Prise en charge partielle par des subventions à MRS est de sciences naturelles et en génie du Canada (CRSNG), le Harry Botterell Fondation pour la recherche en neurosciences, l&#39;ARC, et la recherche cardiovasculaire et Kelly Garfield Fonds de développement. SRA est récipiendaire du Prix de la Reine de la recherche universitaire et la Robert J. Wilson Fellow. PJ est un récipiendaire du Prix d&#39;études supérieures de la Reine et le Robert J. Wilson Fellow. AG et SA sont récipiendaires du Prix d&#39;études supérieures de la Reine. SF a été le récipiendaire de la bourse d&#39;études supérieures en science et technologie (BÉSOST). AG est le récipiendaire de la bourse de la Reine Franklin Bracken supérieures. SA est le récipiendaire de la bourse de la Reine RS McLaughlin supérieures.

1. Ressusciter les cellules macrophages congelés <ol><li> Avant de ressusciter les cellules congelées du congélateur à -80 ° C, on a besoin pour préparer un milieu de culture stérile de l&#39;aide moyenne filtrée Eagle modifié par Dulbecco (DMEM) supplémenté avec 10% sérum de veau fœtal (FBS) et 5 ug / ml de plasmocin. Plasmocin est une solution antibiotique utilisé dans nos recherches pour éliminer et prévenir la contamination des mycoplasmes des cultures cellulaires. </li><li> Pour un flacon de cellules congelées, on ajoute 4 ml, 1 ml du milieu de culture à un 25 cm 2 flacon de culture cellulaire dans une hotte de confinement stériles Biohazard. </li><li> Lorsque le flacon de cellules congelées sont prises dans le congélateur à -80 ° C, ils sont rapidement décongelés par le réchauffement de la main dans la hotte stérile, ce qui prend environ 2-3 min. </li><li> Dès que les cellules sont décongelées, elles sont rapidement transférés dans le ballon contenant le milieu conditionné aide d&#39;une pipette de 1 ml stérile. </li><li> Le flacon contenant les cellules sont placées dans un incubateur de culture tissulaire à 37 ° C et 5% de CO 2. </li><li> Les cellules vérifié pour la croissance et l&#39;adhérence sur une base quotidienne jusqu&#39;à ce qu&#39;ils deviennent 75% à 90% de confluence en utilisant un microscope inversé. </li><li> Durant l&#39;incubation de la culture cellulaire, 12 mm de verre circulaire diapositives sont stérilisés en les couvrant de 70% d&#39;éthanol dans une boîte de Pétri grand verre pendant 1 heure dans le cabinet confinement des biorisques, l&#39;alcool est ensuite retirée et stockée pour reusage, et les parois de verre mouillée sont laissées exposées au flux d&#39;air stérile filtré dans l&#39;armoire sous une lampe UV pendant 24 heures ou jusqu&#39;à ce qu&#39;elles soient sèches. </li></ol> 2. Cellules Placage pour le dosage sialidase <ol><li> Avant de plaquer les cellules, une seule lame de verre stériles circulaire est soigneusement placé dans un puits d&#39;une BD Falcon 24 ainsi à fond plat avec couvercle culture tissulaire plaque de polystyrène traitées en utilisant un stérilisés flamboyante pleine incurvée, 4 &quot;, dentelé, pince en acier inoxydable . Pour un flacon de cellules à confluence de 75%, on utilise habituellement 12 puits de la plaque de culture. </li><li> Pour les cellules macrophages adhérents, le milieu DMEM conditionné est retiré du ballon à l&#39;aide d&#39;une pipette de 5 ml stériles dans l&#39;armoire de confinement stériles. Laver les cellules adhérentes une fois avec 1x stérile tamponnée Tris pH 7,4 salines de supprimer tout milieu contenant du sérum, et ajouter 1 ml de milieu sans calcium (CMF) tampon phosphate salin à pH 7,4 suffisant pour couvrir les cellules. Les cellules sont placées dans l&#39;incubateur 37 ° C pendant environ 10 min ou jusqu&#39;à ce que les cellules commencent à se décoller dans le ballon. Le flacon peut être bercé ou secoué pour desserrer les cellules adhérentes restantes. </li><li> Pour l&#39;mL 1 des cellules en suspension dans le flacon, ajouter 3 mL de milieu DMEM conditionné. Prenez 0,5 ml de cellules en suspension et stérilement les ont transférés à la plaque de culture de tissus contenant les 12 diapositives mm de verre circulaire. Environ, 0,5 x 10 6 cellules sont transférées dans les lames de verre circulaires. </li><li> Les cellules de culture de tissu contenant la plaque sont incubées à 37 ° C dans 5% de CO 2 pendant 24 heures afin de permettre aux cellules d&#39;adhérer à la lame de verre circulaire. </li></ol> 3. Assay sialidase 1. Rendre le contrôle <ol><li> Placer une lame de microscope (dépoli: 1 fin, 1slide, 25 x 75 x 1 mm) dans l&#39;armoire de confinement ou de paillasse et ajouter en mélangeant dans l&#39;ordre séquentiel les composés suivants: 1 ul DAKO + 1 uL de 4-Muñana et enfin + 3 ul de Tris pH tamponné salin 7.4 (SCT). </li><li> Après avoir enlevé les médias d&#39;un puits contenant les cellules, soulevez doucement la lame de verre circulaire, avec les 4 &quot;pince à l&#39;angle du bien. Retirez délicatement la lame de verre avec la pince et placez la lame avec les cellules qui donnent sur la solution du mélange sur la la lame de microscope. </li><li> Immédiatement, prenez la diapositive microcope à un épi-microscopie fluorescente qui a été fixé pour le filtre UV et dispose d&#39;un appareil photo numérique pour capturer les images fluorescentes. </li><li> Focus du microscope à contraste de phase sous jusqu&#39;à ce que vous pouvez voir les cellules, puis passer en mode fluorescent. Prenez des photos de cellules fluorescentes sous à 1 min, 2 min et 3 min. Prenez une photo unique de cellules en contraste de phase. </li></ol> 2. Faire le test positif <ol><li> Découvrir les composés suivants dans l&#39;ordre séquentiel sur une autre lame de microscope: 1 ul DAKO + 1 uL de 4-Muñana + 2 ul de LPS + 1 uL de TBS. </li><li> Répéter les étapes 3.1.2 à 3.1.4 ci-dessus. </li></ol> 3. Faire le test positif avec inhibiteur de la neuraminidase Tamiflu <ol><li> Découvrir les composés suivants dans l&#39;ordre séquentiel suivant sur une autre lame de microscope: 1 microlitre DAKO + 1 microlitre de 4-Muñana + 2 ul de LPS + 1 uL de Tamiflu. </li><li> Les concentrations de Tamiflu sont pré-établi en 1x tampon Tris pH 7,4 salines; la concentration finale des composés en contact avec les cellules vivantes sur la lame de verre circulaire aura un facteur de dilution de 5. </li><li> Répéter les étapes 3.1.2 à 3.1.4 ci-dessus. </li></ol> 4. Détermination de la concentration d&#39;inhibiteur nécessaire pour inhiber 50% de l&#39;activité sialidase (IC 50) <ol><li> Afin de quantifier les fluorescents entourant les cellules, les images sont analysées à l&#39;aide ImageJ 1.38x avec le plugin d&#39;analyser pour mesurer RBG. Prenez 50 lectures aléatoires de la fluorescence entourant les cellules et calcule la moyenne de la fluorescence totale. </li><li> La CI 50 est générée à partir d&#39;un tracé de la concentration de l&#39;inhibiteur convertis en unités log (par exemple, log ng / ml) sur l&#39;axe des x contre la moyenne de fluorescence sur l&#39;axe Y en utilisant la régression non linéaire. </li></ol> 5. Les secrets du succès <ol><li> Assurez-vous que les cellules en culture sont sans contamination par des mycoplasmes. Nous utilisons régulièrement Plasmocin dans un milieu de culture pour contrôler cela. </li><li> Le substrat artificiel de la neuraminidase, 4-Muñana, devrait être utilisé fraîchement préparés. Le substrat préparé peut être utilisé dans une semaine pour le dosage sialidase. </li><li> Si la lignée cellulaire a une faible expression des récepteurs à la surface cellulaire, on peut utiliser une dose plus élevée du ligand pour la stimulation. </li><li> Nous avons également constaté que les cellules repiquées depuis plus de 6 fois risquent de perdre leur phénotype du récepteur. Fraîchement, les cellules doivent être cultivées ressuscités. </li></ol> 6. Les résultats représentatifs Voir le protocole d&#39;animation de l&#39;essai avec des résultats représentatifs sialidase joint dans le <a href="http://www.jove.com/files/ftp_upload/2142/szewczuk_JoVE_sialidase_assay.pptx">fichier 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 et PJ ont contribué également que les premiers auteurs.

En utilisant le dosage nouvellement développée pour détecter l&#39;activité sialidase dans les cellules macrophages vivants 2, nous avons utilisé cette technologie pour détecter l&#39;activité de l&#39;activité sialidase sialidase induite par le ligand en direct BMC-2 cellules macrophages de manière dose-dépendante aussi bien en direct DC-2.4 des cellules dendritiques , HEK-TLR4/MD2, HEK293, les cellules SP1 adénocarcinomes mammaires, WT humaine et 1140F01 et WG0544 de type I des cellules de fibrob...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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detection of  neu1 sialidase activity in regulating toll-like receptor activation

Mammifères récepteurs Toll-like (TLR) sont une famille de récepteurs qui reconnaissent pathogène motifs moléculaires associés. Non seulement les TLR capteurs crucial de microbes (par exemple, virus, bactéries et parasites) des infections, mais elles jouent aussi un rôle important dans la physiopathologie des maladies infectieuses, les maladies inflammatoires et, éventuellement, dans les maladies auto-immunes. Ainsi, l&#39;intensité et la durée des réponses TLR contre les maladies infectieuses doit être étroitement contrôlée. Il s&#39;ensuit que la compréhension de l&#39;intégrité structurale des récepteurs de capteurs, de leurs interactions ligand et éléments de signalisation est essentielle pour la protection immunologique ultérieure. Elle permettrait également d&#39;importantes opportunités de modification de la maladie grâce à la manipulation du capteur. Bien que les voies de signalisation des TLR capteurs sont bien caractérisés, les paramètres contrôlant les interactions entre les capteurs et leurs ligands restent encore mal définies. Nous avons récemment identifié un nouveau mécanisme d&#39;activation TLR par son ligand naturel, qui n&#39;a pas été observé précédemment 1,2. Il suggère que l&#39;activation induite par des ligands de TLR est étroitement contrôlée par l&#39;activation Neu1 sialidase. Nous avons également signalé que Neu1 réglemente rigoureusement les récepteurs TrkA et neurotrophine comme TrkB 3, qui impliquent Neu1 et de la matrice métalloprotéinase-9 (MMP-9) diaphonie dans un complexe avec les récepteurs 4. Le test a été initialement sialidase utiliser pour trouver un ligand roman, thymoquinone, dans l&#39;activation de Neu4 sialidase à la surface cellulaire de macrophages, cellules dendritiques et des cellules de fibroblastes par l&#39;intermédiaire des protéines GPCR Gαi et MMP-9 5. Pour les récepteurs TLR, nos données indiquent que Neu1 sialidase est déjà dans un complexe avec les récepteurs TLR-2, -3 et -4, et est induite lors de la liaison du ligand du récepteur soit. Activé Neu1 s&#39;hydrolyse sialidase sialyl α-2 ,3-β-galactosyl liés résidus éloignés de liaison du ligand pour enlever encombrement stérique au TLR-4 dimérisation, MyD88/TLR4 complexes de recrutement, l&#39;activation de NFkB et les réponses des cellules pro-inflammatoires. Dans un rapport de collaboration, sialidase Neu1 a été montré pour réglementer la phagocytose en 6 cellules macrophages. Pris ensemble, le dosage sialidase nous a fourni des idées puissantes pour les mécanismes moléculaires de l&#39;activation du récepteur induite par le ligand. Bien que la relation précise entre Neu1 sialidase et l&#39;activation des TLR, récepteurs Trk n&#39;a pas encore été totalement élucidé, cela représenterait une nouvelle approche ou pionnière de voies de régulation cellulaire.

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

Le dosage de la sialidase est une approche simple technique qui élucidera roman mécanisme moléculaire (s) de capteurs TLR des infections microbiennes et leur implication dans les maladies inflammatoires au niveau des récepteurs à la surface cellulaire de macrophages vivre.

Détection de Neu1 activité sialidase dans la régulation activation du récepteur Toll-like

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 vues.  Queen's University - Kingston, Ontario. Le dosage de la sialidase est une approche simple technique qui élucidera roman mécanisme moléculaire (s) de capteurs TLR des infections microbiennes et leur implication dans les maladies inflammatoires au niveau des récepteurs à la surface cellulaire de macrophages vivre.

Vidéo: 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).

Combinaison de adhésif à base de bandes d&#39;échantillonnage et de fluorescence In situ</emHybridation> pour la détection rapide des Salmonella Sur les produits frais

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

Recherche

Immunologie et infection

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 d&#39;activation des basophiles pour l&#39;enquête des IgE dans les mécanismes d&#39;hypersensibilité médicamenteuse

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.

Imagerie quantitative de Lineage spécifiques Toll-like Receptor la signalisation médiée par les monocytes et les cellules dendritiques à partir de petits échantillons de sang humain

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 salive, les glandes salivaires, et Collection hémolymphe de tiques 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.

Les méthodes de détection quantitative d&#39;anticorps induite par l&#39;activation du complément sur les globules rouges

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.

Activation et évaluation de NLRP3 Inflammasome activité de l&#39;IL-1β Utilisation dans des cellules dendritiques humaines dérivées de monocytes

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.

Isolement d&#39;anticorps murins péritonéale macrophages pour effectuer Analyse expression génique Sur Toll-like récepteurs Stimulation

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.

Isolement et activation de murins Lymphocytes

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.

Cytométrie en flux Analyse des cellules tueuses naturelles lytique Activité dans le sang humain

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.