Pesquisa relatada nesta publicação foi apoiada pelo Instituto Nacional do Câncer do Instituto Nacional de Saúde sob o número Prêmio R01CA174385. O conteúdo é da exclusiva responsabilidade dos autores e não representam necessariamente a posição oficial do Instituto Nacional de Saúde.

1. Preparação de reagentes <ol><li> Prepare RPMI-PLGH misturando 500 ml ml de RPMI-1640 e 5 de cada um de penicilina-estreptomicina, L-glutamina, HEPES e solução. </li><li> Prepare R10 solução misturando RPMI-PLGH com 10% de Soro Fetal Bovino (FBS). O FBS inactivado pelo calor é a 56 ° C durante 30 min antes da adição. </li><li> Pré-aquecido a 37 ° C de 50 ml de RPMI-PLGH, 15 ml ml de PBS, e 15 de R10 em tubos cónicos estéreis. </li><li> Fabricação de matrizes de nanowells em PDMS: O mestre de silício é fabricado usando fotolitografia, essencialmente como descrito anteriormente 10. Misturar cuidadosamente a base de Sylgard 184 kit elastómero e agente de cura de peso de 10:1 em um copo descartável usando uma faca de plástico. Desgaseificar a mistura, em uma câmara de vácuo durante 1 hr. Despeje a mistura para a matriz nanowell, vedar com uma lâmina de vidro e deixar repousar durante 30 minutos. Transferir o conjunto dentro de um forno ajustado para 80 ° C durante 2 h e deixar arrefecer à temperatura ambiente durante 1 hr. O elastômero vontade atue durante este período e vontade vínculo com a lâmina de vidro. A lâmina de vidro que contém a matriz de PDMS nanowell é cuidadosamente levantada do mestre de silício, cobertos com fita adesiva Scotch e armazenadas até utilização futura. </li></ol> 2. Célula alvo Labeling (T) <ol><li> Conte 5.000.000 células alvo a partir de cultura de células utilizando um hemocitómetro e sedimentar as células em um tubo estéril de 15 mL cónico por centrifugação a 340 xg durante 5 min. A cultura de células é dividida no dia anterior em 5 ml de volume total (densidade = 1 milhão / ml) e cultivadas em frasco T-25 (VWR). Protocolos para contagem de células utilizando um hemocitómetro foram descritos em detalhe anteriormente 11 </li><li> Aspirar a mídia em excesso, deixando para trás cerca de 50 ul de mídia. </li><li> Preparar uma solução de trabalho de Célula tracker Red (CTR) através da mistura de 0,5 ul de estoque CTR (1 mM) a 150 ul de meio RPMI-PLGH (concentração final 2,5 uM). Adicionar 150 ul da solução de trabalho para as células alvo e misturar cuidadosamente com uma pipeta de P200.Alternativamente, PKH 26 (concentração final de 2 mM) pode ser usada como células alvo de etiquetas de acordo com as instruções do fabricante. </li><li> Incubar a 37 ° C / 5% de CO2 durante 20 min. </li></ol> 3. Rotulagem de células efetoras (E) <ol><li> Conte 5.000.000 células efectoras provenientes da cultura de células utilizando um hemocitómetro e sedimentar as células em um tubo estéril de 15 mL cónico por centrifugação a 340 xg durante 5 min. </li><li> Aspirar excesso de mídia </li><li> Preparar uma solução de trabalho de solução DyeCycleViolet Vybrant Stain pela adição de 1 uL de 5 mM estoque Violet Vybrant a 1 ml de meio R10 (concentração final de 5 uM). Adicionar a solução de trabalho para as células e ressuspender P200 usando uma pipeta. Alternativamente, PKH 67 (concentração final de 2 mM) pode ser usada como células alvo de etiquetas de acordo com as instruções do fabricante. Se PKH 67 é utilizado, em seguida, SYTOX Green não deve ser utilizada uma vez que eles estão no mesmo canal de fluorescência. A enumeração de metas apoptóticas é feito exclusivamente através de umnnexinV coloração no presente caso. Nota: Se executar lapso de tempo e medições não terminais, corantes UV como Violeta Vybrant deve ser evitado. </li><li> Incubar a 37 ° C / 5% de CO2 durante 20 min. </li></ol> 4. Separação de células vivas e mortas Usando um gradiente de densidade <ol><li> Adicionar 6,8 ml e 6,0 ml de meio RPMI-PLGH ao alvo e células efectoras, respectivamente, e misture pipetando para cima e para baixo suavemente. </li><li> Camada ml 3,5 de Ficoll-Paque Plus a parte inferior de cada tubo cónico contendo alvo e células efectoras. FICOLL camada lentamente para garantir boa formação de camadas entre FICOLL e mídia. </li><li> Centrifugar os tubos a 340 xg durante 30 min. sem freio e aceleração. </li><li> Durante a centrifugação, transferir 7 mL de PBS pré-aquecido a uma placa de quatro poços. Prepare nanowell array (fabricado em PDMS): inferior de lâmina de vidro limpa com etanol e oxidar o PDMS no ajuste alto RF usando plasma oxidante durante 1 min. Esta etapa irá sterilize matriz nanowell enquanto também tornar o PDMS hidrofílico. Imediatamente mover a matriz nanowell na placa contendo PBS. </li><li> Transfira a placa para uma cabine de segurança biológica e aspirar excesso de PBS. Fazer 10 ml de ágar a 1% por adição de 100 nobre pó de ágar a 10 mg de água DI ml e, em seguida, ela microondas por 30-45 segundos. Aplicar aquecer a 1% de ágar nobre na borda superior e inferior da lâmina de vidro que contém a matriz nanowell para imobilizar a matriz e esperar 10 minutos para agar solidificar. Adicionar 3 ml de PBS e agar excesso aspirado. </li><li> Adicionar 3 ml de R10 para o topo da matriz e deixar nanowell equilibrar durante ~ 10 min. </li><li> Subsequente à centrifugação de Ficoll, aspirar 5 ml de meio a partir da camada superior de cada tubo. Tenha cuidado para não aspirar a camada que contém as células. </li><li> Colher as células (camada branca), recuperando ml 1-2 da camada entre FICOLL e mídia com P1, 000 pipeta e mudança para novos tubos cônicos estéreis. Repita este procedimento para os dois conjuntos de células ao se certificar de que você máximaize das células de recuperação. </li><li> Adicionar 3 ml de RPMI pré-aquecido PLGH-se a cada tubo da mistura, por pipetagem e sedimento por centrifugação a 340 xg durante 5 min em plena aceleração e de travagem. </li><li> Aspirar a mídia em excesso, adicionam-se 5 ml de meio RPMI pré-aquecido PLGH-se a cada tubo e repetir a centrifugação a 340 xg durante 5 min. </li><li> Aspirar o excesso de media e ressuspender o sedimento de células em 500 uL de R10. </li><li> Contar as células vivas utilizando o método de exclusão de azul de tripano em um hemacitómetro. </li></ol> 5. Carregando célula para matriz Nanowell <ol><li> Aspirar o excesso de mídia matriz nanowell e adicionar 2 ml de R10 fresco através da matriz. Aspirar novamente enquanto certificando-se o topo da matriz nanowell não é muito úmido / seco como ele vai afetar a distribuição de células. </li><li> Depósito de 1 x 10 5 células efectoras em 200 ul de R10 para nanowell superfície da matriz (densidade = 5 x 10 5 células / ml). </li><li> Deixe as células assentar durante 5 min e usando um padrão de tecido de verificação microscópio cultura de ense que a distribuição das células é desejável. Adicionar mais células, se necessário. </li><li> Depósito 1 x 10 5 células-alvo em 200 ul de R10 nanowell superfície da matriz (densidade = 5 x 10 5 células / ml). </li><li> Deixe as células assentar durante 5 min e usando um padrão de tecido de verificação microscópio cultura para assegurar que a distribuição das células é desejável. Adicionar mais células, se necessário. </li><li> Lavar cuidadosamente com matriz nanowell 2 ml R10 e aspirado excesso de mídia. </li><li> Preparar 3 ml R10 pré-aquecido num tubo de 15 ml estéril cónico. Adiciona-se 3 uL de 0,5 mM de ácido nucleico SYTOX Verde Stain (concentração final 0,5 uM) e 60 jil de anexina V-Alexa Fluor 647 para preparar a solução de trabalho. Adicionar a solução de trabalho para a placa de quatro poços cuidadosamente assegurando ao mesmo tempo que as células não são deslocadas a partir das cavidades. </li><li> Incubar a 37 ° C / 5% de CO2 durante 15 min. </li></ol> 6. Imagem 1 <ol><li> Adquirir imagens da matriz nanowell usando um micr fluorescênciaoscope: Neste exemplo, usamos um Observer ZEISS Z-1 microscópio equipado com um palco motorizado e Lambda-DG4. </li><li> Inicializar o software e o microscópio e verificar a intensidade do sinal na luz transmitida e os outros canais fluorescentes. De modo geral, haverá cinco canais correspondentes a: luz transmitida, anexina V-647 (Cy5 filtro), CTR (DsRed filtro), SYTOX Green (FITC filtro), e Vybrant Violet (DAPI filtro). Certifique-se que o microscópio que configurar para garantir sinal máximo de ruído, evitando a saturação. </li><li> Definir X e Y posição para a matriz nanowell. Iniciar o processo, definindo a posição zero ea inicialização de foco no centro de 7 x 7 bloco matriz nanowell no canto superior esquerdo do selo matriz nanowell. Definir as posições x, y, para coincidir com o centro de cada bloco e o valor de z para reflectir com precisão a focagem em cada bloco. </li><li> Uma vez que a posição eo foco está tudo pronto, certifique-se de definir a aquisição de imagens em modo linear e adquirir imagens. <sTrong&gt; Nota: Se for necessário, na incubadora de CO 2 e sobre o microscópio, pelo menos, 30 minutos antes da experiência. </li></ol> 7. Pós-imagem Transferir a placa 4 poços contendo a matriz nanowell numa incubadora (37 ° C / 5% CO 2) durante 6 horas. 8. Imagem 2 Adquirir as imagens no segundo ponto do tempo, conforme descrito no Passo 6. 9. Análise de Imagem <ol><li> A ocupação de células dentro da nanowells é determinada através da utilização de programas apropriados de segmentação de imagem (por exemplo, ImageJ, <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> ) essencialmente como descrito anteriormente 9,12. </li><li> A análise do conjunto inicial de imagens de microscópio (t = 0 horas, t 0) é utilizado para gerar um banco de dados das nanowells individuais contendo exactamente uma célula alvo vivo (identificadopor CellTracker Red, vermelho) e sem células mortas (identificados por anexina V coloração, verde) [0 T1t nanowells]. </li><li> Análise de o segundo conjunto de imagens de microscópio (t = 6 horas; t 6) é utilizado para determinar de forma independente um segundo banco de dados de nanowells contendo uma célula alvo (vermelho) [6 T1t nanowells]. Uma verificação de integridade (consulta de banco de dados) é implementada para assegurar que todos os alvos vivos em t 0 são compatíveis com alvos em t 6 para produzir um conjunto final de metas combinadas (implementado como nanowells que têm T1t 0 = T1t 6, designado como T1match) </li><li> O número de células efectoras em nanowells é determinada utilizando coloração violeta Vybrant em t 0 [E1 nanowells] </li><li> Uma consulta de base de dados é implementado para identificar nanowells contendo efectores individuais co-incubado com alvos individuais (definida como nanowells que têm correspondência E1 e T1, designados como E1T1) </li><li> Apoptose induzida efetor é marcado por i<html> dentifying alvos que estão anexina V negativa em t 0 e V Anexina positivo em t 4 (E1T1 com anexina V na coloração de alvo t 6). </li></ol> 10. Imagem Time-lapse opcional de Matar Usando BioStation Nikon IM <ol><li> Pegue uma placa de Petri com o fundo lamela e cortar um pequeno pedaço do chip matriz nanowell (preparado na Etapa 5) que vai caber exatamente na lamela. Certifique-se de que o chip fica molhada durante o corte. </li><li> Misturar 1 x 10 6 alvos e 1 x 10 6 células T em 100 ul de meios de comunicação, e pipeta-lo para a lamela. </li><li> Permitir que cerca de 15-30 minutos para permitir que as células resolver. </li><li> Rapidamente inverter o chip matriz pequena nanowell e colocá-lo sobre a lamela. </li><li> A fim de assegurar que o chip não será capaz de se mover durante a imagiologia lugar, um ou dois 20 milímetros x 20 milímetros lamelas sobre a parte superior do chip. Aplicar uma pressão suave para empurrar o chip para o fundo do prato. </li></ol>tenda &quot;&gt; Nota: bottom O chip matriz nanowell é muito grande para imagens de alta resolução e, portanto, tem que ser virado de cabeça para baixo Durante este, muitas das células são lavadas para fora Uma vez que este não pode ser facilmente controladas, o número de células no Passo 9.2.. e tempo de incubação na etapa 9,3 têm de ser optimizados.

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Não há conflitos de interesse declarados.

Temos descrito o protocolo para um ensaio de elevado rendimento de célula única citolítica activado através de co-incubação de efectores e alvos em matrizes de nanowells (Figura 1). Além disso a taxa de transferência de uma grande vantagem da técnica é a capacidade de monitorizar efectora mediada por citotoxicidade contra células alvo desejadas, sem a necessidade para a engenharia de células alvo, que por sua vez permite o uso de autólogos ou matched / primário de células tumorais, como a...

<table border="1"><tbody><tr><td> Nome do reagente / Material </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> RPMI-1640 w / o L-glutamina </td><td> Cellgro </td><td> 15-040 CV- </td><td></td></tr><tr><td> Penicilina-estreptomicina </td><td> Cellgro </td><td> 30-002 CI- </td><td> 10.000 UI de penicilina estreptomicina 10000 ug / ml </td></tr><tr><td> L-glutamina </td><td> Cellgro </td><td> 25-005 CI- </td><td> 200 mM de solução de </td></tr><tr><td> HEPES </td><td> Sigma Aldrich </td><td> H3537 </td><td> 1M </td></tr><tr><td> De soro fetal bovino (FBS) </td><td> Atlanta Biologicals </td><td> S11150 </td><td> Lote testado </td></tr><tr><td> Rastreador de celular mancha vermelha </td><td> Invitrogen </td><td> C34552 </td><td> 50 ug </td></tr><tr><td> Vybrant DyeCycle Violeta Mancha </td><td> Invitrogen </td><td> V35003 </td><td> 5 mM </td></tr><tr><td> SYTOX Ácido Nucleico mancha verde </td><td> Invitrogen </td><td> S7020 </td><td> 5 mM </td></tr><tr><td> Anexina V-Alexa Fluor 647 </td><td> Invitrogen </td><td> A23204 </td><td> 500 ul </td></tr><tr><td> PBS Dulbecco </td><td> Cellgro </td><td> 21-031 CV- </td><td> 500 ml </td></tr><tr><td> Agar Noble </td><td> DISCO </td><td> 2M220 </td><td> 100 g </td></tr><tr><td> Trypan Blue </td><td> Sigma Aldrich </td><td> T8154 </td><td> 0,4% de líquido, filtrado estéril </td></tr><tr><td> Hemocitômetro </td><td> Hausser Scientifics </td><td> 1492 </td><td> Linha brilhante </td></tr><tr><td> 4-bem chapa </td><td> Thermo Fisher </td><td> 167603 </td><td></td></tr><tr><td> Harrick Cleaner Plasma </td><td> Harrick Plasma </td><td> PDC-32G </td><td> Aspirador de plasma básica </td></tr><tr><td> Observer.Z1 </td><td> ZEISS </td><td&gt; </td><td> Microscópio de fluorescência (funciona com as três partes abaixo) </td></tr><tr><td> Lambda 10-3 </td><td> Instrumento Sutter </td><td></td><td> Filtro controlador </td></tr><tr><td> Lambda DG-4 </td><td> Instrumento Sutter </td><td></td><td> Ultra-High-Speed ​​Wavelength switcher </td></tr><tr><td> Hamamatsu EM-CCD da câmera </td><td> Hamamatsu </td><td> C9100-13 </td><td> Microscópio CCD da câmera </td></tr><tr><td> 15 tubo cônico ml </td><td> BD Falcon </td><td> 352097 </td><td></td></tr><tr><td> 50 ml tubo cónico </td><td> VWR </td><td> 3282-345-300 </td><td></td></tr><tr><td> Nikon Biostation </td><td> Nikon Instruments Inc. </td><td> Biostation IM </td><td></td></tr><tr><td> Vidro prato de cultura de fundo </td><td> MatTek Corporação </td><td> P35G-0 </td><td> 35 milímetros placa de Petri de 10 mm de micropoços </td></tr></tbody></table>

quantitative high-throughput single-cell cytotoxicity assay for t cells

Imunoterapia do cancro pode aproveitar a especificidade da resposta imunitária para alvejar e eliminar tumores. Terapia celular adoptiva (ACT) com base na transferência adoptiva de células T geneticamente modificadas para expressar um receptor de antigénio quimérico (CAR) se mostrou promissora em ensaios clínicos 1-4. Existem várias vantagens de usar CAR + células T para o tratamento de cancros, incluindo a capacidade para alvejar os antigénios do MHC não-limitados e para funcionalizar as células T para a sobrevivência óptima, homing e persistência no interior do hospedeiro, e, finalmente, para induzir a apoptose de CAR + As células T, em caso de toxicidade central 5. Delineando as funções ideais de CAR + células T associados com o benefício clínico é essencial para projetar a próxima geração de ensaios clínicos. Os recentes avanços na imagem animal vivo como microscopia multifotônica revolucionaram o estudo da função de células imunes in vivo 6,7. Embora estes estudos tenham avançado a nossa compreensão da função das células T in vivo, de células T ACT baseada em ensaios clínicos exige a necessidade de ligar as características moleculares e funcionais de células T preparações pré-infusão com eficácia clínica pós-perfusão, utilizando-se, em Os ensaios in vitro de células T de monitorização funções como, citotoxicidade e a secreção de citocinas. Padrão de citometria de fluxo-ensaios baseados foram desenvolvidos para determinar o funcionamento geral das populações de células T ao nível de uma única célula, mas estes não são adequados para a monitorização e a formação de conjugado vidas ou a capacidade da mesma célula para matar alvos múltiplos 8. Matrizes microfabricated projetados em polímeros biocompatíveis como polidimetilsiloxano (PDMS) é um método particularmente atraente para confinar espacialmente efetores e metas em pequenos volumes 9. Em combinação com a microscopia de fluorescência automatizado lapso de tempo, thousands de-alvo efetor interações podem ser monitoradas simultaneamente por poços individuais de imagem de uma matriz nanowell. Apresentamos aqui um método de alto rendimento para a monitorização de células T da citotoxicidade mediada ao nível de uma única célula que pode ser amplamente aplicado para estudar a função de células T citolíticas.

Um exemplo da aplicação do ensaio de elevado débito citolítica é demonstrado na Figura 2. Resumidamente, CD19 marcado específico CAR + células T foram co-incubadas com células EL4 marcadas rato alvo nos poços individuais de uma matriz nanowell (secções 1-5). Uma imagem inicial foi gravado no microscópio fluorescente automatizada para identificar a ocupação (efectores e / ou alvos) de cada nanowell único na matriz (Secção 6). Processamento de imagem foi usada para identificar ...

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Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Descrevemos uma única célula ensaio de elevado débito para medir a citotoxicidade de células T, quando incubado com as células-alvo tumorais. Este método emprega uma matriz elastomérica denso, de sub-nanolitro poços (~ 100000 poços matriz /) para confinar espacialmente as células T e as células alvo em proporções definidas e está acoplado a microscopia de fluorescência para monitorizar alvo efetuador conjugação e apoptose subsequente.

Quantitativo de alta capacidade ensaio de citotoxicidade de célula única para as células T.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

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Research

JoVE Journal

Immunology and Infection

14.9K visualizações.  University of Houston. Descrevemos uma única célula ensaio de elevado débito para medir a citotoxicidade de células T, quando incubado com as células-alvo tumorais. Este método emprega uma matriz elastomérica denso, de sub-nanolitro poços (~ 100000 poços matriz /) para confinar espacialmente as células T e as células alvo em proporções definidas e está acoplado a microscopia de fluorescência para monitorizar alvo efetuador conjugação e apoptose subsequente.

Vídeo: Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Alta capacidade método de detecção de vírus Influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Imunologia e Infecção

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Uma avaliação quantitativa da migração celular pelo ensaio Phagokinetic faixa Motilidade

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Um ensaio fenotípico microscópico para quantificação de micobactérias intracelulares adaptadas para triagem de alto rendimento/alto conteúdo

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

De alto rendimento quantitativo em tempo real de RT-PCR Ensaio para Determinar Perfis de Expressão do tipo I e III do interferão subtipos

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

High Throughput medição de Extracelular DNA lançamento e Formação NET quantitativa em neutrófilos humanos<em&gt; In Vitro</em

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

High Throughput, em tempo real, Teste Dual-leitura dos intracelular Actividade Antimicrobiana e citotoxicidade celular eucariótica

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

A Fluorescência baseada em Linfócitos ensaio adequado para high-throughput screening de pequenas moléculas

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

Um ensaio de alta produtividade para avaliar e quantificar a formação de neutrófilos extracelular armadilha

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Um do elevado-throughput Shigella-ensaio bactericida específico

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

Functionalization dos Cantialavancos do microscópio da força atômica com únicas-células de T ou único-partícula para a espectroscopia imunológica da força da único-pilha

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...