contadas usando hemocitômetro ou umtécnica similar. </li><li> Manter as lâminas de câmara em gelo durante 10 min. NOTA: Arrefecimento é necessário, a fim de bloquear a absorção indesejável, e tem uma fagocitose sincronizado. </li><li> Execute as etapas de 3.1.3 a 3.1.5 </li><li> Pré-aquecer o estágio de microscópio de 37 ° C e equilibrar a câmara com 5% de CO 2. Espera 30-45 min para a temperatura e o CO 2 a estabilizar antes de iniciar a geração de imagens. NOTA: Uma vez que os CO 2 e temperatura níveis são críticos para a fagocitose, é importante ligar o aquecedor do microscópio antes do experimento para equilibrar a câmara ambiental. </li><li> Colocar a lâmina câmara contendo as células no recinto microscópio incubador equilibrado a 37 ° C e 5% de CO 2. </li><li> Realizar imagem ao vivo 17, 18. NOTA: As definições para a potência do laser (ganho, do furo de pino, etc.) podem variar, dependendo do tipo de the microscópio e sonda fluorescente usada. Portanto, recomendamos consultar o manual do microscópio para a condição ideal para a sua imagem ao vivo 17,18. <ol><li> Use microscópio confocal de varredura a laser (eo software associado) para geração de imagens ao vivo. Use objectivo óleo 63X 1.4NA com um furo de 1-1,1 unidades arejado. </li><li> Para iniciar a criação de imagens, utilizado o campo brilhante para encontrar uma região representativa no slide câmara. Em seguida, selecione o campo clicando na opção Posição sobre o software. Para detectar GFP na Pista 1 usado o conjunto de filtro para 488 nm do laser com um divisor de feixe de 582 nm, para detectar comprimentos de onda entre 488 e 582 nm. </li><li> No Track 2 selecionar um conjunto de filtros ideal para mCherry (RFP), utilizando um divisor de feixe de 578 nm e detectar comprimentos de onda entre 578 e 600 nm. Use 488 nm e 578 nm lasers com potência de laser de 50% e uma saída de ganho de 600-700. Obtenção de imagens a cada 30 seg para um total de 90 min. </li></ol></li></ol></li></ol>

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The authors declare no conflicts of interest.

fagócitos profissionais, tais como macrófagos e células dendríticas, engolir e eliminar agentes patogénicos invasores, portanto, fazer a fagocitose um componente importante do sistema de defesa do hospedeiro. Durante este processo de submeter a fagócitos extensa reorganização da membrana e o rearranjo do citoesqueleto na sua superfície celular 8, 19, 20. Para entender melhor este processo dinâmico, a visualização da...

Apesar de a exposição contínua a agentes patogénicos, tais como bactérias, vírus e fungos, o corpo está bem equipado com o mecanismo imunitário que proporcionam protecção contra a infecção. O sistema imune inato é a primeira linha de defesa contra agentes patogénicos invasores e baseia-se principalmente em células fagocíticas que reconhecem e internalizam alvos estranhos. A fagocitose é um processo celular evolutivamente conservada que engloba o englobamento de partículas não desejadas superiores a 0,5 uM. As células fagocíticas expressar uma vasta gama de receptores imunes (também conhecidos como receptores de reconhecimento de padrões, PRRs) na superfície da célula que lhes permitam reconhecer padrões moleculares associados a agentes patogénicos (PAMPs) presentes sobre os agentes patogénicos anteriores para imersão 3. A ligação patógeno é seguido por agrupamento de receptores na superfície da célula e desencadeia a formação de um copo fagocitária. Isso resulta em remodelação membrana impulsionado-actina que se projeta em torno deo alvo, eventualmente, envolvendo-o e beliscando-off para formar um vacúolo phagosomal discreta 2, 5. O fagossomo então amadurece e acidifica por fusão posterior com endossomas tardios e lisossomas que formam fagolisossomo 6. Embora a fagocitose é descrito como um evento mediado por receptores e orientada para a actina, este processo também depende de modificação espacial-temporal de lípidos que compõem a membrana plasmática, como fosfoinositídeos (IPs) e esfingolipídeos 7, 8. Embora a polimerização da actina é ditada por uma acumulação local de fosfoinositol-4,5-bifosfato (PI (4,5) P 2) na base do copo fagocitária, actina despolimerização depende da conversão de (PI (4,5) P 2 a fosfoinositol-3,4,5-bifosfato (PI (3,4,5) P 3) 3, 9. Ambas as modificaçõessão essenciais como os antigos leva a extensão bem sucedida de pseudopods ao redor do alvo e este último permite afundar de partículas no citosol da phagocyte 10. As células que possuem a capacidade de fagocitar ou são fagócitos profissionais, como macrófagos / monócitos, granulócitos neutrófilos, / e células dendríticas (CDs) ou os fagócitos não profissional, tais como fibroblastos e células epiteliais 11. A fagocitose realizada por todos os fagócitos desempenha um papel central na manutenção e remodelação de tecidos, enquanto que a fagocitose realizada pelos fagócitos profissionais é responsável pela coordenação da resposta imune inata e adaptativa contra os agentes patogénicos. fagócitos profissionais que não apenas engolir e matar o patógeno, mas também apresentam antigénios às células linfóides do sistema imunitário adaptativo. Isso contribui para a liberação de citocinas pró-inflamatórias e ao envolvimento de células linfóides, portanto, levando ao bloqueio de sucesso de infecção de 12. técnicas bioquímicas convencionais têm sido fundamentais para ganhar conhecimento sobre o mecanismo molecular de diferentes processos celulares durante a fagocitose, tais como modificações pós-translacionais e diversas associações de alta afinidade entre proteínas. No entanto, é difícil a obtenção de informações sobre as dinâmicas temporais e espaciais de eventos fagocíticas usando os métodos bioquímicos convencionais. imagens de células vivas não só nos permite monitorar eventos celulares de uma forma sensível tempo, mas também nos permite obter informações em um nível única célula. Descrevemos aqui um método para investigar os diferentes estágios de fagocitose, bem como para analisar todo o processo de espaço-temporalmente utilizando microscopia confocal.

<table><tbody><tr><td>&amp;beta;-Mercaptoethanol</td><td>AppliChem</td><td>A1108</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Cell Signaling Technology&amp;nbsp;</td><td>9998</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dilactate)</td><td>ThermoFisher</td><td>D3571</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>ThermoFisher</td><td>BP-231-1</td><td></td></tr><tr><td>DMEM (Dulbecco&amp;rsquo;s Minimal Eagle&amp;rsquo;s medium)</td><td>Gibco</td><td>11965</td><td></td></tr><tr><td>PBS, 1x (Phosphate- Buffered Saline)</td><td>Corning cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>Fetal Bovine serum</td><td>Sigma Aldrich</td><td>12003C</td><td></td></tr><tr><td>FITC-coupled IgG-coated latex beads</td><td>Cayman</td><td>500290</td><td></td></tr><tr><td>L-Glutamine 200 mM (100x)</td><td>ThermoFisher</td><td>25030081</td><td></td></tr><tr><td>Paraformaldehyde Solution (4% in PBS)</td><td>Affymetrix</td><td>19943 1 LT</td><td></td></tr><tr><td>Penicillin-streptomycin (10,000 U/ml)</td><td>ThermoFisher</td><td>&lt;a href=&quot;https://www.fishersci.com/shop/products/gibco-penicillin-streptomycin-10-000-u-ml-3/15140122&quot;&gt;15-140-122&lt;/a&gt;</td><td></td></tr><tr><td>Phalloidin-Alexa Fluor 488</td><td>ThermoFisher</td><td>A12379</td><td></td></tr><tr><td>RAW 264.7 cells</td><td>ATCC</td><td>TIB-71</td><td></td></tr><tr><td>RPMI (Roswell Park Memorial Institute)</td><td>Gibco</td><td>61870</td><td></td></tr><tr><td>Saponin</td><td>Sigma Aldrich</td><td>S7900</td><td></td></tr><tr><td>Trypan blue solution (0.4% (w/v) in PBS)</td><td>Corning cellgro</td><td>MT25900CI</td><td></td></tr><tr><td>Trypsin-EDTA (1x) (0.05%)</td><td>ThermoFisher</td><td>25300054</td><td></td></tr><tr><td>Tween 20 Surfact-Amps Detergent Solution</td><td>ThermoFisher</td><td>&amp;nbsp;85114</td><td></td></tr><tr><td>Zymosan-Alexa Fluor 594</td><td>ThermoFisher</td><td>Z23374</td><td></td></tr><tr><td>Chambered 1.0 Borosilicate Coverglass system (8 chambers)</td><td>ThermoFisher</td><td>155361</td><td></td></tr><tr><td>Glasstic slide 10 with grids</td><td>Hycor</td><td>87144</td><td></td></tr></tbody></table>

visualizing the early stages of phagocytosis

O corpo dos mamíferos está equipado com várias camadas de mecanismos que ajudam a defender de invasões de patógenos. Fagócitos profissionais do sistema imunitário - neutrófilos, tais como células dendríticas, macrófagos e - manter a capacidade inata para detectar e eliminar tais agentes patogénicos invasores através de fagocitose 1. Fagocitose envolve eventos coreografia de reorganização da membrana e remodelação da actina na superfície da célula 2, 3. Fagócitos internalizar com sucesso e erradicar moléculas estranhas somente quando todas as fases de fagocitose são cumpridas. Estas etapas incluem o reconhecimento e a ligação do patogénio pelos receptores de reconhecimento de padrões (SRRS) residem na superfície celular, formação de copo fagocítica através de saliências membranosas enriquecido-actina (pseudópodes) para envolver as partículas, e cisão do fagossoma seguida de maturação fagolisossomo que resulta namatando do patógeno 3, 4. Imagem e quantificação de vários estágios de fagocitose é instrumental para elucidar os mecanismos moleculares deste processo celular. O presente manuscrito relata métodos para estudar as diferentes fases de fagocitose. Nós descrevemos uma abordagem baseada em microscópio para visualizar e quantificar a, formação de copo fagocitária de ligação, ea internalização de partículas pelos fagócitos. Como fagocitose ocorre quando os receptores inatos em células fagocíticas encontrar ligandos-alvo numa partícula maior do que 0,5 fim, os ensaios aqui apresentados compreendem a utilização de fungos patogénicos Cândida albicans e outras partículas, tais como grânulos zimosano e IgG-revestidos.

método baseado em microscópio para monitorizar as diferentes fases da fagocitose é apresentada. Os diferentes eventos durante a fagocitose de várias partículas fluorescentes por células DC2.4 são mostrados. Utilizando as técnicas descritas aqui, foram investigados o papel de esfingolípidos nas fases iniciais de fagocitose. Para este fim, células dendríticas DC2.4 geneticamente deficientes em Sptlc2, a enzima que catalisa o primeiro e limitante da velocidade passo na via de biossíntese dos esfingolípidos, fo...

Watch this Scientific Journal Video about Visualizing the Early Stages of Phagocytosis at JoVE.com

Visualizing the Early Stages of Phagocytosis

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

Visualizando os estágios iniciais da fagocitose

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...

visualizing-the-early-stages-of-phagocytosis

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

16.6K visualizações.  Oregon Health & Science University. Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 µm including zymosan and IgG-coated beads.

Vídeo: Visualizing the Early Stages of Phagocytosis

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Live-célula Microscopia vídeo de Fagocitose patógeno fúngico

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Imunologia e Infecção

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 ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

Técnica High Throughput Fluorometric de Avaliação da macrófago fagocitose e actina Polimerização

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

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 ...

"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.

"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 ...

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 ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Time-Lapse imagem em 3D da fagocitose por macrófagos de rato

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.

Investigando a Faagocytose da Leishmania usando Microscopia Confocal

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up ...

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (M&#934;) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study M&#934; phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance M&#934; phagocytosis of non-opsonized zymosan of both naive and IFN-&#947; treated M&#934;. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (M&#934;) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent molecule.

Regulação de células-tronco mesenquimais da fagocitose macrófago; Quantitação e Imagem

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics ...

Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells

Source: Horsthemke, M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/57566">Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages.</a> J. Vis. Exp. (2018)
This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.

Imagem em tempo real da fagocitose de macrófagos de glóbulos vermelhos opsonizados por IgG

Source: Horsthemke, M., et al. Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages. J. Vis. Exp. (2018)
This video demonstrates an assay to monitor ...