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冰箱冷冻细胞小瓶，他们正在迅速解冻用手变暖，在无菌罩，这大约需要2-3分钟。 </li><li>只要细胞解冻，他们迅速转移到包含的条件培养基的烧瓶中，使用1毫升的无菌移液器。 </li><li>烧瓶中含有细胞被放置在一个组织文化的孵化器在37 ° C和5％的CO 2。 </li><li>细胞增长和坚持每天检查，直到他们成为使用倒置显微镜的75％至90％汇合。 </li><li>在细胞培养孵化，12毫米圆形玻片覆盖70％的乙醇在一个大的玻璃培养皿中的生物危害遏制柜为1小时的菜的消毒酒精后，删除和回用中，与湿玻璃两侧暴露在内阁中的无菌过滤紫外线下的空气流量为24小时或直到他们干。 </li></ol> 2。电镀细胞的唾液酸酶检测<ol><li>电镀前的细胞，单一无菌圆形玻片上精心放置在一个BD猎鹰24孔平底带盖组织培养处理的聚苯板，使用消毒燃烧充分弯曲，4"，呈锯齿状，不锈钢forcep 1瓶细胞75％汇合，我们通常使用12井培养板。 </li><li>对于贴壁巨噬细胞，DMEM条件培养基使用5毫升无菌吸管瓶在无菌遏制柜中删除。洗净的贴壁细胞一旦与1X无菌三缓冲液pH为7.4，以消除任何含药血清的培养基，并添加1毫升的钙中等免费（CMF）磷酸盐缓冲生理盐水在pH值7.4，足以覆盖细胞。这些细胞放置约10分钟，或在37℃培养箱中培养，待细胞开始解除烧瓶中。烧瓶可以轻轻摇晃或动摇，放松任何剩余的贴壁细胞。 </li><li>悬浮细胞1 ml的容量瓶中，加3毫升的DMEM条件培养基。取0.5毫升悬浮细胞和无菌他们转移到组织培养板，含有12毫米圆形玻片。约0.5 × 10 6细胞转移到圆形玻片。 </li><li>组织培养板中含有细胞在37℃，5％的CO 2 24小时，以便为细胞坚持以圆形玻片。 </li></ol> 3。唾液酸酶检测 1。使控制<ol><li>将载玻片（磨砂:1月底，1slide，25 × 75 × 1毫米）在遏制柜或实验室工作台和添加顺序混合下列化合物:1μL，DAKO公司+ 1μL4 - MUNANA，最后+ 3μL的Tris缓冲液pH值7.4公司（TBS）。 </li><li>媒体以及含有细胞取出后，轻轻提起4"forcep到井的角落的圆形玻璃幻灯片。forcep小心取出玻片上，将面临到的混合溶液对细胞的幻灯片在显微镜幻灯片。 </li><li>随即，采取microcope幻灯片落射荧光显微镜已设置紫外线过滤器和一个数码相机捕捉荧光图像。 </li><li>聚焦显微镜下相衬，直到你可以看到细胞，然后切换到荧光灯模式。下1分钟，2分钟，3分钟荧光细胞的照片。以单一的相衬下的细胞图片。 </li></ol> 2。使积极的测试<ol><li>现货另一个显微镜玻片上的顺序下列化合物:1μL，DAKO公司+ 1 4 MUNANA微升+ 2μL的LPS + 1μL的TBS。 </li><li>重复以上步骤3.1.2至3.1.4。 </li></ol> 3。连同神经氨酸酶抑制剂达菲制作的实证检验<ol><li>现货另一个显微镜玻片上的下列化合物在以下顺序:1μLDAKO公司+1μL4 MUNANA + 2μL的LPS + 1μL达菲。 </li><li>达菲浓度是事先准备好的1X的Tris缓冲盐水的pH值7.4;终浓度的圆形玻璃幻灯片的活细胞接触的化合物将有5稀释倍数。 </li><li>重复以上步骤3.1.2至3.1.4。 </li></ol> 4。抑制剂浓度测定需要抑制50％的唾液酸酶的活动（IC 50 ） <ol><li>为了量化周围细胞的荧光图像分析测量的RBG的分析插件使用ImageJ 1.38x。采取随机抽查50周围细胞的荧光读数和计算的平均总荧光。 </li><li> IC 50转换为对数单位（例如，日志毫微克/毫升）对平均荧光X轴Y轴使用非线性回归的抑制剂浓度的情节产生。 </li></ol> 5。成功的秘诀<ol><li>确保在文化细胞支原体污染。我们经常使用在培养基Plasmocin控制。 </li><li>人工神经氨酸酶的底物，4 MUNANA，应使用新鲜配制。准备基板可用于在一个星期内的唾液酸酶检测。 </li><li>如果该细胞系有细胞表面上的低的受体的表达，可以使用一个较高剂量的配体为刺激。 </li><li>我们也经历了6倍以上的传代细胞可能会失去他们的受体表型。新鲜，复活细胞应培养。 </li></ol> 6。代表性的成果见协议与唾液酸酶检测在所附的代表结果<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/2142/szewczuk_JoVE_sialidase_assay.pptx">的动画</a> PowerPoint文件。

<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和PJ作为第一作者同等贡献。

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0.0194μMBMC - 2细胞相比，神经氨酸酶抑制剂德纳（2 -脱氧- 2 ,3 -脱氢酶的19.17微米的集成电路50 DN -乙酰神经氨酸）5。我们还报告说，其他应用程序，如具体的反Neu1，-2和3抗体，没有天晴诱导唾液酸酶的活性，抑制住的BMC - 2和人类的THP - 1巨噬细胞细胞，但反Neu4抗体完全阻止这一活动。唾液酸酶的活性有一个应用程序来检测一场轰轰烈烈的唾液酸酶的活性与天晴对待生活的主要骨髓（BM）会展次要Neu1缺乏症（NeuI科威特第纳尔），但不能从Neu4淘汰赛从WT和hypomorphic蛋白酶派生的巨噬细胞（ Neu4 KO）小鼠1,2,5。此外，百日咳毒素（PTX），G蛋白偶联受体（GPCR）和基质金属蛋白酶（MMP）galardin和哌嗪的广泛抑制剂Gαi蛋白的特异性抑制剂应用到生活的BMC - 2，THP - 1和初级骨髓巨噬细胞完全阻断天晴诱导唾液酸酶的活动 5 。神经节苷脂具体的霍乱毒素B亚基（CTXB）以及与CTX，酪氨酸激酶抑制剂K252a，和广泛的G蛋白偶联受体抑制剂苏拉明不遵守这些相同的抑制作用。 MMP - 9的特异性抑制剂，抗MMP - 9抗体和抗Neu4抗体，但不是特异性抑制剂MMP - 3完全阻止天晴生活唾液酸酶的活性诱导THP - 1细胞Neu4和MMP - 9表达对细胞表面的5。 两者合计，唾液酸酶测定可用于涉及取决于配体的性质，如Neu1或Neu4 sialidases的配体诱导的受体激活的分子机制提供了强大的见解。唾液酸酶的配体的约束受体介导的活性的配体诱导的速度表明，糖化受体TrkA的NGF，BDNF TrkB的Toll样受体形成细胞表面上的膜，涉及分子配体的约束受体的组织平台的信令范式Gαi蛋白，MMP - 9和Neu1或Neu4唾液酸。配体结合各自的受体诱导唾液酸通过G蛋白偶联受体信号的活动，通过膜Gαi蛋白和MMP - 9的激活。 Neu1或Neu4和MMP - 9与细胞表面上的的受体的复杂的串扰，使唾液酸酶的快速激活受体协会，以消除产生功能受体唾液酸空间位阻。

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<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco, Rockville, MD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>4-MUNANA</td>
<td>&nbsp;</td>
<td>Biosynth International Inc., Itasca, IL, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Fetal calf serum</td>
<td>&nbsp;</td>
<td>HyClone, Logan, Utah, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</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 ...

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.5K 次观看.  Queen's University - Kingston, Ontario. 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）。

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

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

激活的B细胞CD40的小鼠模型

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

科研

免疫与感染

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).

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 ...

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.

嗜碱粒细胞激活测试IgE介导的药物过敏机制的调查

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.

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

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.

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

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.

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

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.

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.

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

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.

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

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.

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

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.