Las investigaciones realizadas en esta publicación fue apoyada por el Instituto Nacional del Cáncer de los Institutos Nacionales de la Salud bajo el número Award R01CA174385. El contenido es de exclusiva responsabilidad de sus autores y no representa necesariamente las opiniones oficiales de los Institutos Nacionales de Salud.

1. Reactivos Preparación <ol><li> Preparar RPMI-PLGH mediante la mezcla de 500 ml ml de RPMI-1640 y 5 de cada uno de penicilina-estreptomicina, L-glutamina, y la solución de HEPES. </li><li> Preparar R10 solución mediante la mezcla de RPMI-PLGH con suero fetal bovino al 10% (FBS). El FBS es inactivado por calor a 56 ° C durante 30 min antes de la adición. </li><li> Pre-caliente a 37 º C 50 ml de medio RPMI-PLGH, 15 ml ml de PBS, y 15 de R10 en tubos cónicos estériles. </li><li> Fabricación de matrices de nanowells en PDMS: El maestro de silicio se fabrica utilizando fotolitografía esencialmente como se describe anteriormente 10. Mezclar bien la base de Sylgard 184 kit elastómero y el agente endurecedor a una relación de peso de 10:1 en un vaso desechable usando un cuchillo de plástico. Desgasificar la mezcla en una cámara de vacío durante 1 hora. Vierta la mezcla sobre la matriz nanowell, selle con un portaobjetos de vidrio y se deja reposar durante 30 minutos. Transferir el conjunto en un horno a 80 ° C durante 2 horas y se deja enfriar a temperatura ambiente durante 1 hr. El elastómero se acte durante este período y se adhiere a la placa de vidrio. La lámina de vidrio que contiene la matriz PDMS nanowell es cuidadosamente levantado del maestro de silicio, cubierta con cinta adhesiva y se almacena hasta su uso futuro. </li></ol> 2. Célula diana (T) Etiquetado <ol><li> Contar 5 millones de células diana de cultivo de células utilizando un hemocitómetro y sedimentar las células en un tubo estéril de 15 ml cónico por centrifugación a 340 xg durante 5 min. El cultivo celular se divide el día anterior en el volumen total de 5 ml (densidad = 1 millón / ml) y se cultivaron en matraz T-25 (VWR). Los protocolos para el recuento de células usando un hemocitómetro se han descrito en detalle previamente 11 </li><li> Aspirar el exceso de material dejando atrás aproximadamente 50 l de medios de comunicación. </li><li> Preparar una solución de trabajo de Cell Tracker Red (CTR) mediante la mezcla de 0,5 l de stock CTR (1 mM) a 150 l de RPMI-PLGH (concentración final 2,5 mM). Añadir 150 l de la solución de trabajo a las células diana y mezclar cuidadosamente con una pipeta P200.Alternativamente, PKH 26 (concentración final de 2 mM) se pueden utilizar células de etiqueta de destino de acuerdo con las instrucciones del fabricante. </li><li> Incubar a 37 ° C / 5% de CO 2 durante 20 min. </li></ol> 3. Efector (E) Cell etiquetado <ol><li> Contar 5 millones de células efectoras procedentes de cultivo de células utilizando un hemocitómetro y sedimentar las células en un tubo estéril de 15 ml cónico por centrifugación a 340 xg durante 5 min. </li><li> Aspirar el exceso de material </li><li> Preparar una solución de trabajo de Vybrant solución Stain DyeCycleViolet mediante la adición de 1 l de 5 mM de existencias Violet Vybrant a 1 ml de R10 medios (concentración final de 5 mM). Añadir la solución de trabajo a las células y resuspender usando P200 pipeta. Alternativamente, PKH 67 (concentración final de 2 mM) se pueden utilizar células de etiqueta de destino de acuerdo con las instrucciones del fabricante. Si PKH 67 se utiliza, a continuación, Sytox Green no debería utilizarse ya que están en el mismo canal de fluorescencia. La enumeración de los objetivos de apoptosis se realiza exclusivamente mediante unnnexinV tinción en este caso. Nota: Si se realiza lapso de tiempo y no mediciones de punto final, como tintes UV Violet Vybrant debe ser evitado. </li><li> Incubar a 37 ° C / 5% de CO 2 durante 20 min. </li></ol> 4. Separación de células vivas y muertas por gradiente de densidad <ol><li> Añadir 6,8 ml y 6,0 ml de RPMI-PLGH a diana y células efectoras, respectivamente, y mezclar pipeteando arriba y abajo suavemente. </li><li> Capa de 3,5 ml de Ficoll-Paque Plus a fondo de cada tubo cónico que contenía diana y células efectoras. Capa de la Ficoll lentamente para asegurar una buena formación de capas entre Ficoll y medios de comunicación. </li><li> Centrifugar ambos tubos a 340 xg durante 30 min. sin freno y aceleración. </li><li> Durante la centrifugación, transferir 7 ml de PBS precalentado a una placa de 4 pocillos. Preparar matriz nanowell (fabricado en PDMS): parte inferior de la placa de vidrio limpia con etanol y oxidar el PDMS en ajuste alto de RF utilizando plasma oxidante durante 1 min. Este paso steRilize la matriz nanowell al mismo tiempo haciendo que el PDMS hidrófilo. Mueva inmediatamente la matriz nanowell en la placa que contiene PBS. </li><li> Colocar la placa a una cabina de bioseguridad y aspirar el exceso de PBS. Hacer 10 ml 1% de agar noble por la adición de 100 mg de polvo de agar a 10 ml de agua desionizada y luego el microondas durante 30-45 segundos. Aplicar calentar 1% de agar noble en el borde superior e inferior del portaobjetos de vidrio que contiene la matriz de nanowell para inmovilizar la matriz y esperar 10 minutos para agar para solidificar. Añadir 3 ml de PBS y agar exceso de aspiración. </li><li> Añadir 3 ml de R10 en la parte superior de la matriz nanowell y se deja equilibrar durante aproximadamente 10 minutos. </li><li> Con posterioridad a la centrifugación de Ficoll, aspirar 5 ml de medio de la capa superior de cada tubo. Tenga cuidado de no aspirar la capa que contiene las células. </li><li> Recoger las células (capa blanca) mediante la recuperación de 1-2 ml de la capa entre Ficoll y medios de comunicación con P1, 000 pipeta y se mueven a nuevos tubos cónicos estériles. Repita esto para los dos grupos de células mientras se asegura de que usted máximaize las células de recuperación. </li><li> Añadir 3 ml de pre-calentado RPMI-PLGH a cada tubo, mezclar mediante pipeteo y sedimento por centrifugación a 340 xg durante 5 min en plena aceleración y freno. </li><li> Aspirar el exceso de material, añadir 5 ml de pre-calentado RPMI-PLGH a cada tubo y repetir la centrifugación a 340 xg durante 5 min. </li><li> Aspirar el exceso de material y resuspender el pellet celular en 500 l de R10. </li><li> Contar las células vivas utilizando el método de exclusión de azul tripán en un hemocitómetro. </li></ol> 5. Célula de carga en matriz Nanowell <ol><li> Aspirar el exceso de material de la matriz nanowell y añadir 2 ml de R10 fresco a través de la matriz. Aspire de nuevo mientras se asegura la parte superior de la matriz nanowell no es demasiado húmedo / seco ya que afectará a la distribución de células. </li><li> Depósito 1 x 10 5 células efectoras en 200 l de R10 sobre la superficie de matriz nanowell (densidad = 5 x 10 5 células / ml). </li><li> Vamos células se asienten durante 5 min y utilizando un estándar de cultivo de tejido de verificación para microscopio enAsegúrese de que la distribución de las células es deseable. Agregar más celdas si es necesario. </li><li> Depósito 1 x 10 5 células diana en 200 R10 l en la superficie de matriz nanowell (densidad = 5 x 10 5 células / ml). </li><li> Vamos células se asienten durante 5 min y utilizando un estándar de cultivo de tejido de verificación microscopio para asegurarse de que la distribución de las células es deseable. Agregar más celdas si es necesario. </li><li> Enjuagar cuidadosamente con arreglo nanowell 2 ml R10 y el exceso de material aspirado. </li><li> Preparar 3 ml precalentado R10 en un tubo cónico de 15 ml estéril. Añadir 3 l de 0,5 mM Sytox Nucleic Acid Green Stain (mM concentración final de 0,5) y 60 l de Annexin V-Alexa Fluor 647 para preparar la solución de trabajo. Añadir la solución de trabajo sobre la placa de 4 pocillos cuidadosamente mientras se asegura que las células no son desplazados de los pocillos. </li><li> Incubar a 37 ° C / 5% de CO 2 durante 15 min. </li></ol> 6. Imagen 1 <ol><li> Adquirir imágenes de la matriz nanowell utilizando un micr fluorescenciaoscope: En este ejemplo, se utiliza un Observador ZEISS Z-1 microscopio equipado con una platina motorizada y Lambda-DG4. </li><li> Inicializar el software y el microscopio y comprobar la intensidad de señal en la luz transmitida y los otros canales fluorescentes. En general, habrá cinco canales correspondientes a: luz transmitida, Anexina V-647 (Cy5 filtro), CTR (DsRed filtro), Sytox Green (filtro FITC), y Vybrant Violet (DAPI filtro). Asegúrese de que el microscopio configurarlo para garantizar la máxima señal a ruido y evitar la saturación. </li><li> Establecer ejes X e Y para la matriz nanowell. Comenzar el proceso de ajuste de la posición cero y la inicialización de enfoque en el centro de 7 x 7 nanowell bloque de matriz en la esquina superior izquierda de la marca de gama nanowell. Establecer los ejes X, Y puestos a coincidir con el centro de cada bloque y el valor z para reflejar con precisión la concentración en cada bloque. </li><li> Una vez que la posición y el enfoque está todo listo, asegúrese de establecer la adquisición de imágenes en modo lineal y adquirir imágenes. <strong&gt; Nota: Si giro necesario en la incubadora y CO 2 en el microscopio de al menos 30 min antes del experimento. </li></ol> 7. Mensaje de imágenes Colocar la placa de 4 pocillos que contiene la matriz nanowell en una incubadora (37 ° C / 5% CO 2) durante 6 horas. 8. Imagen 2 Adquirir las imágenes en el segundo punto de tiempo como se indica en el Paso 6. 9. Análisis de Imagen <ol><li> La ocupación de las células dentro de la nanowells se determina por el uso de los apropiados programas de segmentación de imágenes (por ejemplo ImageJ, <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> ) esencialmente como se describe previamente 9,12. </li><li> Análisis del conjunto inicial de imágenes de microscopio (t = 0 h; t 0) se utiliza para generar una base de datos de los nanowells individuales que contienen exactamente una célula diana en vivo (identificadopor CellTracker Rojo, rojo) y no hay células muertas (identificados por tinción con Anexina V, verde) [T1t 0 nanowells]. </li><li> Análisis de la segunda serie de imágenes de microscopio (t = 6 h, t 6) se utiliza para determinar de forma independiente una segunda base de datos de nanowells que contienen 1 célula diana (rojo) [T1t 6 nanowells]. La comprobación de integridad (consulta de base de datos) se implementa para asegurar que todos los objetivos en vivo en t 0 se hacen coincidir con los objetivos en t 6 para producir un conjunto final de objetivos coincidentes (implementada como nanowells que tienen T1t 0 = T1t 6, designada como T1match) </li><li> El número de células efectoras en nanowells se determina mediante tinción con violeta Vybrant en t 0 [nanowells E1] </li><li> Una consulta de base de datos se lleva a cabo para identificar nanowells que contienen efectores individuales co-incubadas con objetivos individuales (definidas como nanowells que tienen partido E1 y T1, designados como E1T1) </li><li> Apoptosis inducida Effector es calificada por i<html> DENTIFICACIÓN objetivos que son Anexina V negativo en t 0 y Anexina V positivo en t 4 (E1T1 con tinción Anexina V en blanco t 6). </li></ol> 10. Opcional Time-lapse de imágenes de la matanza Uso BioStation Nikon IM <ol><li> Tome una cápsula de Petri con fondo cubreobjetos y cortar un pedazo pequeño de la gama de chips nanowell (preparado en el paso 5) que se ajuste exactamente en el cubreobjetos. Asegúrese de que el chip se mantiene húmeda durante el corte. </li><li> Mezclar 1 x 10 6 dianas y 1 x 10 6 células T en 100 medios de comunicación, y mu l pipeta que en el cubreobjetos. </li><li> Espere unos 15-30 minutos a que las células se asiente. </li><li> Rápidamente invertir el pequeño chip nanowell matriz y colóquela sobre el cubreobjetos. </li><li> Con el fin de asegurar que el chip no será capaz de moverse durante la formación de imágenes, el lugar de una o dos cubreobjetos de 20 mm x 20 mm sobre la parte superior del chip. Aplicar presión para empujar suavemente el chip a la parte inferior del plato. </li></ol>tienda de campaña &quot;&gt; Nota: bottom El chip array nanowell es demasiado gruesa para imágenes de alta resolución y por lo tanto tiene que ser al revés Durante este tiempo, muchas de las células se lavan Puesto que esto no puede ser fácilmente controladas, el número de células en el Paso 9.2.. y tiempo de incubación en el paso 9,3 se deben optimizar.

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No hay conflictos de interés declarado.

Hemos esbozado el protocolo para un alto rendimiento de una sola célula ensayo citolítico habilitado a través de co-incubación de los efectores y metas en las matrices de nanowells (Figura 1). Además de rendimiento de una de las principales ventajas de la técnica es la capacidad de controlar efectora mediada por la citotoxicidad contra células diana deseadas sin la necesidad de la ingeniería de las células diana que a su vez permite el uso de células tumorales autólogas o apareadas / primaria...

<table border="1"><tbody><tr><td> Nombre del reactivo / material </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </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 a 002-CI </td><td> 10.000 UI de penicilina 10.000 g / ml estreptomicina </td></tr><tr><td> L-glutamina </td><td> Cellgro </td><td> 25 a 005-CI </td><td> 200 mM de solución </td></tr><tr><td> HEPES </td><td> Sigma Aldrich </td><td> H3537 </td><td> 1M </td></tr><tr><td> Suero fetal bovino (FBS) </td><td> Atlanta Biológicos </td><td> S11150 </td><td> Lote probado </td></tr><tr><td> Cell Tracker mancha roja </td><td> Invitrogen </td><td> C34552 </td><td> 50 g </td></tr><tr><td> Vybrant DyeCycle Violet Stain </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> Annexin V-Alexa Fluor 647 </td><td> Invitrogen </td><td> A23204 </td><td> 500 l </td></tr><tr><td> PBS de Dulbecco </td><td> Cellgro </td><td> 21-031 CV- </td><td> 500 ml </td></tr><tr><td> Agar Noble </td><td> DIFCO </td><td> 2M220 </td><td> 100 g </td></tr><tr><td> Azul de tripano </td><td> Sigma Aldrich </td><td> T8154 </td><td> 0,4% de líquido, se filtró estéril </td></tr><tr><td> Hemocitómetro </td><td> Hausser Científicos </td><td> 1492 </td><td> Bright línea </td></tr><tr><td> 4 pocillos </td><td> Thermo Fisher </td><td> 167603 </td><td></td></tr><tr><td> Harrick Plasma Cleaner </td><td> Harrick Plasma </td><td> PDC-32G </td><td> Basic limpiador de plasma </td></tr><tr><td> Observer.Z1 </td><td> ZEISS </td><td&gt; </td><td> Microscopio de fluorescencia (trabaja con las tres partes abajo) </td></tr><tr><td> Lambda 10-3 </td><td> Sutter Instrument </td><td></td><td> Filtro regulador </td></tr><tr><td> Lambda DG-4 </td><td> Sutter Instrument </td><td></td><td> Ultra-alta velocidad de conmutación de longitud de onda </td></tr><tr><td> Hamamatsu EM-CCD cámara </td><td> Hamamatsu </td><td> C9100-13 </td><td> Microscopio cámara CCD- </td></tr><tr><td> Tubo cónico de 15 ml </td><td> BD Falcon </td><td> 352097 </td><td></td></tr><tr><td> Tubo cónico de 50 ml </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> Fondo de cristal placa de cultivo </td><td> MatTek Corporación </td><td> P35G-0 </td><td> 35 mm placa de Petri de 10 mm micropocillo </td></tr></tbody></table>

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

Inmunoterapia del cáncer puede aprovechar la especificidad de la respuesta inmune para atacar y eliminar tumores. Terapia celular adoptiva (ACT) sobre la base de la transferencia adoptiva de células T modificadas genéticamente para expresar un receptor de antígeno quimérico (CAR) ha mostrado una promesa considerable en los ensayos clínicos 1-4. Hay varias ventajas de utilizar CAR + células T para el tratamiento de cánceres, incluyendo la capacidad de apuntar no-MHC antígenos restringidos y para funcionalizar las células T para la supervivencia óptima, mensajeras y persistencia dentro del huésped, y finalmente para inducir la apoptosis de CAR + células T en el caso de toxicidad para el huésped 5. La delineación de las funciones óptimas de CAR + células T asociados con un beneficio clínico es esencial para el diseño de la próxima generación de ensayos clínicos. Los recientes avances en imágenes de animales vivos como microscopía multifotónica han revolucionado el estudio de la función celular inmune in vivo 6,7. Aunque estos estudios han avanzado nuestra comprensión de las funciones de las células T in vivo, las células T ACT basada en ensayos clínicos, requiere la necesidad de vincular las características moleculares y funcionales de las células T preparaciones de pre-infusión con eficacia clínica después de la infusión, utilizando en ensayos in vitro de vigilancia de células T como las funciones, la citotoxicidad y la secreción de citoquinas. Estándar de citometría de flujo basados ​​en ensayos han sido desarrollados que determinan el funcionamiento general de las poblaciones de células T a nivel de una sola célula, pero estos no son adecuados para el seguimiento de la formación de conjugado y tiempos de vida o la capacidad de la misma célula para matar múltiples objetivos 8. Arrays microfabricados diseñado en polímeros biocompatibles, como el polidimetilsiloxano (PDMS) son un método particularmente atractivo para confinar espacialmente efectores y objetivos en volúmenes pequeños 9. En combinación con la microscopía de fluorescencia automatizado time-lapse, aunqueusands de interacciones efector-objetivo pueden ser controlados simultáneamente por los distintos pozos de imágenes de una gran nanowell. Se presenta aquí una metodología de alto rendimiento para el seguimiento de la citotoxicidad de células T mediada a nivel de una sola célula que puede aplicarse ampliamente para estudiar la funcionalidad de las células T citolítica.

Un ejemplo de la aplicación del ensayo citolítico de alto rendimiento se demuestra en la Figura 2. Brevemente, etiquetado específico CD19-CAR + células T se co-incubaron con células diana marcadas ratón EL4 en los pocillos individuales de una matriz de nanowell (Secciones 1-5). Una imagen inicial se registró en el microscopio fluorescente automatizada para identificar la ocupación (efectores y / o metas) de todos y cada uno nanowell en la matriz (Sección 6). El procesamiento de imág...

Watch this Scientific Journal Video about Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells at JoVE.com

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

Se describe una sola célula de alto rendimiento de ensayo para medir la citotoxicidad de las células T cuando se incuba con las células diana tumorales. Este método emplea una matriz densa, elastómero de sub-nanolitros pozos (~ 100.000 pozos / matriz) para confinar espacialmente las células T y las células diana en proporciones definidas, y está acoplado a la microscopía de fluorescencia para monitorizar efector-diana conjugación y posterior apoptosis.

Cuantitativo de alto rendimiento de una sola célula de ensayo de citotoxicidad de las células T

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

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

Research

JoVE Journal

Immunology and Infection

14.9K vistas.  University of Houston. Se describe una sola célula de alto rendimiento de ensayo para medir la citotoxicidad de las células T cuando se incuba con las células diana tumorales. Este método emplea una matriz densa, elastómero de sub-nanolitros pozos (~ 100.000 pozos / matriz) para confinar espacialmente las células T y las células diana en proporciones definidas, y está acoplado a la microscopía de fluorescencia para monitorizar efector-diana conjugación y posterior apoptosis.

Video: 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.

De alto rendimiento Método de detección de virus de la 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, ...

Investigación

Inmunología e Infección

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.

Una evaluación cuantitativa de la migración celular mediante el ensayo de la canción Motilidad Phagokinetic

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.

Un Ensayo Fenotípico Microscópico Para La Cuantificación De Micobacterias Intracelulares Adaptadas Para La Detección De Alto Rendimiento / Alto Contenido

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 Rendimiento Cuantitativa en tiempo real RT-PCR ensayo para determinar los perfiles de expresión de los tipos I y III de interferón 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.

Alto Rendimiento de medición de ADN extracelular de salida y la formación neta cuantitativa en 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.

De alto rendimiento, en tiempo real, Prueba de doble lectura de Intracelular Actividad antimicrobiana y la citotoxicidad 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.

Una de linfocitos ensayo basado en la fluorescencia adecuado para el cribado de alto rendimiento de pequeñas 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.

Un análisis de alto rendimiento para evaluar y cuantificar la formación de neutrófilos trampa extracelular

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.

Un alto rendimiento Shigella-ensayo 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.

Funcionalización de voladizos de microscopio de fuerza atómica con células de una t o de una sola partícula para espectroscopia de fuerza de una sola célula inmunológica

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