在罗宾逊实验室的研究得到了来自科隆卓越集团的资助, 这是关于衰老相关疾病的细胞应激反应的支持, 德国科隆大学 (CECAD; 由 DFG 在德国联邦的卓越主动性之内资助和州政府) 和赠款从德意志 Forschungsgemeinschaft (SFB 670), 科隆财富和玛丽亚-Pesch 基金会的德国科隆大学。

所有涉及使用致病性的步骤 . 沙门氏菌 必须在 BSL-2 或更高的生物安全级别设施中进行。 S 的区域性和涂层 鼠伤寒, 以及骨髓源性巨噬细胞 (BMDMs) 的感染必须在层流罩下进行, 以防止污染。 S 的隔离含有吞噬的 鼠伤寒杆菌 可在任何 BSL-2 实验室的工作台上执行. 
 从小鼠体内提取骨髓, 将其分化成巨噬细胞, 是按照动物福利机构指南进行的, 并由北莱茵-伐国家自然机构批准,环境和消费者保护 [Landesamt f 和 #252; r Natur、Umwelt 和 Verbraucherschutz (LANUV) Nordrhein-Westfalen;文件编号: 84-02.05.40.14.082 和 84-02.04.2015. A443] 和科隆大学. 
 1. 培养 鼠伤寒杆菌 <ol> <li> 接种 沙门氏菌 (SL1344 菌株) 从一个单一的细菌菌落到5毫升的脑心灌注 (BHI) 汤使用细菌循环. </li>
	<li> 在37和 #176 的孵化箱中孵育细菌悬浮液; C. 一整夜摇晃. </li>
	<li> 在第二天, 将1毫升的细菌悬浮液转化为19毫升的 BHI 汤, 在圆锥烧瓶中孵育, 在37和 #176; C 在带震动的孵化器中. </li>
	<li> 监视 S. 用分光光度计测量 600 nm (od 600 ) 的光学密度 (od), 以 鼠伤寒杆菌 生长。大约每30分钟测量一次. </li>
	<li> OD 600 达到1.0 时, 从孵化箱中取出细菌悬浮液并将其转移到50毫升管中。离心机细菌在 5400 x g 为15分钟在4和 #176; C. </li>
	<li> 在10毫升无菌磷酸缓冲盐 (PBS) 中去除上清和重. </li>
	<li> 5400 x g 离心15分钟在4和 #176; C. </li>
	<li> 去除上清液, 重4.9 毫升无菌 PBS 中的细菌. </li>
</ol> 2。用琥珀酰亚胺-生物素/亲和-共轭磁珠的 S. 鼠伤寒杆菌涂层 <ol> <li> 添加100和 #181; L 10 毫克/毫升生物素连接解决方案 (nhs-生物素溶解在二甲基亚砜) 新鲜制备的细菌悬浮在上一步中准备。例如, 将5毫克的 NHS-生物素稀释为0.5 毫升的无菌亚砜。正确混合移多次. </li>
	<li> 将混合料分成五1.5 毫升的管子. </li>
	<li> 在室温 (RT) 的 thermoblock 上孵育2小时, 并在 350 rpm 时持续晃动. </li>
	<li> 在 RT 中离心管在 1.5万 x g 处10分钟并丢弃上清液. </li>
	<li> 重在1毫升的无菌 PBS, 离心在 1.5万 x g 10 分钟的 RT 和丢弃上清. </li>
	<li> 重复步骤2.5 两次, 以完全删除多余的生物素链接解决方案. </li>
	<li> 最后一次洗涤后, 重1毫升的无菌 PBS 中的颗粒. </li>
	<li> 传输100和 #181; 10 毫克/毫升亲和共轭磁珠解决方案成1.5 毫升管, 并留在磁性机架上5分钟. </li>
	<li> 用吸管取出溶剂, 从磁性齿条上取下亲和共轭磁珠溶液, 并在步骤2.7 中重1毫升的生物素涂层-细菌悬浮液. </li>
	<li> 在 RT 上的 thermoblock 上孵育1小时, 持续晃动 350 rpm. </li>
	<li> 将解决方案放在磁性机架上; 等待5分钟, 然后用吸管移除不附着在墙上的细菌 (将其放在标签为和 #34 的试管中; 涂细菌和 #34;)。附着在与磁体接触的管壁上的分数是生物素/亲和涂层细菌. </li>
	<li> 从1毫升的无菌 PBS 中取出含有标签细菌的重. </li>
	<li> 将其再次放置在磁性机架上, 等待5分钟, 然后使用吸管移除 PBS. </li>
	<li> 重复步骤2.13 和2.14 两次, 以删除所有未涂有亲和共轭磁珠的细菌. </li>
	<li> 最后一次洗涤后, 重500和 #181 中的涂布细菌, 并将管状菌和 #34 贴上标签; 涂层细菌和 #34;. </li>
	<li> 计数和 #34 的菌落形成单位 (cfu); 涂细菌和 #34; 和 #34; 涂层细菌和 #34; BHI 琼脂板上电镀系列稀释的解决方案。大约 2 x 10 8 cfu/毫升的涂层细菌是从20毫升的初始细菌悬浮与 OD 600 1.0。将涂膜细菌悬浮液保持在4和 #176; C 在第二天用于感染巨噬细胞. </li>
</ol> 3。感染 BDMDs <ol> <li> 在同一天, 当细菌被涂上生物素和亲和共轭磁性珠子时, 盘子完全分化 BMDMs 22 在6厘米的菜肴中密度为 5 x 10 6 细胞每道. </li>
	<li> 在第二天, 离心涂层细菌悬浮在 1.5万 x g 为5分钟在4和 #176; C. </li>
	<li> 在罗斯威尔公园纪念研究所 (RPMI) 培养基中除去上清和重, 并辅以10% 胎牛血清 (FBS), 浓度为 10 x 10 6 cfu/毫升. </li>
	<li> 为了感染巨噬细胞在10的多样性, 去除培养基从 BMDMs 和增加5毫升涂层细菌悬浮准备。最佳的教学语言可以评估每一个细胞类型或实验目的的孤立吞噬. </li>
	<li> 在 RT 孵育10分钟以同步吞噬. </li>
	<li> 孵育在37和 #176; C 在 5% CO 2 孵化器为30分钟开始吞噬。其他细胞类型可能需要不同的孵化时间, 以确保细菌内部化. </li>
	<li> 从盘子中取出含有细菌的培养基, 用 RPMI 三次清洗细胞, 去除 non-internalized 细菌. </li>
	<li> 最后添加10毫升的 RPMI, 包含 10% FBS 和 50 #181; 庆大霉素杀死任何 non-phagocytized 细菌. </li>
	<li> 在37和 #176 中孵育感染的 BMDMs; C 与 5% CO 2 直到所需的时间点。所需的时间点必须根据所用的细菌和细胞类型和分析目的进行实验确定。为研究早期体-endosome 融合事件在 S. 沙门氏菌感染的 BMDMs, 30 分钟孵化建议。对于以后的事件的分析, 吞噬可以提取后2小时或 4 h 的孵化。长时间的巨噬细胞孵育与 S. 超过 24 h 的鼠伤寒导致巨噬细胞死亡。在体提取前扩大细胞内感染可能导致生物素分子的降解。然而, 我们并没有观察到24小时前涂层细菌中生物素的损失. </li>
</ol> 4。隔离 S. 沙门氏菌 -包含吞噬 <ol> <li> 准备所需的体隔离缓冲区 A (50 毫米管道, pH 值 7, 50 毫米氯化镁 2 , 5 mm EGTA), 加入糖 (德勤), 以最终浓度1毫米, 素 B 为最终10和 #181; M, 蛋白酶和磷酸酶抑制剂的浓度, 由制造商推荐. 
	注: 必须在使用前立即将素 B 添加到体隔离缓冲区 a 中。素 B 扰乱细胞骨架, 促进细胞膜破裂. </li>
	<li> 在所需的时间点, 从受感染的细胞中取出培养基, 用不育的 PBS 加热到 RT. </li>
	<li> 添加750和 #181; phagosome 隔离缓冲每道菜, 在冰上孵育20分钟。在使用不同的单元格号时, 应按比例调整隔离缓冲区 A 的音量. </li>
	<li> 添加250和 #181; 体隔离缓冲器 B (50 毫米管, pH 7, 50 毫米氯化镁 2 , 5 毫米 EGTA, 220 毫米甘露醇, 和68毫米蔗糖)。岩石的板块, 以确保缓冲区达到整个表面的菜。在使用不同的单元格号时, 应按比例调整隔离缓冲区 B 的音量. </li>
	<li> 通过使用橡皮警察轻轻地刮掉盘子里的细胞, 然后把它们转移到 pre-chilled 1.5 毫升的管子上. </li>
	<li> 通过26G 针通过1毫升注射器至少15次 (一个愿望加上一个弹射的细胞悬浮计数作为一次) 的细胞悬浮。这足以释放 BMDMs 的胞浆含量。通过针的次数必须针对每个单元格类型进行优化. </li>
	<li> 将电池悬浮在磁性机架上, 等待5分钟。附着在磁体上的微粒是含有涂层的吞噬- S. 伤寒.悬浮液中含有其余的细胞成分. </li>
	<li> 将悬浮液转换为标记为和 #34 的1.5 毫升管; 胞和 #34;. </li>
	<li> 在1毫升的无菌 PBS 中, 从磁性机架上取下带有隔离吞噬的管, 并重它们. </li>
	<li> 将体悬浮在磁性机架上, 等待5分钟, 然后卸下 PBS. </li>
	<li> 重复步骤4.9 和4.10 以清洗隔离的 . 沙门氏菌 -包含吞噬. </li>
	<li> 最后删除 PBS 并重所需的缓冲区中的吞噬; 例如, 在 radioimmunoprecipitation 分析 (帕) 的缓冲液中进行蛋白质检测。大约50-200 和 #181; 根据本议定书的每个样本获得的蛋白质 g, 取决于细胞的初始数量和感染的莫伊. </li>
</ol>

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R., Jagannath, C., Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systems+biology+approach+to+study+the+phagosomal+proteome+modulated+by+mycobacterial+infections.">A systems biology approach to study the phagosomal proteome modulated by mycobacterial infections.</a> Int J Clin Exp Med. 2 (3), 233-247 (2009).</li><li>Rogers, L. D., Foster, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+phagosomal+proteome+and+the+contribution+of+the+endoplasmic+reticulum.">The dynamic phagosomal proteome and the contribution of the endoplasmic reticulum.</a> P Natl Acad Sci USA. 104 (47), 18520-18525 (2007).</li></ol>

作者没有什么可透露的。

用于隔离S的新方法本文介绍了用生物素和亲和-共轭磁珠吞噬细菌的方法. 在细胞膜的轻微破坏后, 含有细菌的吞噬可以很容易地用磁性齿条来提取。我们表明, 细菌的标记保留了病原体诱导炎症的能力, 不会改变宿主细胞的吞噬特性。重要的是, 这种方法获得的吞噬丰富的细菌和 endosome/溶标记和缺乏胞浆污染。
与传统的采用蔗糖梯度分离法的体分离方法相比, 该?...

巨噬细胞是循环的专门的吞噬干细胞, 可以检测、吞噬和降解外周组织中的任何外来微粒, 从凋亡细胞到入侵微生物, 如细菌。表面受体介导的病原体特异标记物的识别通常存在于微生物的表面 (称为病原体相关的分子模式或 PAMPs), 巨噬细胞启动一个复杂的细胞膜重组为了包围和吞噬病原体1。
吞噬的病原体然后被巨噬细胞所包含的细胞内囊泡称为体。通过一系列的融合和裂变事件与其他囊泡, 如体和溶酶体, 包含体获得了一套蛋白质, 需要消除 phagosomal 的内容。因此, 体的酶组成在这个过程中是高度可变的, 被称为体成熟的2。
吞噬后不久, multimeric 复合泡 atp 酶 (v atp 酶) 被纳入体膜融合与体3。这个复合体利用 ATP 泵浦质子从胞到腔的体4。体的酸化是必要的为融合事件与其他泡5和为激活许多 pH 依赖性降解酵素6。在体膜上快速组装的另一种 multimeric 酶复合物是氧化酶 (NOX) 复合物。为了产生活性氧 (ROS) 分泌到体腔内, 并大大有助于杀死被吞噬的微生物7, 氮氧化物复合物氧化。
在成熟的初始步骤, 吞噬目前的标记通常如 Rab5 和 Rab7 的早期和晚期体分别与 v 型 atp 酶8的 v-0亚基。吞噬与溶酶体和晚期体的融合导致吞噬病原菌暴露于多种水解酵素, 如蛋白酶蛋白酶、脂肪酶和β-乳糖9。对这些酶的活化也需要管腔的酸化。例如, 蛋白酶 D 的裂解产生活性的短形式是 pH 依赖性的10。这些酶降解病原体, 并调解由巨噬细胞主要组织相容性复合物 (MHC) II 类分子向 T 细胞提出的病原体衍生短肽的产生, 以触发自适应免疫应答11。
因此, 体成熟对先天免疫反应至关重要, 并与免疫系统的先天和适应性武器联系在一起。这并不奇怪, 病原体已经进化的战略, 以克服由巨噬细胞通过上述的体成熟的过程。例如, 胞内细菌结核分枝杆菌和军团肺通过抑制 v atp 酶的组装和相应的流明酸化, 防止体成熟,12,13.其他细菌如李斯特菌或志贺氏杆菌福氏诱导体膜中的孔隙形成, 以逃逸到胞14,15。另一方面,沙门氏菌 enterica螺旋体鼠伤寒杆菌 (S。沙门氏菌)能够修改液泡内体的属性, 将其转换为其复制16的合适位置。此功能使S.沙门氏菌是研究病原体介导的体成熟干预的一个非常有趣的模型。
S. 鼠伤寒是一种在人类中引起胃肠炎的兼胞内细菌。在侵入固有后, S.鼠伤寒杆菌快速检测和吞噬的巨噬细胞, 并包含在吞噬17。一些报告以前曾描述过S.沙门氏菌-包含吞噬目前的制造商为体和溶酶体18, 和其他研究发现体-溶融合阻止后, S.鼠伤寒病毒感染19。
最初, 体成熟于S.用免疫荧光显微镜对鼠伤寒杆菌感染进行了研究。在分离细菌的吞噬技术的发展使更准确的研究体的内容, 从 endosome 和溶标记。迄今为止, 用于分离细菌的吞噬的主要方法是蔗糖梯度的亚细胞分馏18,20。然而, 这种方法需要多个离心步骤, 可以造成机械损伤的吞噬, 可能会影响稳定的 phagosomal 组分 (蛋白质和血脂), 是费时。此外, 它还需要使用 ultracentrifuge: 一个专门的设备, 这是无法进入每个实验室。
最近, 一种新的方法已被用于分离细菌含有吞噬, 其中细菌病原体被标记为化 lipopetide (Lipobiotin), 后来提取使用亲和共轭磁珠21.我们提出了一种替代的补充方法, 标记细菌表面胺类大分子与 NHS-生物素后, 亲和共轭磁珠。该方法获得的吞噬在 endosome 和溶标记物中具有很高的丰富性, 可用于多种测定, 从蛋白质分析到学分析。此外, 它不需要专门的设备, 如超速。此外, 通过消除离心步骤, 吞噬的机械损伤和使用的时间都大大减少。这种方法可以很容易地适应隔离的吞噬含有其他细菌, 如革兰氏阳性的金黄色葡萄球菌, 也包括在这份手稿。总之, 此方法用于隔离S.含有吞噬的鼠伤寒杆菌是一种简单、cost-effective 且耗时较传统的蔗糖梯度分离法, 离心, 呈现高浓度的含菌吞噬。

<table><tbody><tr><td>EZ-Link NHS Biotin</td><td>Thermo Fisher Scientific</td><td>20217</td><td></td></tr><tr><td>FluidMag Streptavidin</td><td>Chemicell</td><td>4205</td><td></td></tr><tr><td>PIPES</td><td>Carl Roth</td><td>9156.2</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Carl Roth</td><td>A537.4</td><td></td></tr><tr><td>EGTA</td><td>Carl Roth</td><td>3054.3</td><td></td></tr><tr><td>Sucrose</td><td>Carl Roth</td><td>4621.1</td><td></td></tr><tr><td>Mannitol</td><td>Carl Roth</td><td>4175.1</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>43816</td><td></td></tr><tr><td>Halt Protease and Phosphatase inhibitor cocktail</td><td>Thermo Fisher Scientific</td><td>1861280</td><td></td></tr><tr><td>Cytochalasin B</td><td>Sigma</td><td>C6762</td><td></td></tr><tr><td>DYNAL or DynaMag Magnet</td><td>Thermo Fisher Scientific</td><td>12321D</td><td></td></tr><tr><td>SmartSpec 3000 Spectrophotometer</td><td>Bio-Rad</td><td>170-2501</td><td></td></tr><tr><td>Bacterial loop (10&amp;micro;l)</td><td>Sarstedt</td><td>86.1562.010</td><td></td></tr><tr><td>Salmonella enterica serovar Typhimurium SL1344</td><td>Leibniz Institute DSMZ-German collection of Microorganisms and Cell Cultures</td><td></td><td></td></tr><tr><td>RPMI</td><td>Biochrom</td><td>FG1415</td><td></td></tr><tr><td>PBS</td><td>Biochrom</td><td>L1825</td><td></td></tr><tr><td>Cy5-streptavidin</td><td>Invitrogen</td><td>SA1011</td><td></td></tr><tr><td>anti-beta-actin antibody</td><td>Santa Cruz Biotechnology</td><td>sc-47778</td><td></td></tr><tr><td>anti-mCherry antibody</td><td>Thermo Fisher Scientific</td><td>PA5-34974</td><td></td></tr><tr><td>anti-Rab5 antibody</td><td>Santa Cruz Biotechnology</td><td>sc-46692</td><td></td></tr><tr><td>anti-Rab7 antibody</td><td>Santa Cruz Biotechnology</td><td>sc-10764</td><td></td></tr><tr><td>anti-v-ATPase (V0) antibody</td><td>Santa Cruz Biotechnology</td><td>sc-28801</td><td></td></tr><tr><td>anti-v-ATPase (V1) antibody</td><td>Santa Cruz Biotechnology</td><td>sc-20943</td><td></td></tr><tr><td>anti-cathepsin D antibody</td><td>Santa Cruz Biotechnology</td><td>sc-6486</td><td></td></tr><tr><td>anti-Tomm20 antibody</td><td>Santa Cruz Biotechnology</td><td>sc-17764</td><td></td></tr><tr><td>anti-calnexin antibody</td><td>Santa Cruz Biotechnology</td><td>sc-46669</td><td></td></tr><tr><td>anti-GAPDH antibody</td><td>Santa Cruz Biotechnology</td><td>sc-20357</td><td></td></tr></tbody></table>

isolation of salmonella typhimurium-containing phagosomes from macrophages

鼠伤寒沙门氏菌是一种在人类中引起胃肠炎的兼胞内细菌。入侵后的叶片固有, S.鼠伤寒杆菌细菌被巨噬细胞快速检测和吞噬, 并被称为吞噬的囊泡, 以使其降解。隔离S.包含吞噬的沙门氏菌已被广泛用于研究S.沙门氏菌感染会改变体成熟的过程, 以防止细菌降解。采用蔗糖梯度离心法对含菌吞噬进行了分离。然而, 这个过程是费时的, 需要专门的设备和一定程度的灵巧。这里描述的是一个用于隔离 S 的简单而快速的方法.鼠伤寒-含有吞噬从巨噬细胞通过涂层细菌与生物素-亲和共轭磁珠。通过这种方法获得的吞噬可以悬浮在任何选择的缓冲液中, 使分离的吞噬的使用范围广泛, 如蛋白质、代谢物和脂质分析。总之, 此方法用于隔离S.含有吞噬的鼠伤寒杆菌是特定的、高效的、快速的, 需要最少的设备, 并且比传统的蔗糖梯度离心方法更具通用性。

通过本议定书隔离含有细菌的吞噬, 需要将细菌的法作为第一步。因此, 我们评估了S.的有效性鼠伤寒杆菌法通过共聚焦显微镜分析 BMDMs 感染化 mCherry-S。沙门氏菌标记为 Cy5-Streptavidin。简言之, BMDMs 在本协议中与 mCherry-S一起被感染。沙门氏菌先前化, 但不与亲和共轭磁珠一起孵育。在37° c 的30分钟孵育后, 细胞用温热的 pbs 洗涤, 用4% ?...

Watch this Scientific Journal Video about Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages at JoVE.com

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

我们在这里描述了一个简单和快速的方法, 以隔离的鼠伤寒沙门氏菌-含有吞噬从巨噬细胞涂层细菌与生物素和亲和。

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

isolation-salmonella-typhimurium-containing-phagosomes-from

Research

JoVE Journal

Immunology and Infection

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

10.6K 次观看.  University of Cologne. 我们在这里描述了一个简单和快速的方法, 以隔离的鼠伤寒沙门氏菌-含有吞噬从巨噬细胞涂层细菌与生物素和亲和。

视频: Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

科研

免疫与感染

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

病原液泡的分离纯化<em&gt;军团</em&gt;感染吞噬细胞

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

高通量检测到的表型<em&gt;沙门氏菌</em&gt;伤寒协会，侵袭和复制的巨噬细胞

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

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. ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

胞浆的定量与液泡<em&gt;沙门氏菌</em&gt;在原发性巨噬细胞差透

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

"吞噬体封闭试验"可视化吞噬体形成的三个维度利用全内反射荧光显微镜（TIRFM）

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection or damage. Currently, the widely used mice models of sepsis include lipopolysaccharide (LPS)-induced endotoxemia, cecal ligation and puncture (CLP), and monobacterial infection model systems. This protocol describes a method to study the host responses during Salmonella Typhimurium infection-induced septic peritonitis in mice. S. Typhimurium, a Gram-negative intracellular pathogen, causes typhoid-like disease in mice.
This protocol elaborates the culture preparation, induction of septic peritonitis in mice through intraperitoneal injection, and methods to study systemic host responses. Furthermore, the assessment of bacterial burden in different organs and the flow cytometric analysis of increased neutrophil numbers in the peritoneal lavage is presented. Salmonella Typhimurium-induced sepsis in mice leads to an increase in proinflammatory cytokines and rapid infiltration of neutrophils in the peritoneal cavity, leading to lower survival.
Every step in this protocol has been optimized, resulting in high reproducibility of the pathogenesis of septic peritonitis. This model is useful for studying immunological responses during bacterial sepsis, the roles of different genes in disease progression, and the effects of drugs to attenuate sepsis.

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

This protocol describes the induction of Gram-negative monobacterial sepsis in a mouse model system. The model is useful in investigating the inflammatory and lethal host responses during sepsis.

沙 门氏菌 斑疹伤寒诱导的小鼠脓毒性腹膜炎的特征

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection ...

使用细菌 LPS 去除 沙门氏菌 培养物中的噬菌体

Bacteriophage Removal from Infected Salmonella Cultures

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and interactions. These organisms are viruses that infect and replicate within bacterial hosts. Phages are ubiquitous on Earth, highly diverse, and very abundant. While bacteriophages have valuable roles in different environments and are a key area of research in microbiology and ecology, their presence can be undesirable in certain industrial processes or products. Considering the abundance and ubiquity of bacteriophages on Earth, the design of procedures for the removal of bacteriophages from bacterial cultures is crucial in diverse laboratory and industrial applications to preserve the integrity of the cultures and ensure accurate experimental results or product quality. Here, we have fine-tuned a protocol to eliminate the bacteriophages from infected Salmonella enterica cultures, using a strategy based on the use of lipopolysaccharides (LPS) located in the outer membrane of Gram-negative bacteria. Bacterial LPS plays an important role in host recognition by phages, and we make use of this property to design an effective procedure for the removal of phages, which use LPS as a receptor, in Salmonella bacterial cultures.

作者聚焦:使用脂多糖有效消除感染沙 门氏菌 培养物中的噬菌体

Bacteriophages, ubiquitous and diverse on Earth, infect and replicate within bacterial hosts, playing a crucial role in microbial ecosystems. Despite their importance, their presence may disrupt industrial processes. We have developed a method using bacterial lipopolysaccharides to eliminate bacteriophages from Salmonella cultures.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

摘要

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一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

病原液泡的分离纯化

高通量检测到的表型

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

胞浆的定量与液泡

"吞噬体封闭试验"可视化吞噬体形成的三个维度利用全内反射荧光显微镜（TIRFM）

沙门氏菌斑疹伤寒诱导的小鼠脓毒性腹膜炎的特征

从感染的沙门氏菌培养物中去除噬菌体

从感染的沙门氏菌培养物中去除噬菌体

从感染的沙门氏菌培养物中去除噬菌体

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

摘要

探索更多视频

此视频中的章节

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

病原液泡的分离纯化

高通量检测到的表型

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

胞浆的定量与液泡

"吞噬体封闭试验"可视化吞噬体形成的三个维度利用全内反射荧光显微镜（TIRFM）

沙 门氏菌 斑疹伤寒诱导的小鼠脓毒性腹膜炎的特征

从感染的沙门氏菌培养物中去除噬菌体

从感染的沙门氏菌培养物中去除噬菌体

从感染的沙门氏菌培养物中去除噬菌体

沙门氏菌斑疹伤寒诱导的小鼠脓毒性腹膜炎的特征