刘健的赠款部分支持是从自然科学和工程研究理事会（NSERC），加拿大，哈利Botterell基金会神经科学的研究，弧，和加菲猫凯利心血管病研究和发展基金。 SRA是一个皇后大学研究奖和罗伯特J.威尔逊奖学金获得者。 PJ是女王的研究生奖和罗伯特J.威尔逊奖学金收件人。 AG和SA接受女王的研究生奖。 SF是科技（OGSST）安大略研究生奖学金的收件人。公司是皇后富兰克林蕨菜研究生奖学金的收件人。 SA是女王的RS麦克劳克林研究生奖学金获得者。

1。复活冷冻的巨噬细胞<ol><li>在复活冷冻细胞在-80 ° C冰箱，需要准备培养基使用无菌过滤贝科改良Eagle培养基（DMEM），辅以10％小牛血清（FBS）和5微克/毫升的plasmocin。 Plasmocin是一种抗生素的解决方案，在我们的研究，以消除和防止细胞培养的支原体污染。 </li><li>一小瓶冷冻细胞，添加4毫升，1毫升培养液，在无菌生物危害防护罩一个25厘米的2细胞培养瓶中。 </li><li>当从-80 ° C冰箱冷?...

<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>

使用新开发的检测，检测现场的巨噬细胞的唾液酸酶的活性，我们用这种技术在现场检测唾液酸酶的活性，配体诱导的唾液酸酶的活性BMC - 2呈剂量依赖性，以及在现场的巨噬细胞的DC - 2.4树突状细胞HEK-TLR4/MD2，HEK293，SP1的乳腺腺癌细胞，人类的WT和1140F01和WG0544型我sialidosis成纤维细胞。抑制神经氨酸酶抑制剂，如达菲（奥司他韦磷酸盐）在现场thymoquinone诱导唾液酸酶的活性的IC 50的0.0194μMBM...

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<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco, Rockville, MD</td>
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<td>4-MUNANA</td>
<td>&nbsp;</td>
<td>Biosynth International Inc., Itasca, IL, USA</td>
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<td>Fetal calf serum</td>
<td>&nbsp;</td>
<td>HyClone, Logan, Utah, USA</td>
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<td>DakoCytomation, Fluorescent Mounting Media</td>
<td>&nbsp;</td>
<td>DAKO North America, Inc., Carpintenia, CA</td>
<td>S3023</td>
<td>15 mL</td>
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<td>Tamiflu (Oseltamivir Phosphate)</td>
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detection of  neu1 sialidase activity in regulating toll-like receptor activation

哺乳动物Toll样受体（TLRs）是一种受体识别病原体相关分子模式的家庭。不仅是TLRs的关键传感器，微生物（如病毒，细菌和寄生虫）的感染，他们也发挥了重要作用，在传染病，炎症性疾病，并可能在自身免疫性疾病的病理生理。因此，对传染病的TLR反应的强度和持续时间必须严格控制。因此，了解传感器的受体，其配体的相互作用和信号元件结构的完整性是至关重要的，为后续的免疫保护。它也将提供重要的机遇，通过传感器操纵的疾病修饰。 TLR的传感器的信号通路虽然是很好的特点，控制参数仍然不明确的传感器及其配体之间的相互作用。最近，我们确定其天然配体，它过去一直没有被观察到1,2的一个新的机制激活的TLR 。这表明，配体诱导的TLR激活紧紧Neu1唾液酸酶的激活控制。我们还报道，Neu1紧紧调节神经营养因子受体TrkA的和TrkB 3，涉及Neu1和基质金属蛋白酶9（MMP - 9）与受体4复杂的串扰。唾液酸酶检测已初步找到Neu4唾液酸酶的激活的巨噬细胞，树突状细胞和成纤维细胞通过G蛋白偶联受体Gαi蛋白和MMP - 9 5的细胞表面上一种新的配体，thymoquinone，。的TLR受体，我们的数据表明，已经在复杂的Neu1唾液酸与TLR - 2，-3和-4受体，无论是受体的配体结合后引起的。激活Neu1唾液酸酶水解唾液酸α- 2 ,3 -联β-半乳糖残基配体遥远约束力删除TLR - 4的二聚体，MyD88/TLR4复杂的招聘，NFKB的激活和促炎症细胞反应的空间位阻。在一个协作的报告中，已被证明Neu1唾液酸酶，调节巨噬细胞的6细胞的吞噬功能。两者合计，唾液酸酶检测配体诱导的受体激活的分子机制提供了强大的见解。虽然Neu1唾液酸酶和激活的TLR之间的确切关系，蒂尔克受体尚未被完全阐明，这将意味着一个新的或创新性解决方案，细胞调控途径。

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

唾液酸酶检测是一个简单的技术方法，将澄清新颖的TLR传感器，微生物感染和参与活巨噬细胞的细胞表面受体水平的炎症性疾病的分子机制（S）。

在调节Toll样受体激活Neu1唾液酸酶活动的检测

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

Research

JoVE Journal

Immunology and Infection

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

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

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

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

科研

免疫与感染

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

定量成像小样本人体血液中单核细胞和树突状细胞系特定的Toll样受体介导的信号

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

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

唾液，唾液腺，淋巴收集肩突硬蜱蜱

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

定量检测抗体诱导补体激活的方法对红血细胞

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

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1ÃƒÅ½Ã‚Â² in Human Monocyte-derived Dendritic Cells

激活和NLRP3炎性活性的人单核细胞来源的树突状细胞用IL-1β测量

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

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

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

Isolation and Activation of Murine Lymphocytes

分离及小鼠淋巴细胞的激活

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

人全血中NK细胞杀伤活性的流式细胞仪分析

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

小鼠骨髓源性巨噬细胞 Inflammasome 活化和 Pyroptotic 细胞死亡的检测

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

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

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

沙门氏菌筛选的高通量平台.

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