Apoyo parcial de subvenciones para MRS de Ciencias Naturales e Ingeniería de Investigación de Canadá (NSERC), el Harry Botterell Fundación para Investigación de Neurociencia, ARC, y el de Investigación Cardiovascular Garfield Kelly y el Fondo para el Desarrollo. SRA es un recipiente del Premio de la Reina de Investigación de la Universidad y el Robert J. Wilson Fellowship. PJ es un recipiente de la concesión de Postgrado de la Reina y el Robert J. Wilson Fellowship. AG y SA son beneficiarios de la concesión de Postgrado de la Reina. SF fue el beneficiario de la Beca de Ontario de Posgrado en Ciencia y Tecnología (OGSST). AG es el receptor de Franklin Becas de la Reina Bracken de Posgrado. SA es el receptor de RS de Becas de la Reina McLaughlin Posgrado.

1. La resurrección de las células congeladas del macrófago <ol><li> Antes de la resurrección de las células congeladas del congelador -80 ° C, hay que preparar el medio de cultivo utilizando filtrado estéril Dulbecco modificado de Eagle (DMEM) suplementado con fetal al 10% de suero bovino (FBS) y 5 mg / ml de plasmocin. Plasmocin es una solución de antibiótico que se utiliza en nuestra investigación para eliminar y prevenir la contaminación por micoplasmas en cultivos celulares. </li><li> Para un vial de células congeladas, se añade 4 ml, 1 ml de medio de cultivo a 25 cm 2 frasco de cultivo celular en una campana de contención de riesgos biológicos estériles. </li><li> Cuando el vial de células congeladas se han tomado de los -80 ° C congelador, se descongelan rápidamente calentando la mano en la campana estéril, que tarda unos 2-3 minutos. </li><li> Tan pronto como las células se descongelan, que se transfieren rápidamente en el frasco que contiene el medio condicionado con una pipeta estéril de 1 ml. </li><li> El frasco que contiene las células se colocan en un cultivo de tejidos incubadora a 37 ° C y 5% CO 2. </li><li> Las células comprobado para el crecimiento y la adhesión a diario hasta que se conviertan en 75% al ​​90% confluente utilizando un microscopio invertido. </li><li> Durante la incubación de cultivo celular, 12 diapositivas mm circular de cristal se esterilizan por cubrir en un 70% de etanol en un vaso grande placa de Petri durante 1 hora en el gabinete de contención de riesgos biológicos, el alcohol es posteriormente retirado y almacenado para la reutilización, y los lados de vidrio mojado quedan expuestos al flujo de aire estéril, filtrada en el armario debajo de un UV durante 24 horas o hasta que se sequen. </li></ol> 2. Las células Revestimiento para el ensayo de sialidasa <ol><li> De las placas, las células, una sola diapositiva de vidrio estériles circular se colocan cuidadosamente en un pocillo de una BD Falcon de 24 pocillos de fondo plano con tapa de la placa de cultivo de tejidos tratados de poliestireno con una curva completa esterilizada de fuego, 4 &quot;, serrado, pinza de acero inoxidable . Para un frasco de células en un 75% de confluencia, por lo general utilizan 12 pozos de la placa de cultivo. </li><li> Para los macrófagos adherentes, el medio DMEM acondicionado se retira del frasco utilizando una pipeta de 5 ml estéril en el gabinete de contención estéril. Lavar las células adheridas, una vez con 1x estéril tamponada de Tris pH 7,4 salina para eliminar cualquier tipo de soporte de suero que contiene, y agregar 1 ml de medio libre de calcio (CMF) tampón fosfato salino a pH 7,4 para cubrir las células. Las células se colocan en la incubadora a 37 ° C durante unos 10 minutos o hasta que las células comienzan a despegar en el frasco. El matraz se mecía suavemente o sacudida para aflojar las células adherentes restantes. </li><li> A la de 1 ml de células en suspensión en el matraz, añadir 3 ml de medio DMEM acondicionado. Tomar 0,5 ml de las células en suspensión y estéril que transfiere a la placa de cultivo de tejidos que contienen los 12 mm diapositivas circular de vidrio. Aproximadamente, 0,5 x 10 6 células se transfieren a las diapositivas de cristal circular. </li><li> Las células de cultivo de tejidos placa que contiene se incuban a 37 ° C en 5% CO 2 durante 24 horas a fin de permitir que las células se adhieran a los portaobjetos de vidrio circular. </li></ol> 3. Sialidasa ensayo 1. Haciendo que el control <ol><li> Colocar un portaobjetos de microscopio (helado: 1 final, 1Deslice, 25 x 75 x 1 mm) en el gabinete de contención o de mesa de laboratorio y añadir, mezclando en el orden secuencial de los siguientes compuestos: 1 l Dako + 1 L de 4-Muñana y, por último, + 3 l de solución salina tamponada de Tris pH 7,4 (TBS). </li><li> Después de la eliminación de los medios de comunicación de un pozo que contiene las células, levante suavemente el portaobjetos de vidrio circular con 4 &quot;pinza a la esquina del pozo. Retire con cuidado el portaobjetos de vidrio con la pinza y colocar la placa con las células que dan a la solución de la mezcla de el portaobjetos del microscopio. </li><li> Inmediatamente, toma la diapositiva microcope a un microscopio epi-fluorescente que se ha fijado para el filtro de los rayos UV y tiene una cámara digital para capturar las imágenes fluorescentes. </li><li> Enfocar el microscopio de contraste de fase bajo hasta que pueda ver las células y luego cambiar al modo de fluorescentes. Tome fotos de las células bajo lámparas fluorescentes en 1 min, 2 y 3 minutos. Tomar una sola foto de las células bajo contraste de fase. </li></ol> 2. Hacer la prueba positiva <ol><li> Lugar los siguientes compuestos en el orden secuencial en otro portaobjetos: 1 l Dako + 1 L de 4-Muñana + 2 l de LPS + 1 l de TBS. </li><li> Repita los pasos 3.1.2 a 3.1.4. </li></ol> 3. Hacer la prueba positiva, junto con inhibidores de la neuraminidasa Tamiflu <ol><li> Lugar de los compuestos siguientes en el orden siguiente secuencia en otro portaobjetos: 1μL Dako + 1μL de 4-Muñana + 2 l de LPS + 1 l de Tamiflu. </li><li> Las concentraciones de Tamiflu son pre-preparados en 1x Tris buffer de pH 7,4 salina, la concentración final de los compuestos en contacto con las células vivas en el portaobjetos de vidrio circular tendrá un factor de dilución de 5. </li><li> Repita los pasos 3.1.2 a 3.1.4. </li></ol> 4. Determinación de la concentración del inhibidor necesaria para inhibir el 50% de la actividad sialidasa (IC 50) <ol><li> Para cuantificar el fluorescente que rodea las células, las imágenes se analizaron con ImageJ 1.38x con el plugin de análisis para medir la RBG. Tomar 50 lecturas de azar de la fluorescencia que rodea las células y se calcula la media de la fluorescencia total. </li><li> El IC 50 se genera a partir de una parcela de la concentración del inhibidor convertido como unidades de registro (por ejemplo, registro ng / mL) en el eje X en contra de la media de fluorescencia en el eje y el uso de regresión no lineal. </li></ol> 5. Secretos para el Éxito <ol><li> Asegúrese de que las células en cultivo son libres de contaminación por micoplasma. Nosotros usamos Plasmocin en un medio de cultivo para el control de este. </li><li> El sustrato artificial de la neuraminidasa, 4-Muñana, se debe utilizar recién preparada. El sustrato preparado se puede utilizar dentro de una semana para el ensayo de sialidasa. </li><li> Si la línea celular tiene una baja expresión del receptor en la superficie celular, se puede utilizar una dosis más alta del ligando para la estimulación. </li><li> También hemos experimentado que las células de pasajes por más de 6 veces puede perder su fenotipo del receptor. Recientemente, las células deben ser cultivadas resucitado. </li></ol> 6. Resultados representante Véase el protocolo de animación de la prueba de sialidasa con resultados representativos en el documento adjunto <a href="http://www.jove.com/files/ftp_upload/2142/szewczuk_JoVE_sialidase_assay.pptx">archivo Powerpoint</a> .

<ol>
	<li>Amith, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neu1+desialylation+of+sialyl+alpha-2,3-linked+beta-galactosyl+residues+of+TOLL-like+receptor+4+is+essential+for+receptor+activation+and+cellular+signaling.">Neu1 desialylation of sialyl alpha-2,3-linked beta-galactosyl residues of TOLL-like receptor 4 is essential for receptor activation and cellular signaling.</a> Cell Signal. 22, 314-324 (2010).</li><li>Amith, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+pathogen+molecule-induced+Toll-like+receptor+activation+and+cell+function+on+Neu1+sialidase.">Dependence of pathogen molecule-induced Toll-like receptor activation and cell function on Neu1 sialidase.</a> Glycoconjugate Journal. 26, 1197-1212 (2009).</li><li>Woronowicz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+neurotrophic+factor+activation+of+Trk+tyrosine+kinase+receptors+on+cellular+sialidase.">Dependence of neurotrophic factor activation of Trk tyrosine kinase receptors on cellular sialidase.</a> Glycobiology. 17, 10-24 (2007).</li><li>Jayanth, P., Amith, S. R., Gee, K., Szewczuk, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neu1+Sialidase+and+Matrix+Metalloproteinase-9+Cross-talk+is+Essential+for+Neurotrophin+Activation+of+Trk+Receptors+and+Cellular+Signaling.">Neu1 Sialidase and Matrix Metalloproteinase-9 Cross-talk is Essential for Neurotrophin Activation of Trk Receptors and Cellular Signaling.</a> Cell. Signal. 22, 1193-1205 (2010).</li><li>Finlay, T. M., Jayanth, P., Amith, S. R., Glimour, A., Guzzo, C., Gee, K., Beyaert, R., Szewczuk, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymoquinone+from+nutraceutical+black+cumin+oil+activates+Neu4+sialidase+in+live+macrophage,+dendritic,+and+normal+and+type+I+sialidosis+human+fibroblast+cells+via+GPCR+G&#945;i+proteins+and+matrix+metalloproteinase-9.">Thymoquinone from nutraceutical black cumin oil activates Neu4 sialidase in live macrophage, dendritic, and normal and type I sialidosis human fibroblast cells via GPCR G&#945;i proteins and matrix metalloproteinase-9.</a> Glycoconj J. 27, 329-348 (2010).</li><li>Seyrantepe, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Phagocytosis+in+Macrophages+by+Neuraminidase+1.">Regulation of Phagocytosis in Macrophages by Neuraminidase 1.</a> Journal of Biological Chemistry. 285, 206-215 (2010).</li></ol>

SRA y PJ han contribuido por igual como primeros autores.

Utilizando el ensayo de nuevo desarrollo para detectar la actividad sialidasa en los macrófagos en vivo 2, se utilizó esta tecnología para detectar la actividad sialidasa en la actividad sialidasa inducida por ligandos en vivo BMC-2 en células de los macrófagos en forma dosis dependiente, así como en vivo DC-2.4 células dendríticas , HEK-TLR4/MD2, HEK293, SP1 células de adenocarcinoma de mama, WT humanos y 1140F01 y WG0544 células de tipo I de fibroblastos sialidosis. Inhibidores de la neuraminidasa...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco, Rockville, MD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>4-MUNANA</td>
<td>&nbsp;</td>
<td>Biosynth International Inc., Itasca, IL, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fetal calf serum</td>
<td>&nbsp;</td>
<td>HyClone, Logan, Utah, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DakoCytomation, Fluorescent Mounting Media</td>
<td>&nbsp;</td>
<td>DAKO North America, Inc., Carpintenia, CA</td>
<td>S3023</td>
<td>15 mL</td>
</tr>
<tr>
<td>Tamiflu (Oseltamivir Phosphate)</td>
<td>&nbsp;</td>
<td>Hoffman La Roche</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

detection of  neu1 sialidase activity in regulating toll-like receptor activation

Mamíferos receptores Toll-like (TLR) son una familia de receptores que reconocen patógenos patrones moleculares asociados. No sólo son los TLR sensores crucial de microorganismos (por ejemplo, virus, bacterias y parásitos), las infecciones, sino que también desempeñan un papel importante en la fisiopatología de las enfermedades infecciosas, enfermedades inflamatorias y, posiblemente, en las enfermedades autoinmunes. Por lo tanto, la intensidad y la duración de las respuestas TLR contra las enfermedades infecciosas deben ser estrictamente controlados. De ello se desprende que la comprensión de la integridad estructural de los receptores de sensor, sus interacciones ligando y componentes de señalización es esencial para la protección inmunológica posterior. También se ofrecen importantes oportunidades para la modificación de la enfermedad a través de la manipulación del sensor. Aunque las vías de señalización de TLR sensores están bien caracterizados, los parámetros que controlan las interacciones entre los sensores y sus ligandos todavía siguen estando mal definidas. Recientemente hemos identificado un nuevo mecanismo de activación de TLR con su ligando natural, que no ha sido observado previamente 1,2. Se sugiere que la activación inducida por ligandos TLR está estrechamente controlada por la activación de sialidasa Neu1. También se ha informado de que Neu1 bien regula los receptores de neurotrofinas como TrkA y TrkB 3, que implican Neu1 y metaloproteinasas de matriz-9 (MMP-9) cross-talk en el complejo con los receptores 4. El ensayo ha sido inicialmente sialidasa utilizar para encontrar un nuevo ligando timoquinona, en la activación de Neu4 sialidasa en la superficie celular de los macrófagos, células dendríticas y células de fibroblastos a través de proteínas GPCR Gαi y 5 de MMP-9. Para los receptores TLR, nuestros datos indican que Neu1 sialidasa ya está en el complejo con los receptores TLR-2, -3 y -4, y se induce a ligando vinculante a cualquier receptor. Activado Neu1 hidroliza sialidasa sialil α-2 ,3-β-ligado galactosil residuos lejos de unión del ligando a quitar impedimento estérico de TLR-4 dimerización, MyD88/TLR4 contratación complejos, la activación de NFkB y pro-inflamatorias respuestas de las células. En un informe de colaboración, sialidasa Neu1 se ha demostrado que regulan la fagocitosis de macrófagos 6. En conjunto, el ensayo sialidasa nos ha proporcionado ideas de gran alcance de los mecanismos moleculares de la activación del receptor inducida por ligandos. Aunque la relación precisa entre Neu1 sialidasa y la activación de TLR, los receptores Trk aún no se ha aclarado completamente, ya que implicaría un nuevo enfoque o pionero de las vías de regulación celular.

Watch this Scientific Journal Video about Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation at JoVE.com

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

El ensayo sialidasa es un enfoque simple técnica que dilucidar nuevo mecanismo molecular (s) de los sensores de TLR de las infecciones microbianas y la participación en las enfermedades inflamatorias a nivel del receptor en la superficie celular de los macrófagos en vivo.

Detección de actividad sialidasa Neu1 en la regulación de Toll-like receptor de activación

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

detection-neu1-sialidase-activity-regulating-toll-like-receptor

Research

JoVE Journal

Immunology and Infection

11.5K vistas.  Queen's University - Kingston, Ontario. El ensayo sialidasa es un enfoque simple técnica que dilucidar nuevo mecanismo molecular (s) de los sensores de TLR de las infecciones microbianas y la participación en las enfermedades inflamatorias a nivel del receptor en la superficie celular de los macrófagos en vivo.

Video: Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

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

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

Test de activación de basófilos para la investigación de mecanismos IgE mediados de Hipersensibilidad a las Drogas

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Imagen cuantitativa de linaje específico Toll-like receptor-mediada por la señalización en los monocitos y células dendríticas de pequeñas muestras de sangre humana

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

La saliva, la glándula salival, y la Colección hemolinfa de las garrapatas Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Los métodos para la detección cuantitativa de anticuerpos inducida por la activación del complemento en los glóbulos rojos

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&amp;#946; in Human Monocyte-derived Dendritic Cells

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Activación y Medición de NLRP3 inflamasoma Actividad Usando IL-1β en las células dendríticas derivadas de monocitos humanos

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Aislamiento de macrófagos peritoneales murinos para llevar a cabo análisis de expresión génica Al Toll-like Receptores Estimulación

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Aislamiento y activación de los linfocitos murinos

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Análisis de citometría de flujo de las células asesinas naturales lítico Actividad en sangre entera humana

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.