Los autores desean reconocer la asistencia técnica de Banes Michelle, así como el Dr. Petrie-Hanson, anguilas y el Dr. Brown Timoteo por su asistencia en el desarrollo y la realización de la inmunohistoquímica. Este proyecto fue financiado por el USDA CRIS proyecto # MISV-0801310 y por la Universidad Estatal de Mississippi Colegio de Medicina Veterinaria.

<html> 1. Ataque bacteriano <ol><li> Crecimiento de las bacterias durante la noche a 30 ° C en agitación de cerebro y corazón de caldo de infusión. </li><li> Detener la circulación del agua en los tanques de peces y añadir el caldo en una concentración de 1 en 100 (10 ml por litro, por ejemplo). </li><li> Después de una hora de incubación, reinicie la circulación del agua en los tanques para eliminar las bacterias restantes. </li></ol> 2. Muestreo <ol><li> Tiempos de muestreo y de los órganos depende del objetivo del estudio, pero en nuestro estudio, hemos utilizado los siguientes puntos de muestreo: 1, 6, 16, 24, 36, 48, 60, 72, 96, 110, 125 y 175 horas post-infección . </li><li> En cada uno de los puntos de tiempo, la eutanasia a seis peces por inmersión en agua que contenga MS222 y muestra los siguientes órganos (los órganos de selección de la muestra se basó en lo que se conoce del proceso de infección): agallas, músculos posteriores a lo largo de la línea lateral, intestino, bazo, hígado, estómago, corazón, riñón cabeza, tronco y los riñones. </li><li> En el muestreo, la carga inmediata de los órganos de casetes histológicos y sumergirlos durante dos horas en formol al 1%. </li><li> Después de dos horas de fijación, la transferencia de las cintas de formol al 50% de etanol, donde permanecen hasta la incrustación. </li><li> Parafina integrar los órganos durante la noche utilizando un pañuelo de papel-Tek VIP 3000 procesador de tejidos (Sakura Tek, Torrance, California) y la sección de 5μm con una Leica RM2255 microtono (Leica Microsystems, Bannockburn, Illinois). </li></ol> 3. Secciones desparafinado <ol><li> Sumerja las secciones de un canal lleno de manchas claras de rito durante aproximadamente 5 minutos para permitir que toda la cera que se disuelva. Levante el bastidor y escurra el exceso de cera solvente. </li><li> Sumerja las partes en un segundo recipiente de claro-rito por 5 minutos. Después de varios usos, sustituir el disolvente en el primer recipiente y se alternan entre los contenedores de primera y segunda. </li><li> Sumerja las secciones en el 100% de alcohol para quitar la cera solvente. </li><li> Sumerja las secciones de un segundo a través de 100% de alcohol. </li><li> Sumerja las secciones de un canal de 70% de alcohol. </li><li> Sumerja las partes con agua del grifo para eliminar todo rastro de alcohol. </li></ol> 4. Búfer de preparación <ol><li> Preparar tampón fosfato salino mediante la disolución de 8,0 g de NaCl, 0,2 g KCl, 1,15 g de Na 2 HPO 4 y 0,2 g KH 2 PO 4 en 1 litro de agua destilada y el pH a 7,4. </li><li> Diluir PBS a 0,9 veces mediante la adición de 900 ml de PBS a 100 ml de agua destilada. </li><li> Preparar suero de bloqueo mediante la adición de 2 ml suero de ratón, 200 l de Triton X-100 y 500 l de BSA a 50 ml de PBS 0.9x. </li><li> Preparar 50 mM de bicarbonato de solución salina tamponada se prepara disolviendo 2,1 g de NaHCO 3 y 4 g de NaCl en 500 ml de H2O destilada y pH a 8.2. </li></ol> 5. Immunhistochemistry <ol><li> Rehidratar secciones por inmersión en PBS 0.9x. </li><li> Incubar los portaobjetos durante 30 minutos en suero de bloqueo. </li><li> Incubar las secciones en ED9 (diluido 1 en 100 en el suero de bloqueo) durante dos horas en una cámara húmeda opaco a temperatura ambiente. </li><li> Enjuague las secciones 4 veces en PBS durante 10 minutos cada uno. </li><li> Incubar las secciones de dos horas en 50 l de la secundaria de anticuerpos (anti-ratón de cabra anticuerpos marcado con FITC, diluido 500 veces en el suero de bloqueo) en una cámara húmeda opaco a temperatura ambiente. </li><li> Enjuague las secciones 3 veces en solución salina tamponada con bicarbonato. </li><li> Enjuague las secciones brevemente en agua destilada. </li></ol> 6. Montaje <ol><li> Dejar el portaobjetos se sequen en la oscuridad durante unos minutos. </li><li> Montaje se realiza mediante permafluor. </li><li> Dejar el medio de montaje para secar, preferentemente durante la noche, en un ambiente fresco y oscuro. </li></ol> 7. Microscopio <ol><li> Los cortes histológicos están listos para observar bajo un microscopio de fluorescencia. En nuestro experimento, se utilizó un microscopio Olympus BX51 equipado con un triple (FITC, Texas-Red MCA) del filtro. El software Picture Frame (MicroFire, Goleta, California) fue utilizado. </li></ol> 8. Los resultados representativos: Debido a que el auto fluorescencia de los tejidos del pez es tan alto, estos tejidos van a aparecer de color naranja en el Tri-filtro, convenientemente proporcionar un fondo para las bacterias. En este contexto, el individuo bacterias teñidas con FITC aparecerá como brillantes puntos verdes y, en su forma de 200X varilla será reconocible (Fig 1). Las secciones defectuosas pueden constituir falsos positivos (debido a un problema en la fase de lavado), en cuyo caso una gran cantidad de bacterias que parecen estar presentes de manera uniforme en la diapositiva. <img src="/files/ftp_upload/2687/2687fig1.jpg" alt="Figura 1"/> Figura 1. Micrografía de una sección del músculo que muestra a dos E. ictaluri (flechas A y B) (200x). igura 2 &quot;/&gt; Figura 2. Esquema general del experimento

<ol>
	<li>Plumb, J. A., Woo, P. T. K., Bruno, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fish Diseases and Disorders. , 479-479 (1998).</li><li>Roberts, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fish Pathology. , (2001).</li><li>Lawrence, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Louisiana State University. , (1997).</li><li>Nusbaum, K. E., Morrisson, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+35S-Labeled+Edwardsiella+ictaluri+into+Channel+Catfish.">Entry of 35S-Labeled Edwardsiella ictaluri into Channel Catfish.</a> Journal of Aquatic Animal Health. 8, 146-146 (1996).</li><li>Karsi, A., Menanteau-Ledouble, S., Lawrence, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+bioluminescent+Edwardsiella+ictaluri+for+noninvasive+disease+monitoring.">Development of bioluminescent Edwardsiella ictaluri for noninvasive disease monitoring.</a> FEMS Microbiology Letters. 260, 286-286 (2006).</li><li>Miyazaki, T., Plumb, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+Edwardsiella+ictaluri+in+channel+catfish,+Ictalurus+punctatus+(Rafinesque).">Histopathology of Edwardsiella ictaluri in channel catfish, Ictalurus punctatus (Rafinesque).</a> Journal of Fish Diseases. 8, 839-839 (1985).</li><li>Areechon, N., Plumb, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Edwardsiella+ictaluri+in+Channel+Catfish,+Ictalurus+punctatus.">Pathogenesis of Edwardsiella ictaluri in Channel Catfish, Ictalurus punctatus.</a> Journal of the World Mariculture Society. 14, 249-249 (1983).</li><li>Dumpala, P. R., Lawrence, M. L., Karsi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+Edwardsiella+ictaluri.">Proteome analysis of Edwardsiella ictaluri.</a> Proteomics. 9, 1353-1353 (2009).</li><li>Thune, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signature-Tagged+Mutagenesis+of+Edwardsiella+ictaluri+Identifies+Virulence-Related+Genes,+Including+a+Salmonella+Pathogenicity+Island+2+Class+of+Type+III+Secretion+Systems.">Signature-Tagged Mutagenesis of Edwardsiella ictaluri Identifies Virulence-Related Genes, Including a Salmonella Pathogenicity Island 2 Class of Type III Secretion Systems.</a> Applied and Environmental Microbiology. 73, 7934-7934 (2007).</li><li>Karsi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+bioluminescence+mutant+screening+strategy+for+identification+of+bacterial+virulence+genes.">High-throughput bioluminescence mutant screening strategy for identification of bacterial virulence genes.</a> Applied and Environmental Microbiology. 75, 2166-2166 (2009).</li><li>Hook, E. W., Mandell, G. L., Douglas, G., Bennett, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles and Practices of Infectious Diseases. , 1700-1700 (1990).</li><li>Ainsworth, J., Capley, G., Waterstreet, P., Munson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+monoclonal+antibodies+in+the+indirect+fluorescent+antibody+technique+(IFA)+for+the+diagnosis+of+Edwardsiella+ictaluri.">Use of monoclonal antibodies in the indirect fluorescent antibody technique (IFA) for the diagnosis of Edwardsiella ictaluri.</a> Journal of Fish Diseases. 9, 439-439 (1986).</li></ol>

Este protocolo detalles de una técnica para la visualización in situ en el bagre patógeno Edwardsiella ictaluri en los cortes histológicos. A lo mejor de nuestro conocimiento, este es el primer protocolo como se describe. El paso más crítico, en nuestra experiencia, es el lavado de los anticuerpos como se describe en el paso 4.4 del presente protocolo como el lavado insuficiente puede dar lugar a falsos positivos. El principal problema con...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> Caldo de infusión cerebro </td><td> Becton Dickinson </td><td> 237200 </td></tr><tr><td> MS222 </td><td> Argent Laboratorios </td><td></td></tr><tr><td> Suero de ratón todo </td><td> Cappel </td><td> 55989 </td></tr><tr><td> Cabra anti-ratón FITC </td><td> Southern Biotech </td><td> 1010-1004 </td></tr><tr><td> Permafluor </td><td> Laboratorio de Visión </td><td> Ta-030-FM </td></tr><tr><td> Cloruro de potasio (KCL) </td><td> Sigma </td><td> P-4504 </td></tr><tr><td> Triton X- </td><td> Sigma-Aldrich </td><td> X100-6X500ML </td></tr><tr><td> Albúmina de suero bovino </td><td> Sigma </td><td> 85040C </td></tr><tr><td> Fosfato de potasio (KH 2 PO 4) </td><td> Sigma </td><td> P-5379 </td></tr><tr><td> Bicarbonato de Sodio (NaHCO 3) </td><td> Sigma </td><td> S-5761 </td></tr><tr><td> Cloruro de sodio (NaCal) </td><td> Sigma </td><td> S-9625 </td></tr><tr><td> Fosfato sódico dibásico (Na 2 HPO 4) </td><td> Sigma </td><td> S-9390 </td></tr></tbody></table>

use of fluorescent immuno-chemistry for the detection of edwardsiella ictaluri in channel catfish (i. punctatus) samples

Mientras Edwardsiella ictaluri es un importante patógeno de bagre de canal Ictalurus punctatus y se ha descubierto hace casi tres décadas 1,2, hasta ahora, a lo mejor del conocimiento de estos autores, no existe ningún método ha sido desarrollado para permitir la visualización en situ de la las bacterias en los cortes histológicos. Mientras que la localización de bacterias se ha determinado in vivo en los estudios previos con el conteo de placas 3, 4 radiométricas etiquetados, o bacterias bioluminiscentes 5, la mayoría de estos estudios se han realizado sólo en el nivel del órgano bruto, con una excepción 6. Esta limitación es particularmente preocupante debido a E. ictaluri tiene un complejo ciclo de infección de 1,7, y tiene una variedad de factores de virulencia 8,9. La compleja interacción de E. ictaluri con su huésped es similar en muchos aspectos a Salmonella typhi 10, que se encuentra en la misma familia taxonómica. A continuación se describe una técnica que permite la detección de bacterias utilizando indirectos de inmunohistoquímica utilizando el anticuerpo monoclonal ED9 anticuerpos descritos por Ainsworth et al. 11. En pocas palabras, un suero de bloqueo se aplica a los cortes histológicos embebidos en parafina para evitar que no específica vinculante. A continuación, las secciones se incuban con el anticuerpo primario: E. ictaluri anticuerpo monoclonal específico ED9. El exceso de anticuerpos se enjuaga y el FITC anticuerpos secundarios marcados se agregan. Después del lavado, las secciones se montan con un fluorescente medio específico de montaje. Esto permitió la detección de E. ictaluri in situ en secciones histológicas de tejidos bagre de canal.

Watch this Scientific Journal Video about Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples at JoVE.com

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

A continuación se describe un procedimiento que permita el etiquetado de los<em&gt; Edwardsiella ictaluri in situ</em&gt; En los cortes histológicos de bagre de canal<em&gt; Ictalurus punctatus</em&gt; Mediante inmunohistoquímica indirecta con anticuerpos monoclonales ED9 como principal, y los anticuerpos fluorescentes FITC etiquetados como secundario. Esto permitió la detección de la bacteria mediante microscopía de fluorescencia.

El uso de fluorescentes inmunoquímica para la detección de Edwardsiella ictaluri En el bagre de canal ( I. punctatus) Muestras

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to ...

use-fluorescent-immuno-chemistry-for-detection-edwardsiella-ictaluri

Research

JoVE Journal

Immunology and Infection

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples

10.4K vistas.  Mississippi State University. A continuación se describe un procedimiento que permita el etiquetado de los Edwardsiella ictaluri in situ En los cortes histológicos de bagre de canal Ictalurus punctatus Mediante inmunohistoquímica indirecta con anticuerpos monoclonales ED9 como principal, y los anticuerpos fluorescentes FITC etiquetados como secundario. Esto permitió la detección de la bacteria mediante microscopía de fluorescencia.

Video: Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinación de adhesivo basado en cinta de muestreo y de fluorescencia In situ La hibridación para la detección rápida de Salmonella En los productos frescos

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Investigación

Inmunología e Infección

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Multiplex detección de bacterias en el complejo de muestras clínicas y ambientales utilizando oligonucleótidos de acoplamiento de microesferas fluorescentes

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

La detección de nanopartículas fluorescentes Interacciones con inmunodeficiencia primaria subpoblaciones celulares por citometría de flujo

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

El uso de fluorescentes Arrays objetivo para la evaluación de las respuestas de células T<em&gt; En vivo</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Método del agitador magnético para la detección de<em&gt; Trichinella</em&gt; Las larvas en muestras de músculo

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Extracción rápido, seguro y sencillo Manual cabecera de ácido nucleico para la detección del Virus en muestras de sangre entera

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Una plataforma de alto rendimiento para la detección de Salmonella spp. /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

La coloración de Immunohistochemical fluorescente multiplexado, por imágenes y análisis en muestras histológicas de linfoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

VirWaTest, un método de punto de uso para la detección de virus en muestras de agua

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.