这项工作是由史密斯一家在生物医学研究和NIH的RO1 AI083426 - 01（星期六）的卓越奖。帕特里克Maschmeyer和梅拉妮Flach支持勃林格殷格翰基金会博士奖学金。

C57BL / 6小鼠，应使用〜20周岁或以上。雄性小鼠，体重25 - 30G的建议使用。星状细胞的产量可以增加喂养小鼠2个月前维生素A丰富的饮食，以星状细胞的分离。纯化约2X10 5肝星状细胞可以从一个C57BL / 6小鼠的肝脏，而星状细胞BALB / C小鼠的产量是相当高的。以下协议是调整至5只小鼠。按照批准的机构动物护理和使用委员会的协议进行了动物保健和实验。 1， 在小鼠肝脏原位灌注消化酶<ol><li>温暖的SC1的缓冲液在37℃水浴和酶灌注解决方案。 </li><li>挂载到蠕动泵硅胶管翅输液。 </li><li>校准泵用PBS获得6.5ml/min，这是将用于肝脏原位灌注流量的层流。 SC1溶液平衡的硅胶管。 </li><li>通过腹腔注射氯胺酮（90毫克/公斤）和甲苯噻嗪（10毫克/公斤）解决方案和测试的反射损失，以确保深narcotization麻醉鼠标。 </li><li>鼠标固定在一个合适的基础上的仰卧姿势。 </li><li>用剪刀和镊子执行纵形切口在腹部皮肤，暴露腹膜。 </li><li>小心打开腹膜，腹腔移动肠子动物的左侧，以揭露门静脉。 </li><li>插入导管进入门静脉输液。对于这一步，立体显微镜的使用建议。 </li><li>启动30毫升SC1溶液的肝脏灌注。打开后，立即启动流腔静脉劣势。表示成功冲洗肝脏的肝组织失去色彩。 </li><li>肝脏灌注30毫升链霉蛋白酶E解决方案。肝叶成功消化后，会膨胀，小叶会出现不同的胶囊通过。 </li><li>肝脏灌注30毫升胶原酶P解决方案。在这一点上的肝脏已经失去了它的形状和外观失张力和无定形。 </li><li>仔细分离隔膜和周围器官的肝脏和存储在冰70毫升SC2的解决方案。 </li><li>额外的小鼠重复的程序。 </li></ol> 2。在体外对小鼠肝脏的消化所有进一步的工作步骤，应在无菌层流罩条件下进行。 <ol><li>肝脏切成约2x2x2mm 3件使用锋利的剪刀。 </li><li>结合70毫升星际暂停包括链霉蛋白酶E -胶原酶P解决方案50毫升肝片和添加的DNA酶I溶液1毫升。 </li><li>精华20min的肝脏在37 ° C，同时搅拌。 </li></ol> 3。密度梯度离心<ol><li>到650毫升猎鹰管通过70μm细胞滤网过滤细胞悬液和SC2的缓冲区添加至50ml。 600克和4 ° C离心10分钟</li><li>小心吸上清液40毫升，再加入150μlDNA酶我的解决方案，以每管和悬浮细胞。 </li><li>池成450毫升猎鹰管细胞悬液和GBSS - B，离心10min后清洗600克和4 ° C。 </li><li>小心吸互不干扰颗粒和我的解决方案，以每管加入150μlDNA酶尽可能上清。重新悬浮于10ml GBSS - B的每管细胞沉淀。 </li><li>池细胞分化成250毫升猎鹰管和GBSS - B的添加的36毫升每管总量。 14毫升Nycodenz解决每管加入拌匀。 </li><li>转移到一个12毫升梯度离心管的细胞悬浮液10ml，共造成10管。轻轻覆盖1.5毫升GBSS - B的每管细胞悬液。 </li><li> 15分钟的梯度离心1500克和4 ° C不带刹车。随后，肝细胞将在试管底部沉淀，而星状细胞的发现为白色环相间。 </li><li>仔细收获相间的星状细胞，并用他们GBSS - B的离心10分钟，在600克和4 ° C。 </li><li>吸出上清液，并辅以10％热灭活胎牛血清，1％青霉素/链霉素，2mm的L -谷氨酰胺，1MM，丙酮酸钠，和10mm HEPES20毫升的DMEM重悬细胞。 2X10 4个细胞/厘米2的浓度，转移到组织培养瓶中的细胞。在37 ° C和5％的CO 2孵育细胞。 </li><li>更改媒体尽快肝星状细胞是贴壁（约2h后编制）洗掉死细胞和细胞碎片。 </li><li>翌日，星状细胞s应该发展自己的星型的形态特征与核周维生素A储存的脂质囊泡。 </li><li>随后的实验中，肝星状细胞可以很容易地脱离非涂布塑料表面使用，如Accutase，如轻度的酶解决方案</li></ol> 4。代表性的成果： 使用协议提供的肝星状细胞的准备，隔离种群的纯度可以考虑这种细胞类型，如明星般的造型，核周脂滴，表达胶质纤维酸性蛋白（GFAP的三大特色测试， ）。代表图片的外观细胞分离后的肝星状细胞2H的特征，以及第1天， 并在体外培养3如图1所示。图2显示了一个代表，已为3天，细胞的分离培养的肝星状细胞GFAP的免疫荧光染色。肝星状细胞分化成肌纤维母细胞在体外培养过程中。这些激活的星状细胞的一个特征标志是α-平滑肌肌动蛋白（αSMA）的表达。图3显示了在激活的星状细胞在体外培养的第7天阿斯玛和肌球蛋白IIA的免疫染色。 <img src="/files/ftp_upload/2710/2710fig1.jpg" alt="图1" /> 图1对肝星状细胞的特性形态。鼠标使用协议提供的肝脏星状细胞分离。图像描绘星状细胞后，细胞分离2H（A，B），以及第1天（c）和（D） 在体外培养3天。肝星状细胞表现出高量的脂质囊泡在核周网站， 并在体外培养的第一天（放大200X）收购其独特的星芒状的形态。 <img src="/files/ftp_upload/2710/2710fig2.jpg" alt="图2" /> 图2肝星状细胞在肝脏中特异性表达GFAP的。星状细胞的分离和immunofluorescently（GFAP红）染色，在体外培养的第3天。细胞核是描绘在蓝色（赫斯特染色）。 <img src="/files/ftp_upload/2710/2710fig3.jpg" alt="图3" /> 图3。肝星状细胞分化成肌纤维母细胞。 C57BL / 6小鼠的肝星状细胞培养7天。随后，他们被转移到玻片和阿斯玛（显示为红色）和肌球蛋白IIA（绿色显示）染色。细胞核用Hoechst counterstained（蓝色）。 <table border="1"><tbody><tr><td colspan="2"> SC1的缓冲区 </td></tr><tr><td> EGTA </td><td> 190mg </td></tr><tr><td>血糖</td><td> 900mg </td></tr><tr><td> HEPES </td><td> 10毫升1M原液</td></tr><tr><td>氯化钾</td><td> 400毫克</td></tr><tr><td> NA 2 HPO 4 × 2 H 2 O </td><td> 151mg </td></tr><tr><td>氯化钠</td><td> 8克</td></tr><tr><td>的NaH 2 PO 4 × H 2 O </td><td> 78mg </td></tr><tr><td>碳酸氢钠3 </td><td> 350毫克</td></tr><tr><td>酚红</td><td> 6mg </td></tr><tr><td> DH 2 O </td><td>填补至1L </td></tr><tr><td colspan="2"> SC2的缓冲区 </td></tr><tr><td>氯化钙2 × 2H 2 O </td><td> 560mg </td></tr><tr><td> HEPES </td><td> 10毫升的1M原液</td></tr><tr><td>氯化钾</td><td> 400毫克</td></tr><tr><td> NA 2 HPO 4 × 2 H 2 O </td><td> 151mg </td></tr><tr><td>氯化钠</td><td> 8克</td></tr><tr><td>的NaH 2 PO 4 × H 2 O </td><td> 78mg </td></tr><tr><td>碳酸氢钠3 </td><td> 350毫克</td></tr><tr><td>酚红</td><td> 6mg </td></tr><tr><td> DH 2 O </td><td>填补至1L </td></tr><tr><td colspan="2"> GBSS的一个缓冲区 </td></tr><tr><td>氯化钾</td><td> 370mg </td></tr><tr><td>氯化钙2 × 2H 2 O </td><td> 225mg </td></tr><tr><td>血糖</td><td> 991mg </td></tr><tr><td> KH 2 PO 4 </td><td> 30毫克</td></tr><tr><td>氯化镁2 × 6 H 2 O </td><td> 210mg </td></tr><tr><td>硫酸镁4 × 7 H 2 O </td><td> 70mg </td></tr><tr><td> NA 2 HPO 4 × 2 H 2 O </td><td> 75毫克</td></tr><tr><td>碳酸氢钠3 </td><td> 227mg </td></tr><tr><td>酚红</td><td> 6mg </td></tr><tr><td> DH 2 O </td><td>填补至1L </td></tr><tr><td colspan="2">GBSS - B的缓冲区 </td></tr><tr><td>氯化钙2 × 2H 2 O </td><td> 225mg </td></tr><tr><td>血糖</td><td> 991mg </td></tr><tr><td>氯化钾</td><td> 370mg </td></tr><tr><td> KH 2 PO 4 </td><td> 30毫克</td></tr><tr><td>氯化镁2 × 6 H 2 O </td><td> 210mg </td></tr><tr><td>硫酸镁4 × 7 H 2 O </td><td> 70mg </td></tr><tr><td> NA 2 HPO 4 × 2 H 2 O </td><td> 75毫克</td></tr><tr><td>氯化钠</td><td> 8克</td></tr><tr><td>碳酸氢钠3 </td><td> 227mg </td></tr><tr><td>酚红</td><td> 6mg </td></tr><tr><td> DH 2 O </td><td>填补至1L </td></tr><tr><td colspan="2"> 链霉蛋白酶发送灌注液 </td></tr><tr><td>链霉蛋白酶Ë </td><td> 100毫克（4000聚氨酯/毫克分钟） </td></tr><tr><td> SC2的缓冲区</td><td> 200毫升</td></tr><tr><td colspan="2"> 胶原酶P灌注液 </td></tr><tr><td>胶原酶P </td><td> 85mg（1.78 U /毫克lyo） </td></tr><tr><td> SC2的缓冲区</td><td> 200毫升</td></tr><tr><td colspan="2"> 链霉蛋白酶E -胶原酶P解决方案 </td></tr><tr><td>链霉蛋白酶Ë </td><td> 50毫克（4000聚氨酯/毫克分钟） </td></tr><tr><td>胶原酶P </td><td> 85mg（1.78 U /毫克lyo） </td></tr><tr><td> SC2的缓冲区</td><td> 50毫升</td></tr><tr><td colspan="2"> DNA酶我解决方案</td></tr><tr><td> DNA酶我</td><td> 6mg（约2000U/mg） </td></tr><tr><td> GBSS - B </td><td> 3毫升</td></tr><tr><td colspan="2"> Nycodenz解决方案 </td></tr><tr><td> Nycodenz </td><td> 8克</td></tr><tr><td> GBSS - A </td><td> 28毫升</td></tr></tbody></table> 表1。缓冲器和肝星状细胞分离所需的酶解决方案。所有的缓冲区的pH值应调整为7.3-7.4。此外，无菌过滤所有缓冲区的建议。重要的是要适应用于根据给定的酶的活性的酶量。

<ol>
	<li>Friedman, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells:+protean,+multifunctional,+and+enigmatic+cells+of+the+liver.">Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.</a> Physiol Rev. 88, 125-172 (2008).</li><li>Geerts, A. H. i. s. t. o. r. y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=heterogeneity,+developmental+biology,+and+functions+of+quiescent+hepatic+stellate+cells.">heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells.</a> Semin Liver Dis. 21, 311-335 (2001).</li><li>Gressner, A. M. &. a. m. p. ;. a. m. p., Weiskirchen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+pathogenetic+concepts+of+liver+fibrosis+suggest+stellate+cells+and+TGF-beta+as+major+players+and+therapeutic+targets.">Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets.</a> J Cell Mol Med. 10, 76-99 (2006).</li><li>Reynaert, H., Thompson, M. G., Thomas, T., Geerts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells:+role+in+microcirculation+and+pathophysiology+of+portal+hypertension.">Hepatic stellate cells: role in microcirculation and pathophysiology of portal hypertension.</a> Gut. 50, 571-581 (2002).</li><li>Winau, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ito+cells+are+liver-resident+antigen-presenting+cells+for+activating+T+cell+responses.">Ito cells are liver-resident antigen-presenting cells for activating T cell responses.</a> Immunity. 26, 117-129 (2007).</li><li>Minicis, S. D. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+during+hepatic+stellate+cell+activation+in+culture+and+in+vivo.">Gene expression profiles during hepatic stellate cell activation in culture and in vivo.</a> Gastroenterology. 132, 1937-1946 (2007).</li><li>Knittel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+liver+myofibroblasts+and+hepatic+stellate+cells+in+normal+and+diseased+rat+livers:+distinct+roles+of+(myo-)fibroblast+subpopulations+in+hepatic+tissue+repair.">Localization of liver myofibroblasts and hepatic stellate cells in normal and diseased rat livers: distinct roles of (myo-)fibroblast subpopulations in hepatic tissue repair.</a> Histochem Cell Biol. 112, 387-401 (1999).</li></ol>

肝星状细胞在肝脏调节必不可少的生理和病理过程。此外，星状细胞具有抗原提呈属性，使其肝免疫反应的一个重要组成部分。虽然肝星状细胞在肝细胞总数占10-15％，这些细胞的分离是具有挑战性的，因为他们在狄氏窦周空间的本地化。 在这里，我们提出了一个简单的方法来隔离 ，就地消化和后续梯度离心法从小鼠肝脏中的肝星状细胞。此协议允许适合免疫分析，?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>产品编号</th></tr><tr><td> 70μm细胞过滤器</td><td>屋宇署</td><td> 352350 </td></tr><tr><td>氯化钙2 × 2H 2 O </td><td> Sigma - Aldrich公司</td><td> C3306 </td></tr><tr><td>胶原酶P </td><td>罗氏公司</td><td> 11249002001 </td></tr><tr><td> DMEM培养液</td><td> Gibco公司</td><td> 11960 </td></tr><tr><td> DNA酶我</td><td>罗氏公司</td><td> 10104159001 </td></tr><tr><td> DPBS </td><td> Cellgro </td><td> 21 - 031 - CV </td></tr><tr><td> EGTA </td><td> Fluka公司</td><td> 3777 </td></tr><tr><td>胎牛血清</td><td> Gibco公司</td><td> 16000 </td></tr><tr><td>血糖</td><td> Sigma - Aldrich公司</td><td> G7528 </td></tr><tr><td>梯度离心管</td><td>格雷纳生物的一</td><td> 163160 </td></tr><tr><td> HEPES </td><td> Gibco公司</td><td> 15630 </td></tr><tr><td> KCL </td><td> Sigma - Aldrich公司</td><td> P9541 </td></tr><tr><td> KH 2 PO 4 </td><td> Sigma - Aldrich公司</td><td> P9791 </td></tr><tr><td> L -谷氨酰胺</td><td> Gibco公司</td><td> 25030 </td></tr><tr><td>氯化镁2 × 6H 2 O </td><td> Fluka公司</td><td> 63068 </td></tr><tr><td>硫酸镁4 x 7H 2 O </td><td> Sigma - Aldrich公司</td><td> M2773 </td></tr><tr><td> NA 2 HPO 4 × 2H 2 O </td><td> Fluka公司</td><td> 71643 </td></tr><tr><td>氯化钠</td><td> Sigma - Aldrich公司</td><td> S3014 </td></tr><tr><td>的NaH 2 PO 4 × H 2 O </td><td> Fluka公司</td><td> 71507 </td></tr><tr><td>碳酸氢钠3 </td><td> Fluka公司</td><td> 71628 </td></tr><tr><td> Nycodenz </td><td> Accudenz公司</td><td> AN7050/BLK </td></tr><tr><td>青霉素/链霉素</td><td> Gibco公司</td><td> 15140 </td></tr><tr><td>蠕动泵</td><td>科尔帕默</td><td> HV - 7523 - 70 </td></tr><tr><td>酚红</td><td> Sigma - Aldrich公司</td><td> P4633 </td></tr><tr><td>链霉蛋白酶Ë </td><td> Calbiochem </td><td> 7433-2 </td></tr><tr><td>硅胶管</td><td>的Masterflex </td><td> HV - 96440 - 14 </td></tr><tr><td>丙酮酸钠</td><td> Gibco公司</td><td> 11360 </td></tr><tr><td>组织培养瓶（25CM 2） </td><td>屋宇署</td><td> 353108 </td></tr><tr><td>翼输液集</td><td> Terumo公司</td><td> 1SV27EL </td></tr></tbody></table>

seven steps to stellate cells

肝星状细胞是明星般形态的肝居民细胞，并在位于肝窦内皮细胞和肝细胞 1,2之间的狄氏空间。星状细胞来源于骨髓前体和存储总体内维生素A 1，2至80％ 。激活后，星状细胞分化成肌纤维母细胞产生细胞外基质，从而促进肝纤维化3。根据合同的能力，肌纤维母细胞星状细胞能调节血管张力与门脉高压症4相关。最近，我们表明，肝星状细胞是有效的抗原提呈细胞，并能激活NKT细胞以及传统的T淋巴细胞5。 在这里，我们提出了一种从小鼠肝的肝星状细胞的高效制备方法。由于他们的窦周本地化，肝星状细胞的分离是一个多步骤的过程。为了呈现星状细胞Disse间隙隔离， 就地消化酶链霉蛋白酶E和胶原酶体育以下灌注小鼠肝脏灌注，肝组织与链霉蛋白酶E和胶原酶P遭受额外的酶处理体外 。随后，该方法需要大量的维生素A储存在肝星状细胞的脂滴的优势。此功能允许从其他类型的肝细胞在8％Nycodenz梯度离心星状细胞的分离。这里描述的协议产生的星状细胞的高纯度和均匀的人口。纯度可评估染色标记分子胶质纤维酸性蛋白（GFAP），荧光显微镜或流式细胞仪分析前的准备工作。此外，光镜下显示星形海港高量的脂滴的肝星状细胞的独特的外观。 两者合计，我们提出一个详细的协议为有效地隔离对肝星状细胞，包括有代表性的图像，其外观形态和GFAP的表达，有助于确定星状细胞实体。

Watch this Scientific Journal Video about Seven Steps to Stellate Cells at JoVE.com

Seven Steps to Stellate Cells

在这里，我们描述了从小鼠肝的肝星状细胞的分离方法。小鼠肝脏星状细胞的纯化，消化<em&gt;原位</em&gt;<em&gt;在体外</em&gt;链霉蛋白酶，胶原酶治疗之前，密度梯度离心法。这种技术产生高纯度的肝星状细胞。

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

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Immunology and Infection

33.5K 次观看.  Harvard Medical School. 在这里，我们描述了从小鼠肝的肝星状细胞的分离方法。小鼠肝脏星状细胞的纯化，消化原位在体外链霉蛋白酶，胶原酶治疗之前，密度梯度离心法。这种技术产生高纯度的肝星状细胞。

视频: Seven Steps to Stellate Cells

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

评估气孔反应活细菌细胞，采用全叶成像

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

科研

免疫与感染

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

一种改进的EPA方法1623使用切向流中空纤维超滤和热解离步骤检测水性<em&gt;隐</em&gt;和<em&gt;贾第虫属。</em

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

抗原驱动结肠炎模型的开发由抗原呈递细胞T细胞抗原研究的介绍

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

精密切割的小鼠肺切片可视化现场肺树突状细胞

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

一种直接从一滴血液中快速中性粒细胞趋化分析的片上方法

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...