The present study was funded in part by a Research Initiation Grant (RIG) from the University of North Texas to Dr. McFarlin. The authors did not receive direct funding for the completion of this study and report no conflict of interest.

NOTA: Todos os procedimentos de coleta de sangue descritos neste método foram realizados em conformidade com a Declaração de Helsinki e aprovado pelo Comitê de Ética UNT (IRB) para seres humanos. Todos os indivíduos deram consentimento por escrito para a recolha de sangue, o qual foi usado no presente método para garantir que eles eram aparentemente saudáveis, de peso corporal normal, e livre de doença. 1. Reagente Fonte e Planejamento <ol><li> Uso CD66b-APC (clone # G10F5; DF = 1: 50) e CD45-APCeFluor780 (clone 2D1 #; DF = 1: 50) para este ensaio. NOTA: Antes do estudo, os anticorpos CD45 e CD66b foram titulados para determinar a diluição óptima que granulócitos claramente resolvidos (CD45 + / + 66b) a partir de outros leucócitos (CD45 + / 66b-) 11. Após a adição do anticorpo diluído para a coloração da diluição final no tubo foi de 875. </li><li> Compra e descongelar estoque S. aureus biopartículas marcado com corante vermelho pHrodo. Suspender a uma concentração de 1 mg de biopartículas por ml em PBS estéril. Biopartículas diluídas e alíquotas armazenamento congelado a -20 ° C até ser utilizada no ensaio. </li><li> Dissolver dihydroethidium (DHE), a qual é convertida em brometo de etídio fluorescente a presença de radicais livres de oxigénio (isto é, o rebentamento oxidativo), em DMSO a uma concentração final de 10 g / ml. </li><li> Misturar N-etilmaleimida (utilizada para impedir a fagocitose adicional na sequência da duração da incubação ensaio especificado) com PBS estéril em uma diluição em 2 passos de 200 mg / ml e 17,5 mg / ml, respectivamente. No ensaio, usar uma concentração final de 15 mM. Quando o descongelamento N-etilmaleimida, utilizar uma temperatura de 37 ° C bloco de calor, tal como a N-etilmaleimida não permanece em solução. </li><li> Após o passo final da fixação, utilizar 7AAD para corar o ADN nuclear. Antes da adição, diluir estoque 7AAD 1:10 com PBS estéril. </li></ol> 2. Coleta de Amostras de sangue <ol><li> Peça sujeitos a chegar ao laboratório após uma O / N rápido (&gt; 8 h) e abstentino de actividade física (&gt; 12 h). </li><li> Após a limpeza da pele com uma compressa embebida em álcool prep, inserir uma agulha estéril de coleta de sangue em uma veia periférica do braço. </li><li> Recolha de sangue em tubos de vácuo que são comercialmente cheios de heparina sódica. </li><li> Após a coleta, aplicar uma bandagem adesiva para o braço do sujeito. </li><li> Inverta tubos de sangue para misturar 10 vezes e, em seguida, colocar em uma cadeira de balanço até a análise. </li></ol> Técnica 3. A fagocitose Assay <ol><li> Descongelar biopartículas S.aureus (RT), de DHE (RT), e N-etilmaleimida (37 ° C). </li><li> Adicionar 20 L de biopartículas S.aureus 4 individuais 1,2 ml tubos, enquanto trabalhava no capô estéril. </li><li> Adicionar 40 L de DHE a cada tubo contendo as biopartículas. </li><li> Bater levemente os tubos sobre a superfície do banco para recolher os reagentes no fundo do tubo. </li><li> Adicionar 100 L de sangue total misturado a cada tubo, que foi preenchido com biopartículas e DHE. </li><li>Limpe a borda interior dos tubos com um aplicador de algodão cotado para remover qualquer vestígio de sangue contaminando. </li><li> Misturar o sangue e os reagentes com uma pipeta electrónica criada para misturar o sangue e reagentes para três ciclos, após a adição do sangue. </li><li> Coloque tubos de ensaio em um balde de gelo e tampa para proteger da luz. </li><li> Incubar tubos de ensaio durante 10, 20 e 40 min. Comece com o tubo 40 min para assegurar que todos os tubos de ensaio terminar ao mesmo tempo. </li><li> Thaw N-ethylmaleimide em 37 ° C banho de pérola. </li><li> Pipetar 15 L de N-etilmaleimida (descrito acima em 1.4) a cada tubo de ensaio, incubar durante 30 min. </li><li> Pipetar 10 l de CD66b-APC e 10 L de CD45-APCeFluor780 anticorpos diluídos (descritos acima em 1.1), incubar durante 60 min. </li><li> Pipetar 750 L de solução de lise correcção WBC / RBC em cada tubo de ensaio, incubar durante 60 min. </li><li> Centrífuga (10 min a 400 xg) tubos de ensaio para recolher o sedimento celular. </li><li> Vacuum aspirar a solução lise WBC / fix RBC, deixando um residual flvolume de uid (100 L) por cima do sedimento celular. </li><li> Pipetar 10 l de solução diluída 7AAD (descrito acima em 1.5) e 50? L de PBS em cada tubo de ensaio. </li><li> Adicione uma gota de contas de calibração (~ 25 L) para cada tubo de ensaio, coloque materiais em tubos de ensaio, tubos embrulhe em papel alumínio e coloque no frigorífico. </li><li> Coloque o tubo de amostra no fluxo baseado em imagem citômetro e coletar um mínimo de 3.000 eventos de granulócitos usando parâmetros pré-definidos: Autosampler, azul (488 nm, 60 mW), vermelho (640 nm, 100 mW),, SSC (785 nm; 8,5 mW) lasers (Figura 1). </li></ol><img alt="Figura 1" src="/files/ftp_upload/52201/52201fig1highres.jpg" /> Figura 1. Aquisição Method &amp; Template. As amostras foram adquiridas em um fluxo com base em imagem citômetro (A). Durante a aquisição, gráficos de pontos de Campo brilhante Proporção vs. CD66b-APC foram gerados para granulócitos separados dos grânulos de calibração perfurantes usin100.2.292.0 software g INSPIRE v. (B). Um mínimo de 5.000 granulócitos foram adquiridos para cada amostra utilizando um amostrador automático, azul (488 nm, 60 mW), vermelho (640 nm, 100 mW),, SSC (785 nm; 8,5 mW) lasers. Por favor, note que um número de histogramas estão presentes durante a aquisição para monitorar as configurações de laser e outros aspectos da coleta de dados. Todos esses histogramas são opcionais e só precisava se os procedimentos operacionais padrão de laboratório exige-los como controle de qualidade. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52201/52201fig1large.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a> 4. Amostra de Aquisição e Análise <ol><li> Use o assistente de compensação software automatizado do software IDEAS para aplicar uma matriz de compensação para arquivos de imagem RAW (RIF) e criar compensar arquivos clonados (CIF). </li><li> Carregar arquivos CIF individuais em software idéias e gerar os seguintes parcelas para identificar os subconjuntos de granulócitos: </li&gt; <li> Estabelecer portões iniciais para identificar as células que foram consideradas em foco usando um histograma para brilhantes RMS gradiente de campo (Figura 2A). Fazer essa determinação para cada amostra usando o controle unstimulated como padrão de referência. </li><li> Use parcelas secundárias às células singleto separadas de detritos e dupletos, utilizando um gráfico de pontos de campo brilhante relação de aspecto (relação entre a altura da célula versus largura) x área do campo brilhante (Figura 2B). Fazer essa determinação para cada amostra usando o controle unstimulated como padrão de referência. </li><li> Uma vez que uma população de células limpo havia sido identificado, estabelecer um gráfico de pontos de CD45 vs. CD66b (Figura 2C) para identificar positivamente granulócitos (CD45 + / 66b +). Criar um enredo filha (Figura 3) de intensidade detalhe brilhante para S. aureus (eixo x) vs. detalhe intensidade luminosa para o burst oxidativo (, eixo y-DHE) para identificar subconjuntos de granulócitos activados. Recolha brimagens de campo ight no canal 1 e 9, biopartículas no canal 2, DHE no Channel 4, 7AAD no Canal 5, CD66b no Canal 11 e CD45 no Canal 12. </li></ol><img alt="Figura 2" src="/files/ftp_upload/52201/52201fig2highres.jpg" /> Figura 2. Modelo de Análise. Este número da série de tramas que foram gerados para identificar as células que estiveram em foco (A), células individuais (B), de granulócitos (C). Parcelas adicionais foram utilizados para identificar os três subconjuntos de granulócitos activados granulócitos vs. inactivos. Toda a análise de arquivos de imagem adquiridos foi completada com software IDEAS v.6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52201/52201fig2large.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a>

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The authors have nothing to disclose.

O presente método representa um refinamento dos métodos existentes para a avaliação da função de granulócitos utilizando citometria de fluxo 1,3,4,12-14. Os passos críticos deste ensaio tendem a ser relacionada com uma mistura apropriada da amostra de sangue com as biopartículas e DHE. A mistura incompleta irá resultar em resultados imprecisos. Enquanto a mistura completa é crítico, o método de mistura deve ser de natureza suave. Sugere-se que a mistura ser realizada utilizando uma pipeta electrónica com uma função de mistura, em vez de um misturador de vórtice. Outro passo crítico no ensaio é para assegurar sempre que não há contaminação do sangue da metade superior do tubo de ensaio. Este sangue residual pode ser removido usando um aplicador com ponta de algodão estéril. A remoção completa é importante porque não fazer isso pode contaminar a preparação final do ensaio com hemácias lisadas-un. Antes de usar este método controles de compensação apropriados devem ser adicionados para controlar spectral sobreposição entre os reagentes utilizados para identificar os vários aspectos da função de granulócitos. Por este método, os controles de compensação envolvem colher amostras de sangue que foram sugeridos no ensaio de incubação de 40 min e, em seguida, a rotulagem com um único marcador (por exemplo, E. coli, a DHE, etc). Após a marcação, eventos positivos individuais são recolhidos e uma matriz de compensação é gerado utilizando um assistente automatizado no software de análise de idéias. É fundamental que se este ensaio é utilizado, os controles de compensação apropriados sejam concluídas para garantir o desempenho adequado do ensaio. A análise é realizada usando o recurso de localizar e co-localização assistentes para identificar esse brilhante intensidade detalhe é a melhor variável para separar as populações e também identificar como existe muita sobreposição entre burst oxidativo e sinais de fagocitose. Especificamente, a utilização de citometria de imagem baseada em fornecida a capacidade de segregar granulócitos activados em três subconjuntos. Tseu colapso subconjunto foi determinada utilizando intensidade detalhe brilhante de fagocitose vs. burst oxidativo. Além de examinar estas funções celulares singulares, foram identificadas as células que exibiram dois eventos ao mesmo tempo no mesmo local anatómico (co-localização). Granulócitos que caíram no subconjunto &quot;high-ativo&quot; foram o único fenótipo que demonstrou co-localização consistente entre fagocitose e burst oxidativo. Esta identificação de subconjuntos de granulócitos ativados é a maior área em que é necessária solução de problemas. É muito importante para um novo usuário a tomar tempo para entender o fluxo de trabalho processo de amostra em idéias e entender a mecânica de modular os populações de células usando o componente de imagem. Outras modificações que um usuário pode procuram fazer incluem a seleção de vezes ensaio de incubação alternativas ou adicionais. O actual método sugere a utilização de durações de 10-40 min; No entanto, dependendo do modelo experimental em que este método épara ser utilizado, pode ser necessário seleccionar as durações mais ensaio. Tal modificação teria de ser avaliado numa base individual. Além disso, foi determinado que a duração do ensaio de incubação tem um efeito significativo sobre a aparência dos três subconjuntos activados. A abordagem descrita neste relatório representa uma extensão do que a informação poderia ser obtido previamente sobre a função de granulócitos 3,4,13,15. Outros laboratórios têm demonstrado a importância de avaliar as alterações na função de granulócitos como parte de uma avaliação abrangente da imunidade e doenças 15-17. Apesar do potencial de este ensaio não é sem limitações. Uma das limitações principais é o custo e tempo de demanda associadas com o processamento elevado de amostras. Quando um desenho de estudo requer um grande número de amostras num determinado dia, estes podem ser difíceis de processar. Processing é simplificada pela utilização de pipetas eletrônicos e dispensadores,mas estes tendem a ser caros e não estão necessariamente disponíveis em cada laboratório. Nossa área de pesquisa concentra-se em um estudo de como o exercício e hábitos alimentares influenciam a saúde do sistema imunológico e funcionar 6,8-10,18-20. Estes objectivos têm implicações práticas importantes para uma variedade de áreas da saúde humana. Além do estudo dos efeitos do exercício e nutricionais o método fagocitose demonstrado neste manuscrito poderia ser útil em outras áreas da imunologia clínica onde a monitorização da função de fagocitose é fundamental para os resultados do tratamento. O presente ensaio é o primeiro de muitos ensaios imunológicos, que, com o potencial de ser re-inventado ao tirar vantagem da informação de imagem única que pode ser possível a partir de um fluxo com base em imagem citómetro.

Granulócitos representam um componente de sistema imune inata do corpo, o que proporciona a primeira linha de defesa contra antigénios invasores. Os métodos anteriores para avaliar a função de granulócitos focado em capacidade de fagocitose ou burst oxidativo utilizando métodos diferentes, o que torna difícil determinar coletivamente como granulócitos mudaram 1-4. Os avanços no campo da citometria de fluxo ter resultado na produção de instrumentos de bancada capazes de alta-resolução, de imagens multi-cor das células de uma forma de alto rendimento 5. A capacidade de combinar imagens com o fluxo tradicional representa citometria de um avanço que fornece a plataforma tecnológica necessária para inovar dentro de citometria de fluxo existente métodos e extrair novas informações sobre o sistema imunológico. Ao longo dos últimos dez anos, o nosso laboratório, entre outros, tem sido intensamente focada sobre o efeito de vários tratamentos patte nutricional e exercíciorns sobre a função imunológica inata 6-9. O método demonstrado neste manuscrito tem implicações práticas no campo da imunologia clínica. O presente método aproveita o poder da citometria de fluxo baseado em imagem para medir simultaneamente a fagocitose de partículas bacterianas e burst oxidativo. Usando essa abordagem, um é capaz de separar os granulócitos ativados utilizando variáveis ​​fornecidas pela parte baseada na imagem da análise. Estes subconjuntos eram identificáveis ​​apenas depois de avaliar as imagens celulares nos granulócitos individuais. Além disso o tempo de incubação do ensaio afectada a transição entre as três subconjuntos de activação 10. Assim, é plausível que o uso de múltiplos tempos de incubação pode permitir um método para testar a mudança na função de granulócitos na sequência de um tratamento experimental específica. O objetivo deste artigo foi demonstrar um método de avaliação da função de granulócitos usando citometria de fluxo baseado em imagem para medir simultaneamente a fagocitose com oxidative estourar.

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			<td style="width:125px;">BD Life Sciences</td>
			<td style="width:119px;"></td>
			<td>used for blood collection</td>
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			<td height="42" style="height:42px;width:216px;">WBC Fixative / RBC Lysis solution</td>
			<td style="width:125px;">eBioscience</td>
			<td style="width:119px;">00-5333-57</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CD66b-APC</td>
			<td style="width:125px;">eBioscience</td>
			<td style="width:119px;">clone G10F5</td>
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			<td style="width:125px;">eBioscience</td>
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			<td height="21" style="height:21px;width:216px;">s.aureus bioparticles</td>
			<td style="width:125px;">Life Technologies</td>
			<td style="width:119px;">A10010</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">dihydroethidium</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">D7008</td>
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			<td height="21" style="height:21px;width:216px;">N-ethylmalemide</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">4259</td>
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			<td height="21" style="height:21px;width:216px;">7AAD</td>
			<td style="width:125px;">EMD Millipore</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">96-channel pipet</td>
			<td style="width:125px;">Integra Biosciences</td>
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			<td height="21" style="height:21px;width:216px;">Bead Bath Incubator</td>
			<td style="width:125px;">LabArmour</td>
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			<td height="21" style="height:21px;width:216px;">Imaging Flow Cytometer</td>
			<td style="width:125px;">EMD Millipore</td>
			<td style="width:119px;">Amnis FlowSight</td>
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			<td height="21" style="height:21px;width:216px;">INSPIRE Software</td>
			<td style="width:125px;">EMD Millipore</td>
			<td style="width:119px;">Amnis INSPIRE</td>
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			<td height="21" style="height:21px;width:216px;">IDEAS Software</td>
			<td style="width:125px;">EMD Millipore</td>
			<td style="width:119px;">Amnis IDEAS</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">X-Pierce Piercable Plate Sealer</td>
			<td style="width:125px;">Excel Scientific, Inc.</td>
			<td style="width:119px;">X-Pierce</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dell Precision Workstation</td>
			<td style="width:125px;">Dell Computers</td>
			<td style="width:119px;">Various</td>
			<td>Used for IDEAS analysis</td>
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image-based flow cytometry technique to evaluate changes in granulocyte function in vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Usando citometria de fluxo baseado em imagem permitiu separar uma população homogênea de granulócitos ativados em três subgrupos de ativação diferentes (Figura 3). Neste método, a forma mais eficaz de resolver as três subconjuntos de activação é brilhante através da representação gráfica da intensidade detalhe para a fagocitose (S. aureus) versus o burst oxidativo (DHE) (Figura 3; Figura 4). Além disso, o uso do assistente de co-localização de software ideias irão permitir a quantificação da presença simultânea de fagocitose e burst oxidativo, que é um sinal típico de granulócitos altamente activadas. <img alt="Figura 3" src="/files/ftp_upload/52201/52201fig3highres.jpg" /> Figura 3. Identificação da Contato granulócitos Subconjuntos. Após a identificação dos granulócitos utilizando marcadores de superfície celular (CD45 + / 66b +), um enredo filha foi gerado com brilhante detalhe intensidade para a fagocitose vs. intensidade detalhe brilhante para burst oxidativo. Usando essa abordagem, o percentual de inativos (roxo), low-ativo (vermelho, A), moderado-ativo (azul, B) e (C) amarelo, granulócitos altos-ativo foi determinada. Esta técnica de propagação também foi usada para avaliar o efeito do tempo de incubação do ensaio sobre a abundância relativa dos diferentes subconjuntos de granulócitos activados. O maior percentual de granulócitos &quot;high-ativo&quot; estava presente após a 40 min de incubação. Toda a análise de arquivos de imagem adquiridos foi completada com software IDEAS v.6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52201/52201fig3large.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a> Além de demonstrar a presença de três subconjuntos distintos de granulócitos activados, demonstrou-se que o tempo de incubação do ensaio influenciaram a abundância relativa de cada subconjunto granulócitos activados. Especificamente, a 40 min incubation resultou no maior percentual de granulócitos &quot;high-activa&quot;. Com a inclusão de, pelo menos, três períodos de incubação do ensaio que pode ser possível determinar a forma como um determinado tratamento clínico altera o estado de activação temporal dos granulócitos. Este é o primeiro método publicado pela citometria de fluxo com base em imagem para identificar diferentes subconjuntos de granulócitos activados como uma função de medições simultâneas de fagocitose e burst oxidativo. <img alt="Figura 4" src="/files/ftp_upload/52201/52201fig4highres.jpg" /> Figura 4. Imagens representativos de marcadores celulares. Uma galeria de imagens de células classificados como de alto-ativa (A), moderado-ativa (B), e de baixo ativo (C) é apresentado nesta figura. Biopartículas S.aureus estão em CH03 , burst oxidativo está em CH04, 7AAD para o núcleo está em CH05, dispersão lateral está em CH06, CD66b está emCH11 e CH12 em CD45 é. Além disso, uma imagem de fusão co-localizada de fagocitose (CH03) vs. burst oxidativo (CH04) é exibido. Áreas de amarelo na imagem merge denotar que a fagocitose e oxidativa explosão estão ocorrendo no mesmo espaço anatômico ao mesmo tempo. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52201/52201fig4large.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro at JoVE.com

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

Baseada em imagem Técnica de Citometria de Fluxo para avaliar as mudanças na função de granulócitos<em&gt; In Vitro</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

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Immunology and Infection

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

12.4K visualizações.  University of North Texas. This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

Vídeo: Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Humano In Vitro Supressão como ferramenta de triagem para o reconhecimento de um Estado precoce de desequilíbrio imunológico

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Imunologia e Infecção

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Quantitativo<em&gt; In vitro</em&gt; Ensaio medir a adesão de neutrófilos ativados primárias células humanas endoteliais microvasculares em condições estáticas

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

In Vitro Ensaio para avaliar o impacto da imunorreguladora Pathways on específica para o HIV CD4 T efetoras celular Função

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Baseada em células de Citometria de Fluxo Ensaio medir citotóxicos Atividade

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

A citometria de fluxo para avaliar o estado de cromatina em células T

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Avaliando Retinal microglia fagocítica Função<em&gt; In Vivo</em&gt; Usando um ensaio baseado em citometria de fluxo

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

Um romance em Vitro ferida cura ensaio para avaliar a migração celular

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolamento e cultura In Vitro de macrófagos alveolares murino e humanos

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

A citometria de fluxo simples com base em ensaio para determinar In Vitro anticorpo dependente do realce do vírus da Dengue, usando soro convalescente vírus Zika

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single cell level and statistical image analysis at a populational level. To overcome this difficulty, we used imaging flow cytometry and transcriptomic profiling (RNA-seq) to determine altered subcellular localization of the cluster of differentiation 1d protein (CD1d) associated with impaired endocytic gene expression in human dendritic cells (DCs), which were exposed to the common lipophilic air pollutant benzo[a]pyrene. The colocalization of CD1d and endocytic marker Lamp1 proteins from thousands of cell images captured with imaging flow cytometry was analyzed using IDEAS and ImageJ-Fiji programs. Numerous cellular images with co-stained CD1d and Lamp1 proteins were visualized after gating on CD1d+Lamp1+ DCs using IDEAS. The enhanced CD1d and Lamp1 colocalization upon BaP exposure was further demonstrated using thresholded scatterplots, tested with Mander&#39;s coefficients for co-localized intensity, and plotted based on the percentage of co-localized areas using ImageJ-Fiji. Our data provide an advantageous instrumental and bioinformatic approach to measure protein colocalization at both single and populational cellular levels, supporting an impaired functional outcome of transcriptomic alteration in pollutant-exposed human DCs.

Imaging flow cytometry provides an ideal approach to detect the morphological and functional alteration of cells at individual and populational levels. Disrupted endocytic function for lipid antigen presentation in pollutant-exposed human dendritic cells was demonstrated with a combined transcriptomic profiling of gene expression and morphological demonstration of protein trafficking.

Integrar a citometria de fluxo de imagem e Transcriptomic de criação de perfil para avaliar alterados CD1d endocítica tráfico

Populational analyses of the morphological and functional alteration of endocytic proteins are challenging due to the demand of image capture at a single ...