El trabajo fue apoyado por el NIH R01-01A1 NS071039 beca de investigación.

<ol>
	<li>Tremblay, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Microglia+in+the+Healthy+Brain.">The Role of Microglia in the Healthy Brain.</a> J. Neurosci. 31, 16064-16069 (2011).</li><li>Graeber, M. B., Li, W., Rodriguez, 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=Role+of+microglia+in+CNS+inflammation.">Role of microglia in CNS inflammation.</a> FEBS Lett. , (2011).</li><li>Ponomarev, E. D., Shriver, L. P., Maresz, K., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cell+activation+and+proliferation+precedes+the+onset+of+CNS+autoimmunity.">Microglial cell activation and proliferation precedes the onset of CNS autoimmunity.</a> J. Neurosci. Res. 81, 374-389 (2005).</li><li>Perruisseau-Carrier, C., Jurga, M., Forraz, N., McGuckin, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNAs+stem+cell+reprogramming+for+neuronal+induction+and+differentiation.">miRNAs stem cell reprogramming for neuronal induction and differentiation.</a> Mol. Neurobiol. 43, 215-227 (2011).</li><li>McCoy, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+miRNAs+in+cytokine+signaling.">The role of miRNAs in cytokine signaling.</a> Front Biosci. 17, 2161-2171 (2010).</li><li>He, M., Xu, Z., Ding, T., Kuang, D. M., Zheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-155+regulates+inflammatory+cytokine+production+in+tumor-associated+macrophages+via+targeting+C/EBPbeta.">MicroRNA-155 regulates inflammatory cytokine production in tumor-associated macrophages via targeting C/EBPbeta.</a> Cell Mol. Immunol. 6, 343-352 (2009).</li><li>Ponomarev, E. D., Veremeyko, T., Barteneva, N., Krichevsky, A. M., Weiner, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-124+promotes+microglia+quiescence+and+suppresses+EAE+by+deactivating+macrophages+via+the+C/EBP-alpha-PU.1+pathway.">MicroRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP-alpha-PU.1 pathway.</a> Nat. Med. 17, 64-70 (2011).</li><li>Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Specific+Cell+Population+by+Fluorescence+Activated+Cell+Sorting+(FACS).">Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS).</a> J. Vis. Exp. (41), e1546-3791 (2010).</li><li>Gabriely, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+21+promotes+glioma+invasion+by+targeting+matrix+metalloproteinase+regulators.">MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators.</a> Mol. Cell Biol. 28, 5369-5380 (2008).</li><li>Caffarelli, E., Filetici, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+in+cancer+development.">Epigenetic regulation in cancer development.</a> Front Biosci. 17, 2682-2694 (2011).</li><li>Cardona, A. E., Huang, D., Sasse, M. E., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+microglial+cells+for+RNA+analysis+or+flow+cytometry.">Isolation of murine microglial cells for RNA analysis or flow cytometry.</a> Nat. Protoc. 1, 1947-1951 (2006).</li><li>Gordon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+magnetic+separation+method+for+high-yield+isolation+of+pure+primary+microglia.">A simple magnetic separation method for high-yield isolation of pure primary microglia.</a> J. Neurosci. Methods. 194, 287-296 (2011).</li><li>Moltzahn, F., Hunkapiller, N., Mir, A. A., Imbar, T., Blelloch, R. <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+MicroRNA+Profiling:+Optimized+Multiplex+qRT-PCR+at+Nanoliter+Scale+on+the+Fluidigm+Dynamic+ArrayTM+IFCs.">High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs.</a> J. Vis. Exp. (54), e2552 (2011).</li></ol>

No hay conflictos de interés declarado.

Recientemente un gran interés fue dado a la microglía y la participación de estas células en muchas patologías asociadas a la neuroinflamación y la neurodegeneración. Además, el papel de los microRNAs en la diferenciación de las células del sistema inmune y el cáncer ha crecido dramáticamente durante los últimos varios años 5,10. Hemos informado anteriormente el papel de miR-124 en el mantenimiento del fenotipo no activado o en reposo de la microglía en el SNC normal 7, pero el papel...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> 1X PBS </td><td> GIBCO </td><td> 14190 </td><td> Puede ser preparado a partir de componentes secos en el laboratorio, pH: 7.2-7.4 </td></tr><tr><td> PBS 10 veces </td><td> Thermo Scientific </td><td> SH30258.02 </td><td> Puede ser preparado a partir de componentes secos en el laboratorio, pH: 7.2-7.4 </td></tr><tr><td> DMEM; FBS </td><td> GBCO </td><td> 11965, 10438 </td><td></td></tr><tr><td> Mirvana kit </td><td> Invitrogen </td><td> AM1561 </td><td></td></tr><tr><td> Percoll </td><td> Sigma </td><td> P4937-500 ml </td><td> 100 ml de 100% de Percoll se prepara mezclando 10 ml de 10X PBS y 90 ml de Percoll de Sigma. 50 ml de 70% de Percoll espreparado por mezcla de 35 ml de 100% de Percoll y 15 ml de DMEM 50 ml de 40% de Percoll se prepara mezclando 20 ml de 100% de Percoll y 30 ml de 1X PBS </td></tr><tr><td> MOG 35-55 péptido </td><td> AnaSpec Inc </td><td> 60130-5 </td><td></td></tr><tr><td> Comité de Libertad Sindical </td><td> DIFCO </td><td> 263810 </td><td></td></tr><tr><td> Toxina pertussis </td><td> Sigma </td><td> P7208 </td><td></td></tr><tr><td> FcR anticuerpos bloqueantes </td><td> BD Biosciences </td><td> 553142 </td><td> Clon 2.4G2 </td></tr><tr><td> Anti-CD11b - PE MAB </td><td> BD Biosciences </td><td> 553311 </td><td> Puede ser utilizado con diferentes fluoróforos (por ejemplo, AF488) </td></tr><tr><td> Anti-CD45-APC-Cy7 mAbs </td><td> BioLegend </td><td> 103116 </td><td> Puede ser utilizado con diferentes fluoróforos (por ejemplo APC) </td></tr><tr><td> Paraformalformaldehído (16%) </td><td> EMS Inc. </td><td> 15710 </td><td> Diluir 1:15 en PBS 1X </td></tr><tr><td> 15% de TBE-gel de urea </td><td> Life Technologies Inc. </td><td> EC68855BOX </td><td></td></tr><tr><td> Micro RNA TaqMan transcripción inversa Kit </td><td> Life Technologies Inc </td><td> 4366596 </td><td></td></tr><tr><td> TaqMan Universal PCR Master Mix </td><td> Life Technologies Inc </td><td> 4304437 </td><td></td></tr><tr><td> Los ensayos TaqMan mmu microARN miR-124a- </td><td> Life Technologies Inc </td><td> 4427975 ID 001182 </td><td></td></tr><tr><td> Los ensayos TaqMan microARN snoRNA-55 </td><td> Life Technologies Inc </td><td> 4427975 ID 001228 </td><td></td></tr><tr><td> Agua libre de nucleasa </td><td> Life Technologies Inc </td><td> AM9937 </td><td> Agua libre de nucleasa </td></tr><tr><td> 384 y placa de reacción clara óptica </td&gt; <td> Life Technologies Inc. </td><td> 4309849 </td><td> Puede ser sustituido con otras placas (por ejemplo, placa de 96 pocillos) en diferentes instrumentos en tiempo real de PCR </td></tr><tr><td> Homogeneizador Dounce (15 ml) </td><td> Wheaton Science Products. </td><td> 358044 </td><td> Teflón / vidrio </td></tr><tr><td> 40 μ de células colador de nylon </td><td> Halcón </td><td> 352340 </td><td></td></tr><tr><td> Centrífuga grande </td><td> Sorval </td><td> RT 6000B </td><td> Diámetro del rotor 18,5 cm </td></tr><tr><td> Centrífuga pequeña </td><td> Eppendorf </td><td> 5415 R </td><td> Diámetro del rotor 6,5 cm </td></tr><tr><td> PCR en tiempo real del instrumento </td><td> Life Technologies Inc. </td><td> AB 7900 HT </td><td> Puede ser sustituido con otros instrumentos </td></tr><tr><td> Citómetro de flujo </td><td> BD Biosciencess </td><td> LSR II </td><td> Puede ser sustituido con otros instrumentos </td&gt; </tr><tr><td> FACS </td><td> BD Biosciences </td><td> FACSAria </td><td> Puede ser sustituido con otros instrumentos </td></tr><tr><td> Espectrofotómetro Nanodrop </td><td> Thermo Scientific </td><td> Nanodrop 1000 </td><td></td></tr></tbody></table>

detection of micrornas in microglia by real-time pcr in normal cns and during neuroinflammation

Microglia son las células del linaje mieloide que residen en el sistema nervioso central (SNC) 1. Estas células juegan un papel importante en patologías de muchas enfermedades asociadas con neuroinflamación tales como la esclerosis múltiple (MS) 2. Microglia en un SNC normales expresan los macrófagos CD11b marcador y exhiben un fenotipo de descanso por los bajos niveles de expresión de marcadores de activación como CD45. Durante los eventos patológicos en el SNC, la microglia se activan según lo determinado por la regulación positiva de CD45 y otros marcadores 3. Los factores que afectan el fenotipo microglia y funciones en el SNC no se ha estudiado bien. Los microARN (miRNA) son una familia creciente de moléculas conservadas (~ 22 nucleótidos de largo) que están involucradas en muchos procesos fisiológicos normales, como el crecimiento y la diferenciación celular 4 y patologías como la inflamación 5. MiRNAs downregulate la expresión de ciertos genes diana por unión secuencias complementarias de laARNm de ir a jugar un papel importante en la activación de las células inmunes, incluyendo los macrófagos y microglia 6 7. Con el fin de investigar las mediadas por miRNA-las vías que definen el fenotipo microglial función biológica, y para distinguir la microglía de otros tipos de macrófagos, es importante para evaluar cuantitativamente la expresión de microARN en particular en distintos subconjuntos de SNC-residente microglia. Los métodos comunes para medir la expresión de miRNAs en el SNC incluyen la PCR cuantitativa a partir de tejido neuronal conjunto y la hibridación in situ. Sin embargo, la PCR cuantitativa a partir de homogeneizado todo el tejido no permite la evaluación de la expresión de miRNA en la microglía, que representan sólo el 15.5% de las células del tejido neuronal. La hibridación in situ permite la evaluación de la expresión de microARN en tipos celulares específicos en las secciones de tejido, pero este método no es completamente cuantitativo. En este informe se describe un análisis cuantitativo y sensitive método para la detección de miRNA por PCR en tiempo real en la microglía aislada de SNC normales o durante la neuroinflamación con encefalomielitis autoinmune experimental (EAE), un modelo de ratón para la EM. El método descrito será útil para medir el nivel de expresión de microRNAs en la microglía en el SNC normal o durante la neuroinflamación asociada a diversas patologías como esclerosis múltiple, derrame cerebral, lesión traumática, la enfermedad de Alzheimer y tumores cerebrales.

Watch this Scientific Journal Video about Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation at JoVE.com

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

La microglía se macrófagos residentes que proporcionan la primera línea de defensa y de vigilancia inmune del sistema nervioso central. Los microARN son moléculas reguladoras que juegan un papel importante en muchos procesos fisiológicos, incluida la activación y diferenciación de los macrófagos. En este artículo se describe el método para la medición de los microRNAs en la microglía.

La detección de microARNs en la microglía por PCR en tiempo real en el SNC normal y durante la neuroinflamación

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

detection-micrornas-microglia-real-time-pcr-normal-cns-during

Research

JoVE Journal

Immunology and Infection

19.3K vistas.  Harvard Medical School. La microglía se macrófagos residentes que proporcionan la primera línea de defensa y de vigilancia inmune del sistema nervioso central. Los microARN son moléculas reguladoras que juegan un papel importante en muchos procesos fisiológicos, incluida la activación y diferenciación de los macrófagos. En este artículo se describe el método para la medición de los microRNAs en la microglía.

Video: Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Imágenes en tiempo real de heterotypic plaquetas neutrófilos Interacciones en el endotelio activado durante la inflamación vascular y la formación de trombos en ratones vivos

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Investigación

Inmunología e Infección

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia cultivo del Sistema Nervioso Central Neonatal y Adultos

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

Imágenes en tiempo real de la célula-célula cruces endoteliales Durante neutrófilos Transmigración Bajo Flujo Fisiológica

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Imágenes en tiempo real de las células mieloides en Dinámica<em&gt; Apc<sup&gt; Min / +</sup</em&gt; Intestinal Tumores de Spinning disco microscopía confocal

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

La cuantificación simultánea de T-Cell Círculos Receptor Excision (TRECs) y Círculos K-Eliminación recombinación Excision (KRECs) por PCR en tiempo real

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

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 ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imaging neutrófilos y monocitos en mesentérica venas por Microscopia intravital en ratones anestesiados en Tiempo Real

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

genotipado de<em&gt; Staphylococcus aureus</em&gt; por Ribosómica espaciador de PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

La detección de alto rendimiento de los patógenos respiratorios de especímenes animales por PCR nanoescala

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

En tiempo real temblando inducida por conversión de ensayo para la detección de priones CWD en materia Fecal

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...