A pesquisa é suportada pelo National Institutes of Health bolsas AI-52381, CA138623 e Lung Cancer Research Board Kentucky e apoio institucional de James Graham Brown Cancer Center.

Metodologia Descrição do Microscópio Experimentos com imagens ao vivo celular realizado TE-FM sistema Epi-Fluorescência anexado ao microscópio invertido Nikon Eclipse TE300. O microscópio equipado com estágio de aquecimento. A HQ digital de pressão fresca B / W CCD (Roper Scientific) câmera e LAMDA 02/10 óptico filtro changer (Sutter empresa instrumento) é anexado ao microscópio. Comprimentos de onda de excitação e emissão são controlados com rodas de filtros e controlado por controlador de roda Lamba 02/10 filtro, Sutter Instruments Co. tempo de exposição de 500 ms deve ser o suficiente para ver RFP ou GFP em células vivas. Controle de hardware e aquisição de imagens são controladas por software Metamorph. A escolha dos filtros para o presente estudo são, filtro define S480/20x, S525/40m e S565/25x, S620/60m para GFP e RFP, respectivamente; EGFP / DsRed dupla Tecnologia, dicróicas Chroma. Esta roda de filtros podem acomodar até seis conjuntos de filtros. Microscópio está ligado com o controlador fase anterior Proscan com vara Joy. Todos esses anexos hardware pode ser controlado por software Metamorph. O microscópio também está ligado com micromanipuladores para segurar o micropipetas. A. Medição dos leucotrienos B 4 Migração de Células Dendríticas Direção Utilizando a definição de microscópio acima descrito, desenvolvemos métodos para seguir ligante migração de células dendríticas dirigido em tempo real. Dois micromanipuladores (Narishige) estão ligados ao microscópio. A quimiotaxia de medula óssea de camundongos derivados células dendríticas (BMDCs) em direção a LTB gradiente 4 foi gravado. Aqui, descrevemos os métodos para preparar as pipetas micro, utilizando micro-pipeta extrator, o carregamento do ligante em micro-pipeta e configurar o experimento quimiotaxia no palco aquecida do microscópio para monitorar a migração direcional das células. Este método pode ser aplicado para testar a eficácia de quimioatrativos distintas na indução de migração, bem como efeito de inibidores da quimiotaxia em células vivas. O isolamento de BMDCs 21 de rato tem sido descrita em várias publicações, incluindo JOVE 22. 1. Preparação de BMDCs <ol><li> Placa do BMDCs (após o cultivo por 10 dias) para a tampa prato Fluoro deslizamento inferior e permitir-lhes crescer por 16 hr antes do experimento. </li><li> Lavar as células com 3 ml de 1X PBS, pelo menos, duas vezes (pela adição de 3 ml de 1X PBS a placa e aspiração para fora buffer). </li><li> Adicionar 2 mL de 1XPBS ao prato. As células estão prontas para o experimento. Manter as células em 37 ° C incubadora antes de experimentar. </li></ol> Preparação do ligante carregado micro pipeta: <ol start="4"><li> Fixar a tubos capilares de vidro (Glass padrão, 0,75 mm x 0,4 mm; 6 &quot;, Cat # 625500, AM Systems, INC) na vertical micropipeta extrator (Narishige Internacional EUA, Inc). </li><li> Manter a temperatura a 52 ° C e deixe o vidro para derreter e se separam para formar dicas borda afiada. </li><li> Corrigir a pipeta no suporte micro-pipeta. </li><li> Suavizar as arestas de micro pipeta usando Micro forjar M-900 (Narishige Internacional EUA, Inc), observando ao microscópio. </li><li> Prepare pelo menos 10-20 pipetas para cada experimento. Nota: As micropipetas também pode ser comprado de Instrumentos de Precisão Mundial (TIP μ, T1801TW1F). </li><li> Tome concentração desejada (500 mL de 100 nM LTB 4 na experiência atual) do ligante em um tubo de effendorf mL e manter a ponta da pipeta micro na solução e permitir que a solução para entrar no micro pipeta (back enchimento). </li><li> Tome 1 mL de ligante e com a seringa (1 cc de tuberculina seringa) e, lentamente, preencher o ligante utilizando Micro-Fill agulha (28G MF-5, instrumentos de precisão World) em micro pipeta de cima. </li><li> Conecte o tubo, que se encaixa bem para a pipeta para pipeta e tubo para agulha (21 G 02/11) acoplado a seringa de 1 mL, que é preenchido com ligante. </li><li> Agora, cuidadosamente mover o ligante cheia micro-pipeta para o estágio do microscópio e corrigir para manipuladores micro. </li><li> Aplique pouca pressão da seringa para fazer ligante certeza é liberando lentamente a partir da ponta. </li><li> Trazer o BMDCs banhado imaturo para o microscópio e mantê-los na chapa aquecida (37 ° C) palco. </li><li> Remova a tampa do prato fluoro. </li><li> Foco as células usando óleo de imersão lente objetiva 60X. </li><li> Cuidadosamente, derrubar o ligante carregado pipeta usando &#39;Coarse manual Manipulator MN-188NE&#39; à proximidade das células. </li><li> O foco da pipeta usando &#39;micromanipulador hidráulico Belas MN-188 NE. </li><li> Aplique pouca pressão da seringa para se certificar de que ligante está lançando no prato. </li><li> Definir os parâmetros de aquisição de imagem para adquirir em brilhante arquivado (10 a exposição ms) em cada 15 segundos durante 2 horas usando software Metamorph. </li><li> Manter monitoramento a cada 30 minutos para progression de migração para ligante. </li><li> Se as células estão lotados na ponta da pipeta, alterar a localização da ponta da placa para continuar a migração de experiência. </li></ol> B. Medição GPCR (BLT1) e proteína citosólica (β-arrestina) Interações, Translocação em células vivas 1. Preparação de DNA plasmidial Constrói Os detalhes de construções de DNA plasmidial foram descritas em Jala et al. (2005) 18. 2. Transfecção de Human BLT1-RFP e β-Arrestina-GFP em Células RBL-2H3 <ol><li> Manter o Rat basofílico Leukomia (RBL-2H3) Cells (60 a 70% confluência) a 37 ° C em atmosfera húmida do ar de 95%, 5% de CO 2 como culturas em monocamada em meio de crescimento (DMEM suplementado com 15% FBS, 2 mM L-glutamina, 100 U / mL de penicilina e 100 mg / mL de estreptomicina) em frascos T75. </li><li> Separar as células do prato / frascos usando 6 mL de tripsina-EDTA (0,05% de tripsina, 0,53 mM EDTA) e incubação por 5 min a 37 ° CO C em atmosfera húmida do ar de 95%, 5% 2. Agite suavemente o frasco para acompanhar o grau de descolamento das células. </li><li> Adicionar 6 mL de meio de crescimento para frasco contendo 6 mL de tripsina-EDTA e coletar as células, misturando-os por pipetagem cima e para baixo várias vezes. Transferência de células em tubos de 15 mL falcão. </li><li> Contar as células usando um hemocitômetro e tomar 4 x 10 6 células em tubos de 15 mL de centrífuga e centrifugar a 480g rpm por 3 minutos e ressuspender em 200 mL de mídia transfecção (DMEM, FBS 20%, 50 mM HEPES). Se você pretende realizar 3 transfections, prepare as células e mídia tranfection nesse sentido pelo número de células e aumentando o volume de meio de transfecção. </li><li> Prepare os plasmídeos a serem transfectadas nas concentrações de, pelo menos, 1,5 mcg / mL. Nós preparamos o DNA plasmidial usando DNA QIAGEN Maxi kit plasmídeo. Adicionar o indivíduo ou a codificação DNAs plasmídeo para humanos BLT1-RFP (25 mg) e β-arrestin1-GFP (15 mg) para cubetas eletroforese estéril, (Bio-Rad # 16552088), que tem 0,4 distância entre os eléctrodos cm. </li><li> Tome 200 mL das células acima ressuspenso e colocá-los em cubetas contendo um ou outro hBLT1-RFP ou β-arrestin1-GFP ou os dois juntos e misture-os suavemente com uma pipeta mL estéril. </li><li> Deixe o cubetas acima em temperatura ambiente por 10 min. </li><li> Electroporate as células do Gene Pulser II com tensão de 250 V e capacidade de 500 mF. </li><li> Depois de deixar o eletroporação células permanecem em temperatura ambiente por 10 min. </li><li> Tome 1 mL de meio de crescimento regular e misturá-lo com células electroporated na cubetas. </li><li> Distribuir 300 mL desta mistura em 35 milímetros de tecido de vidro pratos fundo de cultura (prato Fluoro, instrumentos de precisão World) contendo 2 ml de meio de crescimento regular e incubar por 1 hora a 37 ° C em atmosfera húmida do ar de 95%, 5% de CO 2. Permitir que as células a aderir ao fundo do prato. </li><li> Substituir a mídia após 1 h de incubação com meio de crescimento regular e permitir que eles crescem a 37 ° C em atmosfera húmida do ar de 95%, 5% incubadora de CO 2 por 18-24 horas. </li></ol> 3. Imagens de células vivas Aquisição de imagens <ol><li> Após 18-24 horas, lave a 2 células ou 3 vezes com 2 ml de vermelho de fenol livre quentes RPMI contendo 10 mM HEPES, pH 7.55 (Adicionar 2 mL de mídia quente diretamente a placa e media aspirar. Repita 2 ou 3 vezes). Adicionar 1,8 mL de vermelho de fenol livre quentes RPMI contendo 10 mM HEPES. </li><li> Coloque 30 milímetros de vidro de fundo lamela placas de cultura contendo células RBL-2H3, transfectadas com hBLT1-RFP e β-arrestin1-GFP no palco microscópio aquecida (37 ° C). </li><li> Recolher as imagens das células em aumento de 600x usando 60 X objectiva Nikon Plano de Apo 60X/1.4 numérica abertura da lente de imersão em óleo (disponível em nosso sistema de microscópio). </li><li> Coloque uma gota de óleo na lente objetiva 60 x e foco as células com campo claro regulares de luz transmitida ao tocar o fundo do prato. </li><li> Para monitorar a fluorescência de proteínas transfectadas, mudar de campo claro (luz transmitida) para filtros de fluorescência (RFP filtro, se você quiser ver receptor ou GFP, se você quiser ver β-arrestin1). </li><li> Escolha a célula brilhantes e saudáveis, que expressa hBLT1-RFP na superfície da célula usando RFP filtro e capturar a imagem. </li><li> Em seguida, mudar para GFP filtro e certifique-se que β-arrestina-GFP expressa no citoplasma e capturar a imagem. Então cor pseudo tanto as imagens, antes que o tempo decorrido capturar imagens de células vivas para se certificar de que sangram através da fluorescência não está tomando ocorrendo entre RFP e GFP. </li><li> Uma vez que a escolha de seu celular feito, definir os parâmetros de acordo com seu propósito usando software (Software Metamorph neste caso). </li><li> Na demonstração atual, dezesseis imagens bit são adquiridos com o veiora definir binning a 1 X 1 combinado com 60 X Plano Nikon objetivo Apo 60X/1.4 numérica abertura da lente de imersão em óleo. </li><li> Recolher as imagens de fluorescência para RFP e fluorocromos GFP simultaneamente em intervalos de 30 segundos de tempo (se as interações translocação / são muito rápido, pode-se reduzir os intervalos de tempo) dessas rodas usando filtro controlado por software Metamorph. </li><li> Os tempos de exposição da câmera definido para 1000 ms para RFP e GFP. (Definir o tempo de exposição específica de acordo com a intensidade de fluorescência da GFP e RFP). </li><li> Primeiro coletar as imagens para 1 min, sem adição de ligante, que serve como ponto 0 tempo. </li><li> Adicione o mL 200 de ligante (do estoque de 10 mM para uma concentração final de 1 mM), após 1 min sem perturbar a placa / ou posição da célula. </li><li> As imagens de fluorescência são colhidos por 60 min após a adição do ligante e armazenados como imagens TIFF com a ordem crescente dos nomes de arquivo. </li><li> Todos esses arquivos podem ser feitos como arquivos de pilha com individual / combinado imagens de fluorescência com selos de tempo usando o software. </li></ol> Processo de imagens e quantificação da fluorescência de proteínas e de localização <ol><li> Adquirir as imagens com os meios simples (sem células) com RFP e GFP filtros fluorescentes e salvá-los como imagens de fundo. Usar essas imagens para subtrair a partir de imagens reais de dados obtidos acima. </li><li> Prepare a pilha de imagens individualmente para imagens RFP e GFP. </li><li> Selecione / define as várias regiões (superfície vs citosólica) para medir a intensidade de fluorescência da GFP e RFP (BLT1 e β-arrestina translocação) </li><li> Plot as intensidades de fluorescência quantidade como a função do tempo. Este gráfico irá fornecer as informações sobre a cinética de translocação de determinada molécula. </li></ol> Resultados representante A. Medição dos leucotrienos B 4 Migração de Células Dendríticas Direção O método descrito neste protocolo foi utilizado para determinar a migração de células dendríticas para leucotrieno B 4 (Figura 1 e vídeo em anexo 1) 21. Podemos aplicar metodologia semelhante para determinar a capacidade quimiotática de células para um ligante específico em células vivas. <img src="/files/ftp_upload/2315/2315fig1.jpg" alt="Figura 1" /> Figura 1. Medula óssea do rato derivados da migração de células dendríticas para 100 nM LTB 4. Vídeo 1. Viva a migração celular de BMDCs para LTB 4. <a href="https://www.jove.com/files/ftp_upload/2315/Jala and Boddulrui_Video1.AVI">Clique aqui para ver o vídeo</a> . B. Medição GPCR (BLT1) e proteína citosólica (β-arrestina) Interações, Translocação em células vivas Após a conclusão deste procedimento, pode-se obter as seguintes informações em eventos GPCR sinalização. <ol class="lroman"><li> A localização de GPCR e proteínas citosólicas e núcleo (Figura 2). </li><li> Interação ligante induzida da superfície GPCR (BLT1 neste caso) com proteína citosólica (β arrestina, neste caso) (Fig. 3). </li><li> Cinética da internalização de receptores e β-arrestina translocação para membrana seguida de internalização, juntamente com os receptores (Figura 4) (Jala et al. 2005) 18. </li><li> Pode-se determinar o status de ativação ligante dependente de receptores em células vivas (em anexo video 2) e determinar motivos críticos ou processos envolvidos na ativação do receptor (Jala et al. 2005) 18. </li></ol><img src="/files/ftp_upload/2315/2315fig2.jpg" alt="Figura 2" /> Figura 2. Expressão de BLT1-RFP (A), β-arrestina-GFP (B), pNuc-PCP (C) em células RBL-2H3. Imagem da cor combinada mostrado no painel de D. <img src="/files/ftp_upload/2315/2315fig3.jpg" alt="Figura 3" /> Figura 3. Translocação Ligand induzida do receptor e β arrestina. Scan linha de intensidades de fluorescência nas células são mostrados. <img src="/files/ftp_upload/2315/2315fig4.jpg" alt="Figura 4" /> Figura 4. Cinética de internalização de receptores e β-arrestina translocação com a adição de 1 mM LTB 4. As intensidades de fluorescência foram medidos em função do tempo na membrana e locais citosólicos da célula. Imagens de vídeo ao vivo de duas células que expressam BLT1-RFP e β-arrestina-GFP sobre a adição de 1 mM LTB 4. <a href="https://www.jove.com/files/ftp_upload/2315/Jala and Bodduluri_Video2.AVI">Clique aqui para assistir ao vídeo</a>

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Imagens de células vivas é uma poderosa ferramenta para demonstrar a função e interações de proteínas específicas à medida que ocorrem em tempo real. Os métodos descritos neste manuscrito mostram claramente que a LTB 4 pode induzir a migração rápida de células dendríticas. Estes métodos não só ampliar os aspectos da função LTB 4 a diversos tipos de células, eles permitem que métodos semelhantes para ser aplicado a uma variedade de quimiocinas e outros testes de sua eficácia c...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cell lines:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rat Basophilic Leukomia Cell line (RBL-2H3) or HEK293 cells.</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>CRL-2256</td>
<td>&nbsp;</td>
<tr>
<td>Media:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Delbecco's modified Eagle&#x2019;s Medium (DMEM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11995</td>
<td>&nbsp;</td>
<tr>
<td>Phenol red free RPMI or DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11835-030</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine (200 mM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin (10000 U/mL)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin, 0.05% (1X) with EDTA 4Na, liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25300</td>
<td>&nbsp;</td>
<tr>
<td>HEPES (1M)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Others:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>35 mm sterile glass coverslip-bottomed Fluoro dishes (0.17 mm thick) (WillCo-dish)</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>FD35-100</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Gene Pulser Cuvette (0.4 cm electrode gap) (Bio-Rad)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>16552088</td>
<td>&nbsp;</td>
<tr>
<td>Instruments/software:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser II electroporater </td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>TE-FM Epi-Fluorescence system attached to Nikon Inverted Microscope Eclipse TE300</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Metamorph Software</td>
<td>&nbsp;</td>
<td>Universal Imaging</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vertical Micro-pipette puller</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micro-Forge M-900</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hadraulic Micromanipulator MO-188NE</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Coarse Manual Manipulator, MN-188NE</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>cDNA constructs:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>cDNA of G-Protein coupled receptor tagged with red fluorescence protein at C-terminus (hBLT1-RFP)</td>
<td>&nbsp;</td>
<td>Jala et al 2005</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td> cDNA of cytosolic protein tagged with GFP (&beta;-arrestin1-GFP in present study).</td>
<td>&nbsp;</td>
<td>Jala et al 2005</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

real-time imaging of leukotriene b4 mediated cell migration and blt1 interactions with &beta;-arrestin

Receptores acoplados à proteína G (GPCRs) pertencem à família de proteínas transmembrana sete e mediar a transdução de sinais extracelulares de respostas intracelulares. GPCRs controle de diversas funções biológicas, como quimiotaxia, liberação de cálcio intracelular, regulação de genes de uma forma ligante dependente via heterotrimeric G-proteínas 1-2. Ligante de ligação induz uma série de mudanças conformacionais levando à ativação de heterotrimeric G-proteínas que modulam níveis de segundos mensageiros como a adenosina monofosfato cíclico (cAMP), o trifosfato de inositol (IP3) e glicerol diacyl (DG). Concomitante com a ativação da ligação ligante receptor também inicia uma série de eventos para atenuar o receptor de sinalização por meio de seqüestro de dessensibilização, e / ou internalização. O processo de dessensibilização dos GPCRs ocorre através de fosforilação do receptor de quinases da proteína G-receptor (GRKs) e subsequente ligação dos β-arrestinas 3. β-arrestinas são proteínas citosólicas e translocar para a membrana após a ativação GPCR, ligação aos receptores fosforilados (maioria dos casos) há, facilitando a internalização dos receptores 4-6. Leucotrieno B 4 (LTB 4) é uma molécula de lipídio pró-inflamatórias derivadas de ácido araquidônico via e medeia suas ações através de GPCRs, LTB 4 receptor 1 (BLT1; um receptor de alta afinidade) e receptor LTB 4 2 (BLT2; um receptor de baixa afinidade ) 7-9. A LTB via 4-BLT1 foi mostrado para ser crítico em várias doenças inflamatórias, incluindo, artrite, asma e aterosclerose 10-17. O presente artigo descreve as metodologias desenvolvidas para monitorar LTB 4 induzida migração de leucócitos e as interações de BLT1 com β-arrestina e, a translocação do receptor em células vivas usando técnicas de microscopia de imagem 18-19. Derivadas da medula óssea células dendríticas de camundongos C57BL / 6 foram isoladas e cultivadas como descrito anteriormente 20-21. Essas células foram testados em métodos de imagem ao vivo de células para demonstrar LTB 4 migração celular induzida. O BLT1 humano foi marcado com uma proteína fluorescente vermelha (BLT1-RFP) no C-terminal e β-arrestin1 marcados com proteína fluorescente verde (β-arr-GFP) e os plasmídeos transfectados em ambos os Rat basofílico Leukomia (RBL-2H3) linhas celulares 18-19. A cinética de interação entre essas proteínas e localização foram monitorados através de microscopia de vídeo ao vivo da célula. As metodologias no presente trabalho descrevem o uso de técnicas microscópicas para investigar as respostas funcionais do G receptores acoplados à proteína em células vivas. O presente artigo também descreve o uso de Metamorph software para quantificar a intensidade de fluorescência para determinar a cinética do receptor e interações proteína citosólica.

Watch this Scientific Journal Video about Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with Î²-arrestin at JoVE.com

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

Este artigo descreve a metodologia para determinar a resposta quimiotática de leucócitos para ligantes específicos e identificar as interações entre os receptores da superfície celular e as proteínas citosólicas usando técnicas de imagem de células vivas.

Em tempo real de imagens de leucotrienos B 4 Migração mediada por células e BLT1 Interações com β-arrestina

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

real-time-imaging-leukotriene-b4-mediated-cell-migration-blt1

Research

JoVE Journal

Immunology and Infection

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

12.7K visualizações.  University of Louisville. Este artigo descreve a metodologia para determinar a resposta quimiotática de leucócitos para ligantes específicos e identificar as interações entre os receptores da superfície celular e as proteínas citosólicas usando técnicas de imagem de células vivas.

Vídeo: Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &#946;-arrestin

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Em tempo real de imagens ao vivo de células T Sinalização de Formação de Complexos

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Imunologia e Infecção

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Monitoramento da Migração de Células dendríticas usando<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Ressonância Magnética

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Imagens em tempo real de heterotípica plaquetas neutrófilos Interações sobre o endotélio ativado durante a inflamação vascular e formação de trombos em camundongos vivos

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

Imagens em tempo real de endoteliais junções célula-célula durante Neutrophil Transmigração Sob Fisiológica Fluxo

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Imagens em tempo real de células mielóides em Dynamics<em&gt; Apc<sup&gt; Min / +</sup</em&gt; Intestinal Tumores girando Disk microscopia confocal

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

Migração intersticial imagens de células CD4 T na derme inflamadas

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

Imagem em tempo real e quantificação de fungos biofilme desenvolvimento usando um sistema bifásico de fluxo de recirculação

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

Modelo de doença microfluida in vitro para estudar interações endoteliais de sangue inteiro e dinâmica do coágulo sanguíneo em tempo real

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are either time-consuming or unable to detect small variations. Presented here is a protocol for the precise quantification of viral titer by analyzing electrical impedance variations of infected cells in real-time. Cellular impedance is measured through gold microelectrode biosensors located under the cells in microplates, in which magnitude depends on the number of cells as well as their size and shape. This protocol allows real-time analysis of cell proliferation, viability, morphology and migration with enhanced sensitivity. Also provided is an example of a practical application by quantifying the decay of influenza A virus (IAV) submitted to various physicochemical parameters affecting viral infectivity over time (i.e., temperature, salinity, and pH). For such applications, the protocol reduces the workload needed while also generating precise quantification data of infectious virus particles. It allows the comparison of inactivation slopes among different IAV, which reflects their capacity to persist in given environment. This protocol is easy to perform, is highly reproducible, and can be applied to any virus producing cytopathic effects in cell culture.

Reported here is a protocol for the quantification of infectious viral particles using real-time monitoring of electrical impedance of infected cells. A practical application of this method is presented by quantifying influenza A virus decay under different physicochemical parameters mimicking environmental conditions.

Monitorando a sobrevivência do vírus da influenza fora do hospedeiro usando análise celular em tempo real

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration through the blood-brain barrier are indispensable steps for the development of E. coli meningitis. Reactive oxygen species (ROS) represent the major bactericidal mechanisms of neutrophils to destroy the invaded pathogens. In this protocol, the time-dependent intracellular ROS production in neutrophils infected with meningitic E. coli was quantified using fluorescent ROS probes detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in mammalian cells during pathogen-host interactions.

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis. During the bacterial infection, reactive oxygen species produced by neutrophils play a major bactericidal role. Here we introduce a method to detect the reactive oxygen species in neutrophils in response to meningitis E. coli.

Quantificação em tempo real de espécies de oxigênio reativo em neutrófilos infectados com meningite escherichia coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...