Damos las gracias al personal del Anatómico laboratorios de patología de la Royal Prince Alfred y Hospitales de Concordia repatriación de recogida de muestras frescas de la CRC y la mucosa intestinal normal. El trabajo fue financiado por una beca del Instituto del Cáncer de Nueva Gales del Sur Programa traslacional.

<img src="/files/ftp_upload/3322/3322fig1.jpg" alt="Figura 1" /> Figura 1. Flujo de trabajo para la preparación de una suspensión de células vivas de una muestra quirúrgica del CRC. 1. Desagregación de la muestra clínica Todas las muestras fueron recolectadas en el Hospital Royal Prince Alfred (Camperdown, NSW, Australia) y el Hospital Concord de Repatriación (Concord West, NSW, Australia) con el consentimiento informado en el Protocolo N º X08-164. <ol><li> Recoger el cáncer fresco colorrectal (CCR) o muestras de adenoma, y ​​en la mucosa intestinal normal por lo menos 10 cm del tumor. Almacene las muestras en Hank equilibrado de pH de la solución salina 7,3 (HBSS) a 4 ° C durante 12 h después de la resección. </li><li> Siga las normas de seguridad de los patógenos humanos, procesar todas las muestras clínicas en un gabinete de seguridad biológica de clase II. Diseccionar las muestras en dos cubos mm en una placa de Petri con dos hojas de bisturí. </li><li> Incubar tumor y el tejido normal por separado en tubos Eppendorf con ocasionales mezcla suave durante 60 minutos a 37 ° C con un volumen igual de medio RPMI 1640 que contiene 2% (v / v) colagenasa tipo 4 (Worthington, Lakewood, NJ, EE.UU.) y 0,1 % (w / v) desoxirribonucleasa I de páncreas bovino (DNAsa I, Sigma-Aldrich). </li><li> Fuerza semi-digeridos de tejidos a través de un colador de alambre de malla fina con un émbolo de una jeringa de 10 ml, células de lavado a través de HBSS. </li><li> Pase resultante suspensión de células a través de 200 micras y 50 micras filtros Filcon (BD Biosciences) para eliminar los agregados celulares. La mayoría de los agregados de ADN, moco y células se extraen de esta serie de filtraciones. </li><li> Centrifugar las suspensiones de células a 400 xg a 20 º durante 5 minutos. </li><li> Pellets resuspender celular en FCS inactivado por calor con 10% dimetilsulfóxido (DMSO), la congelación lenta en crioviales y almacenar a -80 °. El proceso de congelación tiende a reducir la mucosidad en la muestra y la lisis de glóbulos rojos. </li></ol> 2. Preparación de muestras para la captura de células <ol><li> Descongelar las muestras de forma rápida en un baño de agua a 37 ° y suspender las células en 10 ml de HBSS para lavar el DMSO. </li><li> Centrifugar las suspensiones de células a 410 xg a 20 º durante 5 minutos. </li><li> Se decanta el sobrenadante y resuspender el botón celular en 500 l de HBSS. </li><li> Tratar la muestra con 0,1% (w / v) ADNasa I durante 20 minutos a temperatura ambiente. </li><li> Mezcla de 10 l de cada suspensión celular con un volumen igual de azul de tripano y de carga de 10 l de la mezcla en un hemocitómetro. El uso de un microscopio de luz a 100 veces de aumento, el recuento de células viables, lo que parece claro debido a la exclusión azul de tripano, mientras que las células muertas de tomar el colorante. Un mínimo de 4 x 10 6 células viables se requiere para la captura de células en el microarray. </li><li> Después del tratamiento con DNAsa, resuspender la suspensión celular en 10 ml de HBSS y centrifugar a 410 xg a 20 º durante 5 minutos. </li><li> Decantar el sobrenadante y resuspender pellets de células en RPMI 1640 con un volumen final de 200 mL. </li></ol> 3. Microarrays de anticuerpos de captura de la célula <ol><li> Humedezca los microarrays de anticuerpos DotScan por inmersión de la sección de nitrocelulosa en tampón fosfato salino (PBS) durante unos 20 s. Limpie con cuidado los bordes del vidrio de la micromatriz con Kimwipes cruzados, evitando tocar la sección de nitrocelulosa. </li><li> Agregue el agua a la bandeja de incubación de microarrays para proporcionar una cámara húmeda. Coloque los microarrays en la cámara y la pipeta la suspensión de células en RPMI 1640 en la sección de nitrocelulosa húmeda. Pipeta gotas en cada esquina de la nitrocelulosa para garantizar un reparto equilibrado de las células. </li><li> Incubar los microarrays a 37 ° C durante 1 h. El período de incubación permite que las células puedan establecerse y entrar en contacto con los anticuerpos en el microarray. Las células que expresan antígenos de superficie que corresponde a los anticuerpos que la tierra en que va a capturar. </li><li> Después de la incubación, sumerja los microarrays suave y verticalmente en tres pilas que contienen al menos 15 ml de PBS para lavar las células no unidas (20 s por lavado). </li><li> Prepare un 3,7% (w / v) de formaldehído en PBS para fijar las células y anticuerpos de entrecruzamiento. Pipeta suavemente alrededor de 1 mL para cubrir la sección de nitrocelulosa de la micromatriz. Incubar durante 20 min a temperatura ambiente. </li><li> Próxima inmersión microarrays en 3 cambios de PBS (15 ml, 30 s cada uno) para vaciar el exceso de formaldehído. </li><li> Limpie los bordes y la parte posterior de la lámina de vidrio con Kimwipes y explorar el uso de microarrays del escáner mientras DotScan, la sección de nitrocelulosa está húmedo. La tomografía óptica proporciona el patrón de expresión de los antígenos de una mezcla de células las células de la población por ejemplo, CRC, leucocitos y otras células del estroma del tumor. </li></ol> 4. Fluorescencia de multiplexación <ol><li> Quitar el microarray del escáner y se aplican 200 l de tampón de bloqueo (2% w / v BSA, 2% inactivado por calor suero humano AB, PBS, pH 7,3). Incubar en la bandeja de microarrays a temperatura ambiente durante 20 min. </li><li> Prepare la multíparaléxico solución en un tubo Eppendorf, cubiertas con papel de aluminio: 20 l ficoeritrina anti-CD3 (Beckman Coulter, Gladesville, NSW, Australia, # IM12824; 1/7.5 dilución final), 10 l Alexa Fluor 647-anti-EpCAM (Biolegend, San Diego, California, EE.UU., 1 / 15 de dilución), 2 l de suero inactivado por calor humano AB (Sigma-Aldrich, Castle Hill, NSW, Australia) y 118 l de tampón de bloqueo. </li><li> Escurrir el exceso de tampón de bloqueo de los microarrays y la pipeta la solución de multiplexación en la sección de nitrocelulosa, extendiendo uniformemente. Incubar durante 30 minutos en la oscuridad a temperatura ambiente. </li><li> Sumerja los microarrays verticalmente en tres canales de 15 ml de PBS fresco (30 s cada uno). </li><li> Deje secar los microarrays en la oscuridad y se almacenan a 4 ° C en una caja de diapositivas. Los microarrays se pueden almacenar en la oscuridad durante un máximo de 3 meses sin pérdida de fluorescencia. </li><li> Busque en la micromatriz seco con un tifón FLA 9000 escáner (GE Healthcare, Rydalmere, NSW, Australia) con la resolución ajustada a 50 (532 nm láser, 580 BP30 emisión de filtro para el PE. 633 nm láser y 670 BP30 emisión de filtro para Alexa 647). Los microarrays son escaneados con el lado de nitrocelulosa hacia abajo en la bandeja de cristal del escáner. </li><li> Guardar las imágenes fluorescentes como archivos TIFF y Tamaño de la imagen de conjunto de Photoshop 17 x 25 cm y una resolución de 72 píxeles / cm. Importar la imagen en el software de análisis DotScan para analizar la intensidad de los puntos. </li><li> El DotReader captura una imagen digital del patrón de unión de puntos y cuantifica la densidad de la unión de células en cada punto de anticuerpos en una escala de gris de 8 bits (1-256 U). Vez en cuando la unión no específica de control de isotipo se restará de los valores de unión de los anticuerpos con los isotipos de inmunoglobulinas correspondientes. La intensidad de fluorescencia de puntos para cada uno de microarrays se normalizaron en contra de la más brillante punto fijado en la intensidad del 100%. Señal / spot fuerza fue grabado en un archivo. Xml (datos brutos) o representado como un gráfico de barras en un archivo. Pdf (informe final) </li><li> Microarrays heatsmaps y la agrupación jerárquica se realizaron utilizando Multiexperiment Viewer (MeV) la versión 4.4 de la suite TM4 Microarrays Software ( <a href="http://www.tm4.org/mev.html">http://www.tm4.org/mev.html</a> ). Agrupación jerárquica se realizó en los antecedentes-ajustado por los MeV con el análisis de ligamiento completo. Distancia euclídea fue utilizado para medir la similitud. El estudiante de 2 colas de la t-test con una variación igual se utiliza para determinar la significación estadística de los resultados. </li></ol> 5. Los resultados representativos: Los resultados de los microarrays DotScan debe mostrar patrones consistentes de células de unión entre los conjuntos de duplicados. Fuerte alineación de puntos de unión (CD44/CD29) permite a una red que se coloca sobre el área de la matriz. La figura 2 muestra un ejemplo de captura óptima de la célula y la multiplexación. La Figura 3 muestra algunos problemas comunes encontrados durante la captura de células y las posibles soluciones. Los resultados de microarrays de células de unión se puede cuantificar mediante la medición de la intensidad del punto expresado en una escala de gris que van de 1 a 256. La Figura 4 muestra los datos numéricos a partir de 58 muestras quirúrgicas CRC, se tiñeron con EpCAM-Alexa 647 anticuerpo, como un mapa de calor con la agrupación jerárquica. A pesar de que el número de muestras es limitado, CRC de la misma etapa tienden a agruparse en el mismo grupo. <img src="/files/ftp_upload/3322/3322fig2.jpg" alt="Figura 2" /> Figura 2. Patrón de unión de células tumorales clínicos del cáncer colorrectal (Australia Clínica Patológica Andamios, ACP escenario B1). (A) DotScan clave de anticuerpos con la ubicación de los anticuerpos de la mitad izquierda de la micromatriz duplicado (contorno). La sección superior contiene el original de 82 anticuerpos de los microarrays de la leucemia DotScan. Un adicional de 40 anticuerpos, lo que corresponde a los antígenos de superficie específica resultó ser regulado hasta en la literatura, se han añadido como los microarrays de &quot;satélite&quot; de un CRC. La sección inferior se compone de anticuerpos isotipo de control (b) la imagen óptica de las células CRC vinculante para los microarrays. (C) CD3 imagen fluorescente que muestra células-T. (D) EpCAM fluorescencia imagen que muestra las células CRC. <img src="/files/ftp_upload/3322/3322fig3.jpg" alt="Figura 3" /> Figura 3. Ejemplos de los pobres resultados DotScan y sus posibles soluciones. (A) de células de baja obligatoria; solución: asegúrese de que al menos 4x106 células viables se encuentran en la matriz (b) células control de isotipo vinculantes y no vinculantes específicos; solución: añadir inactivado por calor suero humano AB a la muestra antes de la incubación de microarrays para reducir al mínimo isotipo de control obligatorio. De vez en cuando, una pequeña cantidad de la unión no específica de las células de la nitrocelulosa que ocurre con las muestras de CRC y no afectan significativamente los resultados. (C) La nitrocelulosa se seque durante la incubación, la solución: asegurar la muestra cubre toda la sección de nitrocelulosa y microarrays se incuba en una superficie plana. (D) los artefactos de alta de fondo, solución: asegurar lamicroarrays se lava bien después de incubación. <img src="/files/ftp_upload/3322/3322fig4.jpg" alt="Figura 4" /> Figura 4. DotScan software de análisis genera gráficos de barras que representan las densidades de células de unión en una escala de gris que van de 1 a 256. Los números del eje se refieren a los antígenos CD. Otras abreviaturas han sido TCR, receptor de células T, κ, λ, cadenas ligeras de inmunoglobulinas; sIg, inmunoglobulina de superficie, DCC, eliminado en la proteína del cáncer colorrectal, el EGFR, receptor del factor de crecimiento epidérmico; FAP, la proteína de activación de fibroblastos, HLA-A, B, C HLA-DR, DR antígenos de leucocitos humanos y A, B y C, respectivamente, MICA, MHC de clase I de la cadena relacionados con la proteína A, MMP-14, metalopeptidasa matriz de 14; pIgR, el receptor de inmunoglobulina polimérica; TSP-1, trombospondina-1; Mabthera, anti-CD20 humanizado. <a href="http://www.jove.com/files/ftp_upload/3322/3322fig4large.jpg">Haga clic aquí para ampliar la imagen</a> .

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No hay conflictos de interés declarado.

En este video, que demuestran cómo los microarrays de anticuerpos DotScan se puede utilizar en una forma simple, semi-cuantitativo de manera de estudiar los perfiles de antígeno de superficie de las poblaciones de células del tejido CRC. La obtención de una suspensión de células viables solo a partir de tejido es esencial para el éxito del experimento, ya que depende de la energía los procesos (por ejemplo, el antígeno de recubrimiento y / o la formación de pseudópodos) parecen se...

The author's email has been corrected in the publication of <a href="http://www.jove.com/video/3322">Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing</a>. There was an error with the author, Jerry Zhou's, email. The author's email has been updated to:
j.zhou@uws.edu.au
from:
jzho7551@mail.usyd.edu.au

Erratum: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

<table border="1"><tbody><tr><th> Nombre del reactivo o equipo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> Solución salina balanceada de Hanks </td><td> Sigma-Aldrich </td><td> H6136-10X1L </td><td> Tamponado con Hepes 25 mM (Sigma # H3375) </td></tr><tr><td> Airpure gabinete de seguridad biológica de clase II </td><td> Westinghouse </td><td> 1687-2340/612 </td><td></td></tr><tr><td> Hojas de bisturí </td><td> Livingstone </td><td> 090609 </td><td> Paquete de 100 </td></tr><tr><td> RPMI 1640 con 2 mM Hepes </td><td> Sigma-Aldrich </td><td> R4130-10X1L </td><td></td></tr><tr><td> Colagenasa tipo 4 </td><td> Worthington </td><td> 4188 </td><td></td></tr><tr><td> Desoxirribonucleasa 1 </td><td> Sigma-Aldrich </td><td> DN25-1G </td><td></td></tr><tr><td> Terumo jeringa (10 ml) </td><td> Terumo </td><td> SS 10 L </td><td> Caja de 100 </td></tr><tr><td> Filcon filtro (200 m) </td><td> BD Biosciences </td><td> 340615 </td><td></td></tr><tr><td> Filcon filtro (50 micras) </td><td> Filcon filtro (50 micras) </td><td> Filcon filtro (50 micras) Filcon filtro (50 micras) y 340.603 </td><td></td></tr><tr><td> Suero fetal bovino </td><td> Gibco / Invitrogen </td><td> 10099-141 </td><td></td></tr><tr><td> Centrífuga 5810 R </td><td> Eppendorf </td><td> 7017 </td><td></td></tr><tr><td> Dimetilsulfóxido </td><td> Sigma-Aldrich </td><td> D2650 </td><td></td></tr><tr><td> El azul tripán </td><td> Sigma-Aldrich </td><td> T8154 </td><td></td></tr><tr><td> Hemocymeter Technocolor Neubar </td><td> Hirschmann </td><td> no está disponible </td><td></td></tr><tr><td> Microscopio de luz </td><td> Nikon </td><td> Nikon TMS </td><td></td></tr><tr><td> Tubos Cyrovial </td><td> Greiner Bio-One </td><td> 121278 </td><td></td></tr><tr><td> Cryo contrainer congelación </td><td> Nalgene </td><td> 5100-0001 </td><td></td></tr><tr><td> DotScan anticuerpos microarrays kit </td><td> Medsaic </td><td> no está disponible </td><td></td></tr><tr><td> DotScan microarrays bandeja de lavado </td><td> Medsaic </td><td> no está disponible </td><td></td></tr><tr><td> Kimwipes </td><td> Kimberly-Clark </td><td> 4103 </td><td></td></tr><tr><td> Formaldehído 37% </td><td> Sigma-Aldrich </td><td> F1635-500ML </td><td></td></tr><tr><td> DotReaderTM </td><td> Medsaic </td><td> no está disponible </td><td></td></tr><tr><td> Albúmina de suero bovino </td><td> Sigma-Aldrich </td><td> A9418-10G </td><td></td></tr><tr><td> Inactivado por calor suero AB 2% </td><td> Invitrogen </td><td> 34005100 </td><td></td></tr><tr><td> Ficoeritrina conjugado CD3 </td><td> Beckman Coulter </td><td> ET386 </td><td></td></tr><tr><td> AlexaFluor647 conjugado EpCAM </td><td> BioLegend </td><td> 324212 </td><td></td></tr><tr><td> Tifón FLA 9000 </td><td> GE Healthcare </td><td> 28-9558-08 </td><td> 532 nm láser, 580 BP30 emisión de filtro para el PE. 633 nm láser y 670 BP30 emisión de filtro para Alexa647 </td></tr><tr><td> Multiexperiment Visor v4.4 </td><td> TM4 Microarrays Software Suite </td><td> Abierto - el software de código (Ref 11) </td><td></td></tr></tbody></table>

colorectal cancer cell surface protein profiling using an antibody microarray and fluorescence multiplexing

El pronóstico actual y la clasificación de la Convención se basa en la puesta en escena de sistemas que se integran los hallazgos histopatológicos y clínicos. Sin embargo, en la mayoría de los casos de CCR, disfunción de las células es el resultado de numerosas mutaciones que modifican la expresión de proteínas y modificaciones post-traduccionales 1. Una serie de antígenos de superficie celular, incluyendo cluster de diferenciación (CD) antígenos, han sido identificados como potenciales de pronóstico o marcadores metastásicos en el CCR. Estos antígenos hacer biomarcadores ideal como su expresión cambia a menudo con la progresión del tumor o interacciones con otros tipos de células, como linfocitos infiltrantes de tumor (TIL) y los macrófagos asociados al tumor (TAM). El uso de la inmunohistoquímica (IHC) para el cáncer de sub-clasificación y el pronóstico está bien establecido para algunos tipos de tumores 2,3. Sin embargo, no solo &quot;marcador&quot; ha demostrado importancia pronóstica superior clínico-patológicas de ensayo o de ganado amplia aceptación para su uso en la presentación de informes de rutina de todos los casos la patología CRC. Un enfoque más reciente de la estratificación pronóstica de los fenotipos de la enfermedad se basa en los perfiles de proteínas de superficie con varios &quot;marcadores&quot;. Mientras que los perfiles de expresión de los tumores mediante técnicas proteómicas como iTRAQ es una poderosa herramienta para el descubrimiento de biomarkers4, no es el óptimo para su uso rutinario en los laboratorios de diagnóstico y no se pueden distinguir diferentes tipos de células en una población mixta. Además, grandes cantidades de tejido tumoral se requieren para la elaboración de perfiles de las glicoproteínas de la membrana plasmática purificada por estos métodos. En este video se describe un método simple para crear perfiles de la superficie del proteoma de células viables de muestras desglosados ​​CRC utilizando un microarray de anticuerpos DotScan CRC. Los microarrays 122-anticuerpo consiste en un estándar de 82-anticuerpo reconoce una amplia región del linaje de los marcadores específicos de leucocitos, las moléculas de adhesión, los receptores y los marcadores de inflamación y respuesta inmune 5, junto con una región por satélite para la detección de 40 marcadores potencialmente pronóstico de CRC . Las células son capturados sólo en los anticuerpos para los que expresan el antígeno correspondiente. La densidad de las células por punto, determinado por lectura óptica, refleja la proporción de células que expresan ese antígeno, el nivel de expresión del antígeno y la afinidad de los anticuerpos 6. Para el tejido de la Convención o en la mucosa intestinal normal, las exploraciones ópticas reflejan el inmunofenotipo de la población mixta de células. Multiplexación de fluorescencia se puede utilizar con el perfil seleccionado sub-poblaciones de células de interés capturado en la matriz. Por ejemplo, Alexa molécula de adhesión celular 647-anti-epiteliales (EpCAM, CD326), es un antígeno de diferenciación pan-epiteliales que se usa para detectar células CRC y también las células epiteliales de la mucosa intestinal normal, mientras que ficoeritrina anti-CD3, se utilizó para detectar la infiltración de células T 7. El CRC DotScan microarrays debe ser el prototipo de una alternativa de diagnóstico para el sistema de clasificación anatómica basada en CRC.

Watch this Scientific Journal Video about Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing at JoVE.com

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

Se describe un procedimiento para la desagregación de cáncer colorrectal (CCR) para producir células viables único, que son capturados en microarrays de anticuerpos a medida el reconocimiento de los antígenos de superficie (CRC DotScan microarrays). Subpoblaciones de células obligado a los microarrays pueden ser perfiladas por multiplexación de fluorescencia utilizando anticuerpos monoclonales marcados con colorantes fluorescentes.

Celular del cáncer colorrectal superficie de la proteína perfiles con un microarray de anticuerpos y la multiplexación de fluorescencia

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

colorectal-cancer-cell-surface-protein-profiling-using-an-antibody

Research

JoVE Journal

Immunology and Infection

13.9K vistas.  University of Sydney. Se describe un procedimiento para la desagregación de cáncer colorrectal (CCR) para producir células viables único, que son capturados en microarrays de anticuerpos a medida el reconocimiento de los antígenos de superficie (CRC DotScan microarrays). Subpoblaciones de células obligado a los microarrays pueden ser perfiladas por multiplexación de fluorescencia utilizando anticuerpos monoclonales marcados con colorantes fluorescentes.

Video: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

El uso de un ensayo de microarrays Pan-viral (Virochip) a la pantalla de muestras clínicas de los patógenos virales

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Investigación

Inmunología e Infección

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular localization can provide insights into the functional role of bacterial proteins. Surface localization of bacterial proteins is a key step towards identification of virulence factors involved in mechanisms of pathogenicity. 
Methods for fractionating leptospiral membranes 2-5 may be selective for a certain class of outer-membrane proteins (OMPs), such as lipoproteins vs. transmembrane OMPs, and therefore lead to misclassification. This likely is due to structural differences and how they are associated to the outer membrane. Lipoproteins are associated with membranes via a hydrophobic interaction between the N-terminal lipid moiety (three fatty acids) and the lipid bilayer phospholipids 6, 7. In contrast, transmembrane OMPs are typically integrated into the lipid bilayer by amphipathic &beta;-sheets arranged in a barrel-like structure 8, 9. In addition, presence of a protein in the outer-membrane does not necessarily guarantee that the protein or its domains are exposed on the surface. Spirochetal outer membranes are known to be fragile and therefore necessitate methods involving gentle manipulation of cells and inclusion of sub-surface protein controls to assess the integrity of the outer membrane.
Here, we present an immunofluorescence assay (IFA) method to directly assess surface exposure of proteins on intact leptospires. This method is based on recognition of leptospiral surface proteins by antigen-specific antibodies. Herein, antibodies specific for OmpL5410 are detetcted aftero binding to native, surface exposed epitopes. Comparison of antibody reactivity to intact versus permeabilized cells enables evaluation of cellular distribution and whether or not a protein is selectively present on leptospiral surface. The integrity of outer membrane should be assessed using antibody to one or more subsurface proteins, preferably located in the periplasm.
The surface IFA method can be used to analyze surface exposure of any leptospiral protein to which specific antibodies are available. Both the usefulness and limitation of the method depends on whether the antibodies employed are able to bind to native epitopes. Since antibodies often are raised against recombinant proteins, epitopes of native, surface-exposed proteins may not be recognized. Nevertheless, the surface IFA method is a valuable tool for studying components of intact bacterial surfaces. This method can be applied not only for leptospires but also other spirochetes and gram-negative bacteria. For stronger conclusions regarding surface-exposure of OMPs, a comprehensive approach involving several cell localization methods is recommended 10.

An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.

Inmunofluorescencia ensayo de leptospiras superficie expuesta proteínas

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Sola célula de análisis de Bacillus subtilis Las biopelículas mediante microscopía de fluorescencia y citometría de flujo

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

La visualización de las respuestas inducidas por toxinas bacterianas mediante microscopía de fluorescencia de células en vivo

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Un enfoque comparativo para caracterizar el paisaje del Huésped-Patógeno interacciones proteína-proteína

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Un ensayo de microarrays miniaturizada glicanos para la evaluación de avidez y especificidad del virus de la gripe A Hemaglutininas

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

The on-bead method for labeling antibodies with small molecules enables labeling of a small amount of antibodies directly from cell media. This method is compatible with amine and thiol chemistry, and can handle multiple samples in parallel, manually or using automated platforms.

Antibody Labeling con tintes fluorescentes utilizando proteína magnética A y Proteína G cuentas

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

Un análisis de Microarray de proteínas para la determinación serológica de anticuerpos las respuestas siguientes infección por Clostridium difficile específica de antígeno

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Sola célula cuantificación del mRNA y la expresión de la proteína de superficie de CD4 infectados por el Virus de la inmunodeficiencia de los simios+ T las células aisladas de macaques del macaco de la India

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.