This work was supported by the Singapore National Research Foundation (grant NRF-RF2010-06 to J.Y.Y.).

Preparação 1. Amostra <ol><li> Cruzar a linha de Gal4 3,22 motorista para UAS-GCaMP5G 19 moscas (w 1118; P {20XUAS-IVS-GCaMP5G} attP40). Permitir que a cruz de crescer a 25 ° C. Nota: Generating voa com várias cópias dos transgenes Gal4 ou UAS-GCaMP pode ajudar a melhorar a intensidade do sinal fluorescente. Uma versão anterior do transgene UAS-GCaMP (UAS-GCaMP3) mostraram muito fraca intensidade de fluorescência quando expressa sob o controlo do condutor Gr68a-Gal4. <ol><li> Coletar e separada por sexo recém-eclosed moscas adultas e levantar a 25 ° C. Nota: O nível básico de GCaMP5G florescimento é o ideal em moscas com idade entre 14-28 dias. Isolando moscas por sexo exclui a possibilidade de interações sociais que podem influenciar as respostas neurais. </li></ol></li><li> Anestesiar uma mosca sob uma corrente suave de CO 2. </li><li> Fixe a fly a uma lamela de vidro, colocando primeiro uma pequena gota de unha polonês no centro de uma lamela de 0,17 mm. Suavemente colocar uma mosca no seu lado sobre a gota de unhas polonês claro e assegurar que toda a queda é coberto pela mosca. </li><li> Execute os passos 1.5 e 1.6 sob um microscópio estereoscópico de dissecação, usado em 8x ampliação. </li><li> Use um pincel molhado para estender uma pata dianteira da mosca. Fixar a pata dianteira em posição pela colocação de duas tiras delgadas de fita ao longo dos primeiro e quinto segmentos do tarso (Figura 2A). Isto irá expor segmentos do tarso T2-T4 para imagiologia (Figura 2B). </li><li> Para neurônios imagem na tromba, coloque uma fina tira de fita adesiva sobre a tribuna das tromba para expor o labelo (Figura 2C, D). </li><li> Desenhar uma barreira hidrofóbica ao redor da mosca com uma caneta PAP para evitar solução de fluir sobre a superfície da lamela. Fazer medições no prazo de 30 min de montagem. </li></ol> 2. Preparação de EstímuloSolução <ol><li> Diluir ligandos lipofílicos (tais como CH503, utilizada neste estudo) em uma solução de PBST: 137 mM de NaCl, 2,7 mM de KCl, 10 mM de Na 2 HPO 4, 1,8 mM de KH 2 PO 4, 0,1% de Triton X-100 (v / v ); pH 7,4. <ol><li> Produzir uma solução stock de 1 mg / ml de ligando utilizando PBST. Prepare todas as diluições posteriores da solução utilizando PBST. Nota: Dependendo da solubilidade do ligando de interesse, podem ser necessários diferentes solventes para dissolver o ligante. A solubilidade do CH503 em vários solventes é descrita na Tabela 1. Ao contrário de outros solventes, PBST não produzir um aumento na fluorescência. </li></ol></li></ol> 3. Estimulação da Gustativa Neurônios e Aquisição de Imagem <ol><li> Utilizando um pipetador de 10 ul, 10 ul de colocar PBST para os segmentos do tarso. Nota: Esta etapa umedece a perna e impede que os segmentos do tarso de drifting. </li><li> Use um sistema óptico que possui uma câmara de suvelocidade ficiente e sensibilidade para alcançar um bom sinal de fluorescência de resolução temporal alta. Nota: Para este estudo foi utilizado um microscópio confocal disco giratório e uma câmera scmos (veja Lista de Materiais). </li><li> Escolha do segmento e neurônios tarsal específica a ser trabalhada. Adquirir três pré-estimulação confocal Z-stacks. <ol><li> Idealmente, as configurações de aquisição de uso que são rápidos o suficiente para permitir a resolução de tempo máximo enquanto ainda captar o volume da célula inteira. Configurações de aquisição de exemplo são: 200 exposição ms, binning 2 x 2 e 6 x 0,5 m 2 secções de óptica, capturados a cada 2 s. Nota: as pernas imobilizadas ter sido fotografados com sucesso para até 10 minutos, em intervalos de 30 seg, sem branqueamento discernível do sinal GCaMP. Para maiores tempos de gravação, verifique se as pernas são mantidas firmemente no lugar na lamela. </li><li> Optimizar a potência do laser para a mais alta relação sinal-ruído possível com fotodegradação mínima durante toda a imaging regime. Para os experimentos aqui descritos, usar um 491 nm diodo laser (100 mW) a transmissão de 30% para exames ambos os neurônios Gr68a e gustativos ppk23 nas pernas e tromba. Use 2 x 2 binning para permitir tempos de exposição mais curtos enquanto ainda alcançar resolução espacial suficiente. Nota: As configurações de energia do laser foram otimizados para permitir a detecção da mudança fluorescente que vão desde um aumento de 1,2 a 4,5 vezes. </li></ol></li><li> Pipetar 10 ml da solução para o estímulo segmentos do tarso. Para experiências de controle, adicionar mais 10 mL de PBST. imediatamente adquirir 117 imagens pós-estimulação. Nota: o número de imagens de pós-estimulação é adquiridos com base na quantidade de tempo em que foi alcançado alteração máxima na fluorescência (aproximadamente 2 min). <ol><li> Passo crítico: Enquanto pipetando solução sobre a preparação, não para tocar a mosca com a ponta da pipeta uma vez que este fará com que a pata dianteira para mover fora de posição. Nota: Como alternativa, useum micromanipulador para posicionar com precisão um capilar de vidro contendo estímulo perto do segmento tarsal a ser trabalhada. Fornecer volumes controlados de estímulo, utilizando um dispensador de microinjecção intracelular de acordo com as instruções do fabricante. </li></ol></li></ol> 4. Processamento de Imagem e Análise: Quantificação da resposta <ol><li> Abra uma série de Z-pilhas confocal usando Fiji ( <a href="http://fiji.sc/Fiji" target="_blank">http://fiji.sc/Fiji</a> ) 23 ou ImageJ ( <a href="http://imagej.nih.gov/ij/" target="_blank">http://imagej.nih.gov/ij/</a> software) análise e processamento de imagem 24. </li><li> Adicione uma projecção soma de intensidade de todas as seis fatias Z para cada um dos 120 pontos de tempo. <ol><li> Selecione a opção &quot;Pilha&quot; em &quot;Imagem&quot;. Além disso, selecione a opção &quot;Z projecção&quot; e digite &quot;1&quot; como a fatia de início e &quot;4&quot;, como fatia stop. Para o tipo de projeção, selecione R20; fatias sum &quot;no menu suspenso e escolha&quot; Todos os prazos &quot;. </li></ol></li><li> Definir a região de interesse (ROI) usando a ferramenta de seleção à mão livre para desenhar um limite em torno de um corpo celular ou projeção neuronal. Clique na etiqueta &quot;Analisar&quot; e selecione a opção &quot;Gerenciador de ROI&quot; em &quot;Ferramentas&quot;. </li><li> Quantificar a fluorescência do ROI selecionando a opção &quot;Definir medidas&quot;, no menu &quot;Analisar&quot; e marcando as caixas para a densidade integrada, área, valor médio e valor máximo de cinza e etiqueta. Em seguida, selecione a opção &quot;Mais&quot; no menu gerenciador de ROI e selecione ainda a opção &quot;medida Multi&quot;. Nota: Isto vai gerar uma caixa pop-up com valores para cada um dos parâmetros seleccionados. </li><li> Verificar os valores de cinzento máximos para assegurar que nenhum dos pixels ter atingido a saturação (por exemplo, 65535 para uma câmara de 16 bits). NOTA: Para as experiências mostradas na tseus papel, as respostas foram medidas quer a partir de corpos celulares ou projecções neuronais, dependendo de onde foi observado o aumento máximo em Af / F. </li><li> Calcula-se a alteração máxima na fluorescência relativa (Af / F) no ponto de tempo t é o seguinte: <img alt="equação 1" src="/files/ftp_upload/53392/53392eq1.jpg" /><ol><li> Subtrair o fundo de fluorescência a partir de cada valor de F (célula). Para quantificar a fluorescência de fundo, medir a densidade integrada de uma ROI de tamanho e forma equivalente a partir de uma área da pata dianteira (ou tromba) destituída de neurónios detectáveis. </li><li> Para quantificar a pós-estimulação de fluorescência, F (célula) t, medir a densidade integrada do ROI do corpo da célula ou processo no ponto de tempo &quot;t&quot;, após a estimulação. </li><li> Para quantificar a fluorescência pré-estimulação, F (célula) t = 0, análise de imagens pré-estímulo do corpo da célula ou processo da mesma forma comoo sinal pós-estimulação. Use a densidade integrada média de 3 imagens para determinar F (celular) t = 0. </li></ol></li></ol>

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The authors declare that there are no competing financial interests.

Descrevemos aqui um método para realizar imagem de cálcio ao vivo de neurônios periféricos Drosophila em 2 órgãos sensoriais diferentes. O Ca 2+ -evoked respostas fluorescentes GCaMP em Gr68a-neurônios induzida pelo ligando feromônio CH503 foram dose-dependentes e quantitativa. Também foi possível discernir diferentes padrões de resposta neural, tais como fásicas e tônicas respostas. Neurônios que mostram respostas fásicas são acreditados para permitir uma...

Animais confiar na informação olfativa e gustativa para mediar decisões essenciais para a sobrevivência e reprodução. Entender como pistas quimio são detectados e processados ​​pelo sistema nervoso exige a identificação do receptor sensorial (s) e os ligandos químicos correspondentes. Drosophila detectar uma variedade impressionante de compostos voláteis e não-voláteis e são um excelente modelo para estudar o fisiológica mecanismos subjacentes chemosensation. Enquanto os órgãos olfactivos perceber moléculas voláteis, os órgãos gustativas são especializados para detectar compostos de baixa volatilidade. Aqui, apresentamos um método para medir diretamente as respostas neuronais dos órgãos gosto de Drosophila melanogaster a baixa volatilidade, ligantes lipofílicos. órgãos gustativos da mosca incluem os anteriores, tromba, e asas. Distribuído sobre a superfície dos órgãos gustativas são estruturas semelhantes a cabelos conhecidos como sensilas que respondem a sugars, bitters, sais, água e feromônios 8. O sensilla foram classificados morfologicamente em cerdas de gosto e sabor pinos 9. Há cerca de 31 cerdas gosto no labelo que são classificados em A longo (tipo L), curto (s-tipo) e morfologias intermediários (-tipo I). Sensilla casa 4 neurônios sensoriais Os &#39;L&#39; e &#39;S&#39; que respondem fisiologicamente ao açúcar (a célula S), baixo teor de sal (a célula L1), alta sal e compostos amargos (a célula L2) e água (a célula W) 10 , 11. O &quot;eu&quot; casa 2 neurônios sensilla sensoriais, uma das quais responde tanto à baixa sal e açúcar, enquanto que outro que responde à elevada de sal 12. Há aproximadamente 41 sensilas sabor em machos e fêmeas em 26 sensilas distribuídos em cada uma das pernas dianteiras. Para ambos os sexos masculino e feminino, há 21 sensilas na midleg, e cerca de 22 sensilas na hindleg 13. Os neurônios gustativos fechados pela sensilla gosto nas pernas também são classified em tipos de L1, L2, W e S. Um método padrão para medir a atividade elétrica dos neurônios individuais utiliza eletrodos extracelulares ou intracelulares para gravar o fluxo de íons. Medições dos eléctrodos permitem função neuronal a ser estudado em organismos não-modelo como as espécies Drosophila, mariposas, abelhas e que não possuem extensas ferramentas genéticas para a rotulagem neural. No entanto, enquanto os métodos eletrofisiológicos têm sido rotineiramente usado para medir a atividade de Drosophila gosto sensilla 4,13,14, aplicando esta abordagem apresenta vários desafios técnicos. Primeiro, gosto cerdas precisam ser identificados com base na morfologia e localização espacial. Após a identificação, medidas eletrofisiológicas pode ser prejudicado pela pequena dimensão do sensilla, limitando a acessibilidade devido à posição e dificuldade em aplicar volumes controladas de um estímulo químico a um gosto de cerdas. Além disso, a estimulação de sensilas podem gerar sinais a partir de mais do que umneuronal tipo 15. Em segundo lugar, a detecção de sinais elétricos podem ser confundidos com o ruído de fundo resultantes das vibrações mecânicas e ruído de equipamentos eletrônicos. Em terceiro lugar, a utilização de um eléctrodo afiado pode danificar a preparação mosca, se utilizado incorrectamente 14. Finalmente, a montagem de uma plataforma de eletrofisiologia requer componentes eletrônicos especializados para a entrega de estímulo, gravação de sinal e análise de dados e pode ser caro. Em D. melanogaster, a disponibilidade de ferramentas geneticamente codificados tem facilitado o desenvolvimento de técnicas de imagiologia que permitem que as respostas de pequenas populações de neurónios a ser estudado. Uma tal abordagem é a utilização do repórter CaLexA 16. Neste método, as sequências de gene que codifica o factor de transcrição VP16-LexA e uma proteína NFAT sensível ao cálcio são fundidos em conjunto. activação de cálcio da calcineurina fosfatase proteína catalisa a fosforilação de de-NFAT e, por sua vez, a FacilitaTES sua importação para o núcleo. No interior do núcleo, o domínio de LexA se liga a um motivo de ligação a DNA-LexAop que dirige a expressão de uma proteína fluorescente verde repórter (GFP), permitindo assim a identificação de neurónios sustentada funcionalmente activado. A abordagem tem sido utilizada com sucesso para medir a resposta de glomérulos olfactiva específica no lobo antenal após a exposição das moscas ao vivo a um odorante 16. Recentemente, as respostas fisiológicas de neurônios receptores gustativos IR52c foram medidos a partir moscas ao vivo usando CaLexA 7. No entanto, para este estudo, foi necessário moscas de idade para até 6 semanas para aumentar a transcrição GFP. Enquanto a imunocoloração com um anticorpo anti-GFP podem ser usadas para aumentar o sinal CaLexA, este método exige a fixação do tecido, evitando, assim, possibilidade de processamento de imagem de células vivas. O cálcio GCaMP proteína indicadora geneticamente codificado também tem sido amplamente utilizado para estudar as respostas neuronais em váriosespécies 17. O GCaMP proteína fluorescente com baixa intensidade antes da estimulação neuronal. Aplicação de um estímulo desencadeia um potencial de acção no neurónio, resultando no influxo de Ca 2+. GCaMP, ligado ao Ca2 +, sofre uma alteração conformacional, fazendo com que a fluorescência com intensidade mais brilhante (Figura 1). Uma abordagem de imagem de cálcio para neurónios único recentemente desenvolvido foi utilizado para identificar a glicose como o ligando para os neurónios pata dianteira gustativos Gr61a específicos 18. Neste estudo, pernas dianteiras dissecados de Drosophila transgênicos expressando GCaMP em neurônios gustativos Gr61a foram cobertos com uma camada de agarose antes da imagem. No entanto, a utilização de tecido dissecado pode causar eventual degradação de expressão GCaMP, limitando, assim, o tempo de medição e detecção de sensibilidade. Além disso, agarose pode limitar a sensibilidade devido aos altos níveis de fundo de fluorescência e suas propriedades de dispersão de luz. To abordar alguns destes inconvenientes, descrevemos o uso de imagem de cálcio GCaMP mediada para gravar as respostas fisiológicas de pata dianteira e tromba neurônios gustativas de animais intactos. Mostramos as respostas fisiológicas de Gr68a e neurónios que expressam ppk23 geneticamente codificado GCaMP5G 19 a uma feromona de lípidos de Drosophila, (3R, 11 Z, 19, Z) -3-acetoxi-11,19-octacosadien-1-ol (CH503) 20, 21. respostas neuronais são medidos por quantificação do aumento na fluorescência do sinal GCaMP5G durante a estimulação de feromonas. Neste protocolo, os neurônios são gravadas para uma duração total de 120 segundos, o que é suficiente para diferenciar padrões de ativação neuronal em células individuais.

<table>
	<tbody>
		<tr>
			<td>Gr68a-Gal4</td>
			<td></td>
			<td></td>
			<td>Gift from H. Amrein (Texas A&#38;M Health Science Center, TX, USA) and J. Carlson (Yale University, CT, USA)</td>
		</tr>
		<tr>
			<td>ppk23-Gal4</td>
			<td></td>
			<td></td>
			<td>Gift from K. Scott (Univ. of California, Berkeley, CA, USA)</td>
		</tr>
		<tr>
			<td>UAS-GCaMP5&#160;</td>
			<td></td>
			<td>42037</td>
			<td>Bloomington Drosophila&#160; Stock Center</td>
		</tr>
		<tr>
			<td>0.17 mm coverslip (Gold-Seal coverslip)</td>
			<td>Electron Microscopy Services</td>
			<td>63790-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail polish, &#34;Hard as Nails Clear&#34;&#160;</td>
			<td>Sally Hansen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Z377821</td>
			<td></td>
		</tr>
		<tr>
			<td>Paint brush</td>
			<td></td>
			<td></td>
			<td>fine-tipped brush</td>
		</tr>
		<tr>
			<td>Tape&#160;</td>
			<td>Scotch brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>13021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, lab grade</td>
			<td>Merck</td>
			<td>10094</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane, HPLC grade</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>H303SK-4</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr>
			<td>PBST</td>
			<td></td>
			<td></td>
			<td>Recipe described in the protocol section</td>
		</tr>
		<tr>
			<td>CH503</td>
			<td></td>
			<td></td>
			<td>Synthesis described in Mori et al., 2010</td>
		</tr>
		<tr>
			<td>sCMOS Camera (ORCA Flash4.0)</td>
			<td>Hamamatsu&#160;</td>
			<td>C11578-22U</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope (Ti-Eclipse)</td>
			<td>Nikon</td>
			<td>Ni-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning Disk Scan head&#160;</td>
			<td>Yokogawa</td>
			<td>CSU-X1-A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquistion Software (MetaMorph Premier)</td>
			<td>Molecular Devices</td>
			<td>40002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td>open source</td>
			<td></td>
			<td><a href="http://fiji.sc/Fiji">http://fiji.sc/Fiji</a></td>
		</tr>
	</tbody>
</table>

measuring physiological responses of drosophila sensory neurons to lipid pheromones using live calcium imaging

Ao contrário dos mamíferos, insetos, como Drosophila têm múltiplos órgãos gustativas. Os neurônios quimio nas pernas, tromba, asas e ovipositor de Drosophila expressam receptores gustativos 1,2, canais iônicos 3-6 e receptores ionotrópicos 7 que estão envolvidos na detecção de sinais sensoriais voláteis e não-voláteis. Esses neurônios contactar directamente tastants, como alimentos, substâncias nocivas e feromonas e, portanto, influenciar muitos comportamentos complexos, tais como alimentação, postura de ovos e acasalamento. gravações dos eléctrodos e imagem de cálcio têm sido largamente utilizados em insectos para quantificar as respostas neuronais evocados por estes saborizantes. No entanto, eletrofisiologia exige equipamento especializado e medições obtenção de um único sensillum gosto pode ser tecnicamente desafiador, dependendo do tipo de célula, tamanho e posição. Além disso, a única resolução neurônio em Drosophila pode ser difícil de alcançar, uma vez hou gosto sensillase mais de um tipo de neurônio quimio. O método de imagem de cálcio ao vivo descrito aqui permite respostas dos neurônios gustativas de solteiro em moscas ao vivo a ser medido. Este método é especialmente adequado para imagiologia de respostas neuronais a feromonas de lípidos e outros tipos de ligandos que têm baixa solubilidade em solventes à base de água.

O indicador de cálcio GCaMP foi geneticamente expressa usando drivers ppk23-Gal4 Gr68a-Gal4 ou. Populações distintas de neurónios e células perna dianteira de suporte não neurais são rotulados por cada controlador (Figura 3A-C). Respostas específicas do ligando ao CH503 feromona lipofílico foram observadas em Gr68a-Gal4 e células expressando Gal4-ppk23 GCaMP (Figura 3D). A mudança relativa na intensidade de fluorescência do sinal GCaMP5G <...

Watch this Scientific Journal Video about Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging at JoVE.com

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

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

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

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

measuring-physiological-responses-drosophila-sensory-neurons-to-lipid

Research

JoVE Journal

Immunology and Infection

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

8.5K visualizações.  Temasek Life Sciences Laboratory. 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. 

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

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Avaliar a resposta estomática ao vivo células bacterianas usando imagens Folha Inteira

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Imunologia e Infecção

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Medir a carga bacteriana e resposta imune em camundongos infectados com Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

Imagens de células vivas de Bacillus subtilis E Streptococcus pneumoniae Usando Microscopia lapso de tempo Automated

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

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 ...

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

De células vivas de Alphaherpes Vírus Transporte anterógrada e Spread

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Phenomics fago: Abordagens fisiológicas para Caracterizar Novel Proteínas Virais

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Um<em&gt; In Vitro</em&gt; Modelo para medição de respostas imunes a Malária no Contexto do HIV co-infecção

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

Métodos de estudo de lipídios Alterações nos neutrófilos ea subsequente formação de neutrófilos Extracelular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Medição da gripe neutralizando Antibody Responses to A(H3N2) vírus em soros humanos por Microneutralization ensaios usando as células MDCK-SIAT1

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.