The authors are financially supported by the Swiss National Science Foundation through an operating grant N&#176; 31003A-149566 (to N.D.), and The Sir Jules Thorn Charitable Overseas Trust through a Young Investigator Subsidy (to P.N.).

Declaração de Ética: Todas as manipulações em animais foram realizados em estrita conformidade com as diretrizes do Comitê da Universidade de Genebra Research Animal. 1. Preparação de Metas fagocíticas <ol><li> Adicionar 20 mg de zimosan secou-se para 10 ml de solução salina tamponada com fosfato estéril (PBS). Agitar em vórtice e aquecimento num banho de água a ferver durante 10 min. Arrefecer e centrifugar a 2000 xg durante 5 min. </li><li> Remover o sobrenadante, ressuspender em 1 ml de PBS e sonicado durante 10 minutos num banho de água sonicador. Transferir 500 uL de dois tubos de 1,5 ml. Centrifugar a 11.000 xg durante 5 min. </li><li> Repita a lavagem com 500 mL PBS. O zimosan fervida pode ser dividido em alíquotas, congelada e armazenada a -20 ° C. </li><li> Lavar duas vezes zimosan em 500 ul preparadas de fresco 0,1 M Na 2 CO 3, pH 9,3 como anteriormente (passo 1.2). </li><li> Ressuspender em 490 mL após a lavagem final. Adicionar 10 ul de 10 mg / ml de FITC dissolvido em dimetil sulfoxidE (DMSO), vortex, cobrir com folha, e incuba-se com agitação durante 4 h à TA. </li><li> Para remover o corante não ligado, lavar como indicado acima (passo 1.2), em primeiro lugar com 0,5 mL de DMSO + 150 ul de PBS, em seguida, 0,5 ml de DMSO + 300 ul de PBS, em seguida, 0,5 ml de DMSO + 450 ul de PBS, em seguida, apenas PBS. </li><li> Contagem de zimosan utilizando um hemocitómetro. Adicionar 1 ml de zymosan a 500 ul de PBS, vórtice, em seguida, adicionar 10 ul para a extremidade da câmara de hemocitómetro, onde se encontra com a lamela colocada sobre a grelha central. Montar o hemocitómetro em um microscópio de campo brilhante equipado com uma objectiva 20X. </li><li> Concentre-se no canto superior esquerdo contendo 16 quadrados e contam todas as partículas de zimosan nesta área, utilizando um contador de contagem mão. Repita com o canto inferior direito. Calcular a concentração de zimosan com a seguinte equação: concentração Zymosan = Contagem / 2 x 500 x 10 4 zimosan / ml. Zimosan marcado pode ser dividido em alíquotas, congelada e armazenada a -20 ° C. </li><li> Opsonizar o wi zymosan rotuladath um anticorpo de coelho anti-zimosan na diluição 1: 100. Vortex e incubar durante 1 hora em um banho de água a 37 ° C. Lava-se com 500 ul de PBS como acima (passo 1.2). Nota: A sonicação é altamente recomendada, particularmente depois de opsonização, como zimosan tende a formar grumos que podem tornar mais difícil a análise mais tarde. A opsonização com 65% v / v de soro de rato ou humano podem ser utilizados como uma alternativa. </li><li> Contagem de zimosan utilizando um hemocitómetro como descrito nos passos de 1,7-1,8, e diluir para 100 x 10 6 de partículas / ml. A opsonização com outros estimuladores fagocíticas, tais como o complemento ou nenhuma opsonização, pode ser alternativamente realizada. </li></ol> 2. Live Video Microscopia <ol><li> Isolar neutrófilos primários de ratinho, tal como descrito em detalhe noutro local 38. <ol><li> Como alternativa, use qualquer tipo de célula fagocitária. Manter em gelo neutrófilos no Roswell Park Memorial Institute (RPMI) 1640 suplementado com 5% de soro de vitelo fetal (v / v), 200 U de penicilina / streptomycin, a uma concentração de 10 x 10 6 células / ml até estar pronto para utilização. Outros tipos de células podem ser semeadas 1-4 dias antes. </li></ol></li><li> Adicionar 2 x 10 5 (20 ul) de neutrófilos para o centro de um prato de 35 mm com fundo de vidro, gota a espalhar pela adição de 50 uL de meio e incubar a 37 ° C durante 5 min para permitir que as células a aderirem. </li><li> Adicionar 1 ml de solução salina equilibrada de Hank (HBSS) aquecido a 37 ° C contendo o inibidor de MPO específico hidrazida 4-aminobenzóico (4-ABH, 10 uM). Os inibidores não-específicos de MPO, tais como azida de sódio (5 mM), também podem ser usados, mas têm sido sugeridos recentemente para exercer efeitos não-específicos adicionais de pH 39. </li><li> Montar o prato num microscópio de campo amplo de fluorescência equipado com um sistema de aquecimento de 37 ° C e uma objectiva de óleo de 40X. Incubar as células sem imagem durante 10 min para permitir que as células e material para equilibrar. </li><li> Ajuste as configurações de microscópio para adquirir uma imagem de transmissão de campo brilhante [phase ou contraste de interferência diferencial (DIC) se disponível] com 440 nm ou 490 nm de excitação e 535 nm de emissão a cada 30 seg. </li><li> Ajuste o tempo de exposição e freqüência de aquisição de acordo com o sistema de microscópio para evitar o excesso de fotodegradação e fototoxicidade e, dependendo das questões experimentais sejam colocadas. Comece a aquisição de imagem. </li><li> Após 2 minutos, adicionam-se 10 x 10 6 (50 ul) pelo zimosan opsonizado com FITC para o centro do prato. Ajuste alvo: rácios de células, dependendo do tipo de fagócitos e alvo utilizado. </li><li> Continuar imagiologia durante 30 min. Após 30 min parar a aquisição de lapso de tempo e iniciar um temporizador. Mova o palco e capturar 10 ou mais instantâneos em diferentes campos de visão a imagem outros phagosomes, tudo dentro de 5 min. </li></ol> 3. Calibração e Controle de Experimentos <ol><li> Prepare as soluções de calibração pH (receitas estão descritos na Tabela 1) antes do dia de experimento e congelar em alíquotas de 10 ml.Descongelar as soluções de calibração e aquecer até 37 ° C num banho de água. Verificar o pH com um medidor de pH e registrar o pH real de soluções. </li><li> Montar uma bomba peristáltica na parte inferior prato de vidro antes da aquisição da imagem e realizar microscopia de vídeo ao vivo como descrito acima (seção 2). </li><li> No final do período de aquisição 30 min ligar a bomba para remover a solução dentro do prato, adicionar 1 ml da primeira solução de calibração e parar a bomba. </li><li> Aguarde até que o sinal estabilize, geralmente 5 min. Repita com cada solução de calibração. <ol><li> Executar, pelo menos, uma calibração por dia experimental, calibrar e começando com o mais elevado pH e trabalhando sequencialmente para o mais baixo, bem como a partir do pH mais baixo e trabalhar sequencialmente para o mais alto. </li></ol></li><li> Como um controlo, adicionar 100 uL de 100 uM de inibidor de NADPH-oxidase iodeto difenil propionium (DPI), diluída em HBSS para as células em 900 ul de HBSS no prato de vidro de fundo, eincube durante 10 min. </li><li> Comece aquisição e adicionar zymosan como acima (passo 2.7). Após 15 min de fagocitose, adicionar 10 ul de 10 mM de inibidor V-ATPase concanamycin A (ConcA) diluídos em HBSS e continuar imagiologia durante mais 15 minutos adicionais. </li></ol> 4. Análise <ol><li> Análise de filmes de lapso de tempo e instantâneos fagosoma. Nota: As instruções a seguir são específicos para ImageJ. Passo-a-passo as instruções podem variar muito, dependendo do software utilizado, mas as funções descritas nesta seção estão disponíveis na maioria dos softwares de análise de imagem profissional. <ol><li> Abra as imagens com software de análise de imagem profissional. Subtrair o fundo no canal 490 por desenhar um pequeno quadrado ROI com a ferramenta de seleção polígono quadrado em uma área onde não há células e clicando em Plugins&gt; BG Subtração de ROI. Em seguida, fazer o upload do mesmo ROI no canal 440, clicando em Editar&gt; Seleção&gt; Restaurar seleção e repita. </li><li>Faça uma imagem de relação de 32-bit do 490 canal dividido pelo canal 440, clicando em Processo&gt; Calculadora da imagem ..., selecionando as imagens apropriadas nos menus drop-down Image1 e Image2, em seguida, selecionando &#39;Divide&#39; no menu drop-down Operação e verificando resultado caixa de seleção de 32 bits (float). </li><li> Alterar tabela de consulta a codificação de cores para uma codificação de cores proporção compatível clicando Image&gt; Tabelas de Pesquisa&gt; Rainbow RGB. Limite a relação da imagem para eliminar a 0 e infinito pixels clicando Image&gt; Adjust&gt; Threshold .... Ajuste as barras de rolagem para que zymosan aparece em vermelho e clique em Aplicar, certificando-se os Set pixels de fundo caixa de seleção para NaN é selecionado. </li><li> Combine isso pilha relação com o canal de campo brilhante em um composto multi-canal clicando em Imagem&gt; Cor&gt; Mesclar Canais, e seleccionando a imagem de campo brilhante no menu canal suspenso cinza ea imagem relação no menu dropdown canal verde, e seleccionando oMantenha imagens de origem check-box. Digitalizar os filmes de lapso de tempo ou instantâneos para determinar quais phagosomes estão a ser analisadas. O canal de campo brilhante é útil para este. </li><li> Desenhe regiões de interesse (ROI) usando a ferramenta de seleção polígono oval em torno phagosomes, e adicioná-los para o gerente ROI clicando Analisar&gt; Ferramentas&gt; ROI Manager&gt; Adicionar. Meça o seu rácio de intensidade zymosan clicando em Mais&gt; Multi-Measure e de-selecionar o Uma linha fatia check-caixa por. </li><li> Para filmes de lapso de tempo, trilha fagossomos um de cada vez, desenhando um ROI, digitando Ctrl + M para medir cada ponto de tempo, e movendo o ROI quando necessário para rastrear valores de intensidade ao longo do tempo. Copie as medidas da janela de resultados em um software de planilha. Nota: Análise de 15 (5 cursos de tempo por experimentação x 3) experiências independentes é ideal, mas mais ou menos pode ser necessária, dependendo da variabilidade observada. Guardar as ROIs é sempre recomendável. Alguns pacotes de software permitem que regi imagemstration e atualização dinâmica de ROIs, facilitando dessa parte da análise. </li></ol></li><li> A conversão de fluorescência a valores de pH <ol><li> Medir a razão 490/440 para phagosomes na calibração filmes de lapso de tempo como descrito na seção 4.1. </li><li> Em uma planilha, traçar os valores da relação com o tempo, e calcular os valores médios da relação de todos os phagosomes dentro do campo de visão (geralmente, entre 5-20) a partir de 5 pontos temporais no meio do período de tempo correspondente a cada calibração de pH solução. (Veja também as caixas vermelhas na Figura 5A.) </li><li> Traçar estas médias de 490/440 valores da razão contra o pH de medição atual. Combinar pelo menos 3 calibrações independentes num único gráfico. Use um pacote de software estatístico ou numérica para executar uma regressão não linear (curva de melhor ajuste), geralmente a uma equação sigmoidal. Nota: Por exemplo, na Figura 5B a equação utilizada foi uma sigmoidal de Boltzmann [Y = Fundo + ((Top-Bottom)/ (1 + EXP ((V50-X) / Slope))], onde valores Y são as relações 490/440, X são os valores de pH e parte superior, inferior, parâmetros V50 e inclinação são calculados pelo software. </li><li> Use a equação obtida para transformar valores da relação de pH. Nota: Por exemplo, na Figura 5B da equação obtida para a curva de calibração na presença de azida de sódio é Rácio 490/440 = 1,103 + [(2,89-1,103) / (1 ​​+ EXP ((6,993-pH) /0.9291)] . Resolvendo para pH dá a seguinte equação: pH = 6,993-0,9291 * [LN ((1.178 / (Ratio-1.103)) - 1))]. </li></ol></li></ol>

<ol>
	<li>Yeung, T., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+signaling+and+the+modulation+of+surface+charge+during+phagocytosis.">Lipid signaling and the modulation of surface charge during phagocytosis.</a> Immunol Rev. 219, 17-36 (2007).</li><li>Flannagan, R. S., Jaumouille, V., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+phagocytosis.">The cell biology of phagocytosis.</a> Annu Rev Pathol. 7, 61-98 (2012).</li><li>Fairn, G. D., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+nascent+phagosomes+mature+to+become+phagolysosomes.">How nascent phagosomes mature to become phagolysosomes.</a> Trends Immunol. 33 (8), 397-405 (2012).</li><li>Nunes, P., Demaurex, N., Dinauer, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+NADPH+oxidase+and+associated+ion+fluxes+during+phagocytosis.">Regulation of the NADPH oxidase and associated ion fluxes during phagocytosis.</a> Traffic. 14 (11), 1118-1131 (2013).</li><li>Binder, B., Holzhutter, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hypothetical+model+of+cargo-selective+rab+recruitment+during+organelle+maturation.">A hypothetical model of cargo-selective rab recruitment during organelle maturation.</a> Cell Biochem Biophys. 63 (1), 59-71 (2012).</li><li>Hurtado-Lorenzo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V-ATPase+interacts+with+ARNO+and+Arf6+in+early+endosomes+and+regulates+the+protein+degradative+pathway.">V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway.</a> Nat Cell Biol. 8 (2), 124-136 (2006).</li><li>Weisz, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acidification+and+protein+traffic.">Acidification and protein traffic.</a> Int Rev Cytol. 226, 259-319 (2003).</li><li>Huynh, K. K., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+vacuolar+pH+and+its+modulation+by+some+microbial+species.">Regulation of vacuolar pH and its modulation by some microbial species.</a> Microbiol Mol Biol Rev. 71 (3), 452-462 (2007).</li><li>De Vito, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sodium/hydrogen+exchanger:+a+possible+mediator+of+immunity.">The sodium/hydrogen exchanger: a possible mediator of immunity.</a> Cell Immunol. 240 (2), 69-85 (2006).</li><li>Moreland, J. G., Davis, A. P., Bailey, G., Nauseef, W. M., Lamb, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anion+channels,+including+ClC-3,+are+required+for+normal+neutrophil+oxidative+function,+phagocytosis,+and+transendothelial+migration.">Anion channels, including ClC-3, are required for normal neutrophil oxidative function, phagocytosis, and transendothelial migration.</a> J Biol Chem. 281 (18), 12277-12288 (2006).</li><li>Winterbourn, C. C., Kettle, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+reactions+and+microbial+killing+in+the+neutrophil+phagosome.">Redox reactions and microbial killing in the neutrophil phagosome.</a> Antioxid Redox Signal. 18 (6), 642-660 (2013).</li><li>Seredenina, T., Demaurex, N., Krause, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-Gated+Proton+Channels+as+Novel+Drug+Targets:+From+NADPH+Oxidase+Regulation+to+Sperm+Biology.">Voltage-Gated Proton Channels as Novel Drug Targets: From NADPH Oxidase Regulation to Sperm Biology.</a> Antioxid Redox Signal. , (2014).</li><li>Kotsias, F., Hoffmann, E., Amigorena, S., Savina, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+production+in+the+phagosome:+impact+on+antigen+presentation+in+dendritic+cells.">Reactive oxygen species production in the phagosome: impact on antigen presentation in dendritic cells.</a> Antioxid Redox Signal. 18 (6), 714-729 (2013).</li><li>Grimm, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte-+and+macrophage-targeted+NADPH+oxidase+mediates+antifungal+host+defense+and+regulation+of+acute+inflammation+in+mice.">Monocyte- and macrophage-targeted NADPH oxidase mediates antifungal host defense and regulation of acute inflammation in mice.</a> J Immunol. 190 (8), 4175-4184 (2013).</li><li>West, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR+signalling+augments+macrophage+bactericidal+activity+through+mitochondrial+ROS.">TLR signalling augments macrophage bactericidal activity through mitochondrial ROS.</a> Nature. 472 (7344), 476-480 (2011).</li><li>Bloomfield, G., Pears, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+signalling+required+for+multicellular+development+of+Dictyostelium.">Superoxide signalling required for multicellular development of Dictyostelium.</a> J Cell Sci.. 116 (Pt 16), 3387-3397 (2003).</li><li>Ramsey, I. S., Moran, M. M., Chong, J. A., Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+voltage-gated+proton-selective+channel+lacking+the+pore+domain.">A voltage-gated proton-selective channel lacking the pore domain.</a> Nature. 440 (7088), 1213-1216 (2006).</li><li>El Chemaly, A., Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+Hv1+proton+channels+regulate+the+ionic+and+redox+homeostasis+of+phagosomes?.">Do Hv1 proton channels regulate the ionic and redox homeostasis of phagosomes?.</a> Mol Cell Endocrinol. 353 (1-2), 82-87 (2012).</li><li>El Chemaly, A., Nunes, P., Jimaja, W., Castelbou, C., Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hv1+proton+channels+differentially+regulate+the+pH+of+neutrophil+and+macrophage+phagosomes+by+sustaining+the+production+of+phagosomal+ROS+that+inhibit+the+delivery+of+vacuolar+ATPases.">Hv1 proton channels differentially regulate the pH of neutrophil and macrophage phagosomes by sustaining the production of phagosomal ROS that inhibit the delivery of vacuolar ATPases.</a> J Leukoc Biol. , (2014).</li><li>Decoursey, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-gated+proton+channels.">Voltage-gated proton channels.</a> Compr Physiol. 2 (2), 1355-1385 (2012).</li><li>Ramsey, I. S., Ruchti, E., Kaczmarek, J. S., Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hv1+proton+channels+are+required+for+high-level+NADPH+oxidase-dependent+superoxide+production+during+the+phagocyte+respiratory+burst.">Hv1 proton channels are required for high-level NADPH oxidase-dependent superoxide production during the phagocyte respiratory burst.</a> Proc Natl Acad Sci U S A. 106 (18), 7642-7647 (2009).</li><li>Sturgill-Koszycki, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+acidification+in+Mycobacterium+phagosomes+produced+by+exclusion+of+the+vesicular+proton-ATPase.">Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase.</a> Science. 263 (5147), 678-681 (1994).</li><li>Clemens, D. L., Lee, B. Y., Horwitz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulent+and+avirulent+strains+of+Francisella+tularensis+prevent+acidification+and+maturation+of+their+phagosomes+and+escape+into+the+cytoplasm+in+human+macrophages.">Virulent and avirulent strains of Francisella tularensis prevent acidification and maturation of their phagosomes and escape into the cytoplasm in human macrophages.</a> Infect Immun. 72 (6), 3204-3217 (2004).</li><li>Alpuche-Aranda, C. M., Swanson, J. A., Loomis, W. P., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+typhimurium+activates+virulence+gene+transcription+within+acidified+macrophage+phagosomes.">Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes.</a> Proc Natl Acad Sci U S A. 89 (21), 10079-10083 (1992).</li><li>Lukacs, G. L., Rotstein, O. D., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+the+phagosomal+pH+in+macrophages.+In+situ+assessment+of+vacuolar+H(+)-ATPase+activity,+counterion+conductance,+and+H++'leak'.">Determinants of the phagosomal pH in macrophages. In situ assessment of vacuolar H(+)-ATPase activity, counterion conductance, and H+ 'leak'.</a> J Biol Chem. 266 (36), 24540-24548 (1991).</li><li>Watanabe, K., Kagaya, K., Yamada, T., Fukazawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+for+candidacidal+activity+in+macrophages+activated+by+recombinant+gamma+interferon.">Mechanism for candidacidal activity in macrophages activated by recombinant gamma interferon.</a> Infect Immun. 59 (2), 521-528 (1991).</li><li>Ip, W. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+and+phagosome+acidification+are+required+for+pathogen+processing+and+MyD88-dependent+responses+to+Staphylococcus+aureus.">Phagocytosis and phagosome acidification are required for pathogen processing and MyD88-dependent responses to Staphylococcus aureus.</a> J Immunol. 184 (12), 7071-7081 (2010).</li><li>Clarke, M., Maddera, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocyte+meets+prey:+uptake,+internalization,+and+killing+of+bacteria+by+Dictyostelium+amoebae.">Phagocyte meets prey: uptake, internalization, and killing of bacteria by Dictyostelium amoebae.</a> Eur J Cell Biol. 85 (9-10), 1001-1010 (2006).</li><li>Jankowski, A., Scott, C. C., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+the+phagosomal+pH+in+neutrophils.">Determinants of the phagosomal pH in neutrophils.</a> J Biol Chem. 277 (8), 6059-6066 (2002).</li><li>Segal, A. W., Geisow, M., Garcia, R., Harper, A., Miller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+respiratory+burst+of+phagocytic+cells+is+associated+with+a+rise+in+vacuolar+pH.">The respiratory burst of phagocytic cells is associated with a rise in vacuolar pH.</a> Nature. 290 (5805), 406-409 (1981).</li><li>Savina, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOX2+controls+phagosomal+pH+to+regulate+antigen+processing+during+crosspresentation+by+dendritic+cells.">NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells.</a> Cell. 126 (1), 205-218 (2006).</li><li>Rybicka, J. M., Balce, D. R., Chaudhuri, S., Allan, E. R., Yates, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosomal+proteolysis+in+dendritic+cells+is+modulated+by+NADPH+oxidase+in+a+pH-independent+manner.">Phagosomal proteolysis in dendritic cells is modulated by NADPH oxidase in a pH-independent manner.</a> EMBO J. 31 (4), 932-944 (2012).</li><li>Mantegazza, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADPH+oxidase+controls+phagosomal+pH+and+antigen+cross-presentation+in+human+dendritic+cells.">NADPH oxidase controls phagosomal pH and antigen cross-presentation in human dendritic cells.</a> Blood. 112 (12), 4712-4722 (2008).</li><li>Balce, D. R., Allan, E. R., McKenna, N., Yates, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma-Interferon-inducible+lysosomal+thiol+reductase+(GILT)+maintains+phagosomal+proteolysis+in+alternatively+activated+macrophages.">gamma-Interferon-inducible lysosomal thiol reductase (GILT) maintains phagosomal proteolysis in alternatively activated macrophages.</a> J Biol Chem. 289 (46), 31891-31904 (2014).</li><li>Sanjurjo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+scavenger+protein+apoptosis+inhibitor+of+macrophages+(AIM)+potentiates+the+antimicrobial+response+against+Mycobacterium+tuberculosis+by+enhancing+autophagy.">The scavenger protein apoptosis inhibitor of macrophages (AIM) potentiates the antimicrobial response against Mycobacterium tuberculosis by enhancing autophagy.</a> PLoS One. 8 (11), e79670 (2013).</li><li>Yuan, Z., Zhi, L., Li-Juan, Z., Hong-Tao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Utility+of+Chloroquine+in+Cancer+Therapy.">The Utility of Chloroquine in Cancer Therapy.</a> Curr Med Res Opin. , 1-12 (2015).</li><li>Ohkuma, S., Poole, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+probe+measurement+of+the+intralysosomal+pH+in+living+cells+and+the+perturbation+of+pH+by+various+agents.">Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents.</a> Proc Natl Acad Sci U S A. 75 (7), 3327-3331 (1978).</li><li>El Chemaly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VSOP/Hv1+proton+channels+sustain+calcium+entry,+neutrophil+migration,+and+superoxide+production+by+limiting+cell+depolarization+and+acidification.">VSOP/Hv1 proton channels sustain calcium entry, neutrophil migration, and superoxide production by limiting cell depolarization and acidification.</a> J Exp Med. 207 (1), 129-139 (2010).</li><li>Levine, A. P., Duchen, M. R., Segal, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HVCN1+channel+conducts+protons+into+the+phagocytic+vacuole+of+neutrophils+to+produce+a+physiologically+alkaline+pH.">The HVCN1 channel conducts protons into the phagocytic vacuole of neutrophils to produce a physiologically alkaline pH.</a> bioRxiv. , (2014).</li><li>Al-Fageeh, M. B., Smales, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+and+regulation+of+the+cellular+responses+to+cold+shock:+the+responses+in+yeast+and+mammalian+systems.">Control and regulation of the cellular responses to cold shock: the responses in yeast and mammalian systems.</a> Biochem J. 397 (2), 247-259 (2006).</li><li>Yui, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basolateral+targeting+and+microtubule-dependent+transcytosis+of+the+aquaporin-2+water+channel.">Basolateral targeting and microtubule-dependent transcytosis of the aquaporin-2 water channel.</a> Am J Physiol Cell Physiol. 304 (1), C38-C48 (2013).</li><li>Griffiths, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+phagosome+individuality+and+membrane+signalling+networks.">On phagosome individuality and membrane signalling networks.</a> Trends Cell Biol. 14 (7), 343-351 (2004).</li><li>Poburko, D., Santo-Domingo, J., Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+regulation+of+the+mitochondrial+proton+gradient+during+cytosolic+calcium+elevations.">Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations.</a> J Biol Chem. 286 (13), 11672-11684 (2011).</li></ol>

The authors have nothing to disclose.

Apesar de ser mais demorada do que os métodos alternativos, tais como a espectroscopia e FACS, que empregam uma estratégia semelhante de um corante utilizando sensível ao pH acoplado a alvos mas medir o pH médio de uma população de fagossomas, microscopia oferece várias vantagens. Primeiro é que interno e externo ligado, mas não internalizadas, as partículas podem facilmente ser distinguidos sem ter que adicionar outros produtos químicos, tais como azul de tripano ou anticorpos, para extinguir ou rotular part...

Phagocytosis, the process through which innate immune cells engulf large particles, evolved from the eating mechanism of single-celled organisms, and involves binding to a target, enveloping it with a membrane and pinching the membrane off to form a vacuole within the cytosol called a phagosome. While the phagosomal membrane is derived from the plasma membrane, active protein and lipid sorting, as well as fusion with endomembranes during phagosome formation, transform the phagosome into a distinct organelle within the cell with degradative properties that allow the killing, neutralization and breakdown of the ingested material1-3. This process, called phagosomal maturation, relies on the delivery of a host of proteolytic and microbicidal enzymes, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which transfers electrons into phagosomes producing the strong oxidant O2- and its derivative reactive oxygen species (ROS) 2,4. 
The luminal pH of phagosomes has a profound influence on several events required for phagosome function. First, pH influences trafficking of endomembranes in general, as pH-dependent conformational changes of transmembrane trafficking regulators alters the recruitment of trafficking determinants such as Arfs, Rabs and vesicular coat-proteins, which in turn define which vesicles may fuse with phagosomes 5-8. Second, the ionic composition of the phagosomal lumen is also transformed as phagosomes mature, and some ion transporters, such as the Na+/H+ exchanger or ClC family Cl-/H+ antiporters, which promote phagocytic function, rely on H+ translocation 9,10. Similarly, ROS production is intimately linked with phagosomal pH. ROS and its toxic oxidant byproducts have long been recognized as crucial for phagosomal killing in neutrophils 4,11,12, and have been shown to play critical roles in other phagocytes including macrophages, dendritic cells (DCs) and amoeba 13-16. The NADPH oxidase is an electrogenic enzyme that releases H+ in the cytosol as NADPH is consumed, and that requires the simultaneous transfer of H+ through companion HVCN1 channels alongside the transported electrons into the phagosomal lumen, in order to alleviate the massive depolarization that would otherwise lead to self-inhibition of the enzyme 17-21. Finally, several proteolytic enzymes have optimal activity at different pH, so time-dependent phagosomal pH changes can influence which enzymes are active and when. The importance of phagosomal pH is highlighted by organisms such as Mycobacterium tuberculosis, Franciscella tularensis and Salmonella typherium that subvert phagocytic killing at least in part by altering phagosomal pH 22-24.
In mammals the main phagocytes are neutrophils, macrophages and dendritic cells, and depending on cell type, time-dependent phagosomal pH changes can vary widely, and appear to play different roles. In macrophages a strong and rapid acidification mediated by the ATP-dependent proton pump vacuolar ATPase (V-ATPase) is one of the key factors mediating killing 25-27, resembling the mechanisms present in amoeba that use phagocytosis as an eating mechanism 28. In these cells activation of acidic proteases is thought to play a key role. In contrast, neutrophil killing relies more on ROS as well as HOCl produced by myeloperoxidase (MPO)11, and the pH remains neutral or alkaline during the first 30 min acidifying only later 29,30. Neutral pH has been suggested to favor the activity of oxidative proteases such as certain cathepsins. In DCs phagosomal pH is controversial, with some reporting acidification and others neutral or alkaline pH 31,32, but ROS and pH may profoundly influence the ability of these cells to present antigens to T cells, one of their main functions 33. 
Importantly, hormones, chemokines and cytokines may produce signaling events that induce maturation and changes in phagocyte behavior, and in turn influence phagosomal pH 34,35. Similarly, drugs, for example the antimalarial chloroquine, which is also considered for anti-cancer therapies 36, may affect phagosomal pH and therefore immune outcomes. Thus, a variety of cell biologists, immunologists, microbiologists and drug developers may be interested in measuring phagosomal pH when seeking to understand the mechanisms underlying the effects of a particular genetic disruption, bioactive compound or microorganism on innate and adaptive immune responses. 
Here we describe a method for measuring phagosomal pH in neutrophils that allowed us to show the importance of the HVCN1 channel in regulating neutrophil phagosomal pH 19. The method is based on the ratiometric property of fluorescein isothiocyanate (FITC) whose fluorescence emission at 535 nm is pH sensitive when excited at 490 nm but not 440 nm 37. When this dye is chemically coupled to a target, in this case zymosan, it can be followed using wide-field fluorescence microscopy, where cells are imaged as they phagocytose, and phagosomal pH changes are measured in real time as the phagosome matures. The actual pH is then gleaned by performing a calibration experiment where cells that have phagocytosed are exposed to solutions of different pH that contain the ionophores nigericin and monensin, that allow the rapid equilibration of the pH within phagosomes with the external solution. Ratio values are then compared to the known pH of solutions, a calibration curve is constructed by nonlinear regression and the resulting equation used to calculate pH from the ratio value.

<table>
	<tbody>
		<tr>
			<td>Zymosan A powder</td>
			<td>Sigma-Aldrich</td>
			<td>Z4250</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate</td>
			<td>Sigma-Aldrich</td>
			<td>F7250</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Anti-zymosan antibody (Zymosan A Bioparticles opsonizing reagent)</td>
			<td>Life Technologies</td>
			<td>Z2850</td>
			<td>Sigma-Aldrich O6637 is an equivalent product. Alternatively 25% serum can be used as an opsonizing reagent.</td>
		</tr>
		<tr>
			<td>4-Aminobenzoic hydrazide (4-ABH)</td>
			<td>Santa Cruz</td>
			<td>sc-204107</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Diphenyleneiodonium chloride (DPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D2926</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Concanamycin A (ConcA)</td>
			<td>Sigma-Aldrich</td>
			<td>27689</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Nigericin</td>
			<td>Sigma-Aldrich</td>
			<td>N7143</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Monensin</td>
			<td>Enzo</td>
			<td>ALX-380-026-G001</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>14200-075</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Hank&#39;s balance salt solution</td>
			<td>Life Technologies</td>
			<td>14025092</td>
			<td>Ringer&#39;s balanced salt solution or other clear physiological buffers may be substituted.</td>
		</tr>
		<tr>
			<td>Sodium carbonate (Na2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S7795</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>2-(N-Morpholino)ethanesulfonic acid (MES)</td>
			<td>Sigma-Aldrich</td>
			<td>M3671&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>N-Methyl-D-glucamine (NMDG)</td>
			<td>Sigma-Aldrich</td>
			<td>M2004&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td>3777</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>&#160;Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td>Sigma-Aldrich</td>
			<td>T1503&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Absolute Ethanol (EtOH)</td>
			<td>Sigma-Aldrich</td>
			<td>2860</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Glass-bottom 35 mm petri dishes (Fluorodish)</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td>Ibidi &#181;-clear dishes or coverslips with appropriate imaging chambers may be sustituted</td>
		</tr>
		<tr>
			<td>Sonicating water bath</td>
			<td>O. Kleiner AG</td>
			<td></td>
			<td>A sonicator may be used instead, various instrument providers exist</td>
		</tr>
		<tr>
			<td>Heamocytometer</td>
			<td>Marienfeld GmbH</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>Widefield live imaging microscope</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td>Various instrument providers exist, but the microscope must be able to image 440/535 and 490/535 excitation/emission respective. Spinning disk confocal set-ups with brightfield capabilities may substituted, but zymosan tend to go out of focus more often.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Peristaltic pump (Dynamax RP-1)</td>
			<td>Rainin</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Schott Gerate GmbH</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>Manual Counter</td>
			<td>Milian SA</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
	</tbody>
</table>

measuring phagosome ph by ratiometric fluorescence microscopy

A fagocitose é um processo fundamental através do qual as células imunes inatas engolir bactérias, células apoptóticas ou de outras partículas estranhas, a fim de matar ou neutralizar o material ingerido, ou apresentá-lo como antigénios e iniciar respostas imunes adaptativas. O pH de fagossomas é um parâmetro crítico de fissão regulação ou fusão com endomembranas e activação de enzimas proteolíticas, eventos que permitem que o vacúolo fagocítica de amadurecer em um organelo degradativa. Além disso, a translocação de H + é necessária para a produção de níveis elevados de espécies reactivas de oxigénio (ROS), as quais são essenciais para a morte eficiente e de sinalização a outros tecidos do hospedeiro. Muitos patógenos intracelulares subverter assassinato phagocytic limitando phagosomal acidificação, destacando a importância do pH em biologia phagosome. Descrevemos aqui um método para a medição raciométrica phagosomal pH em neutrófilos usando isotiocianato de fluoresceína (ITCF) marcado com zimosano como fagocítica targets e geração de imagens de células vivas. O ensaio baseia-se nas propriedades de fluorescência do FITC, que é extinta por pH acídico, quando excitado a 490 nm, mas não quando excitado a 440 nm, permitindo a quantificação de uma relação dependente do pH, em vez de fluorescência absoluta, de um único corante. Um protocolo detalhado para a realização do corante calibração in situ e a conversão de relação a valores de pH verdadeiro também é fornecido. -Dye único raciométrica métodos são geralmente considerado superior ao comprimento de onda único ou protocolos pseudo-ratiometric dual-dye, como eles são menos sensíveis a perturbações tais como o branqueamento, o foco alterações, variações laser e rotulagem irregular, que distorcem o sinal medido. Este método pode ser facilmente modificado para medir o pH em outros tipos de células fagocíticas, e zymosan pode ser substituído por qualquer outra partícula contendo amina, a partir de drageias inertes para os microorganismos vivos. Finalmente, este método pode ser adaptada para fazer uso de outras sondas fluorescentes sensíveis a diferentes gamas de pH ou outro phagosomatividades de al, tornando-se um protocolo generalizado para a imagiologia funcional de phagosomes.

A seguir, são resultados representativos para um experimento onde o pH phagosomal de neutrófilos primários de rato isolados da medula óssea de tipo selvagem ou Hvcn1 - / - ratinhos foram comparados. Para uma experiência bem sucedida, é importante para obter phagosomes suficientes dentro do campo de visão durante toda a duração do filme de lapso de tempo, evitando muitas phagosomes, que mais tarde serão mais difíceis de segmento durante a análise de imagem. A Figura 1 mo...

Watch this Scientific Journal Video about Measuring Phagosome pH by Ratiometric Fluorescence Microscopy at JoVE.com

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

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 ...

measuring-phagosome-ph-by-ratiometric-fluorescence-microscopy

Research

JoVE Journal

Immunology and Infection

13.8K visualizações.  University of Geneva. 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. 

Vídeo: Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

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

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

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

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

Imunologia e Infecção

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Visualização das respostas toxina bacteriana induzida por microscopia de fluorescência de células vivas

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

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 ...

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 ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

Imagem raciométrica de pH extracelular no biofilme dentário

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

"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.

&quot;Phagosome Closure Assay&quot; 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 ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Visualizando intracelular SNARE tráfico pela vida de fluorescência, microscopia de imagem

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Medição de deformabilidade e heterogeneidade de glóbulos vermelhos no sangue por Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

In Vitro Formação canina neutrófilos extracelular armadilha: Análise dinâmica e quantitativa por microscopia de fluorescência

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Methods Article

Medindo phagosome pH Raciometrico por microscopia de fluorescência