Name: assessing bacterial invasion of cardiac cells in culture and heart colonization in infected mice using listeria monocytogenes
Uploaded: 2015-05-27T14:00:00.000+00:00
Duration: 9 min 18 s
Description: Watch this Scientific Journal Video about Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes at JoVE.com

这项工作得到了公共卫生服务机构AI41816和AI099339（N.E.F.）和F31AI094886-01（P.D..M）的支持。 内容完全由作者负责，不一定代表资金来源的官方观点。

1. L. 单细胞菌株的 储存和文化条件
<ol><li>通过自动切割脑心输液（BHI）和将熔融介质倒入培养板来准备固体介质。让盘子在室温下干燥过夜，然后在37°C处再通宵干燥。</li>
	<li>使用无菌循环，从以前制造的冰柜储存（BHI液体介质中20%甘油中悬浮的细菌，储存在-80°C）或含有以前被隔离的菌落的藻类板中获取少量 L.单细胞基因 样本。</li>
	<li>使用不化技术，对样本进行单个菌落隔离。请务必对交叉条纹之间的循环进行消毒，以防止过度结转，并确保对正在评估的菌株的单个菌落进行隔离。在 37 °C 处隔夜孵化盘子。</li>
	<li>使用无菌循环，去除分离物的一个菌落，并在14毫升聚苯乙烯管中接种2毫升BHI汤（无阿加）。</li>
	<li>对于检测和感染，在 37 °C 孵化器中静态地（不摇晃）在一夜之间孵育肉汤培养。对于培养隔离物的放养，在 37 °C 的汤培养孵育，在 180-200 rpm 的夜间摇晃。培养物可以通过将甘油添加到 20% 的最终浓度并无限期地将密封管储存在 -80 °C 来储存。</li>
</ol>2. H9c2 心脏肌细胞的储存和培养条件
<ol><li>购买或获得冷冻库存的H9c2细胞，以及杜贝尔科的改良鹰的介质（DMEM）含有高葡萄糖和聚氨酯，胎儿牛血清（FBS），L-谷氨酰胺（谷胱甘肽），和青霉素-链霉素-谷氨酰胺混合物（PSG）。 FBS、胶粘和PSG应储存在-20°C，而DMEM可存储在4°C。 在冷冻前将 FBS 引用到 50 毫升圆锥管中是有帮助的。</li>
	<li>在层压流罩中，解冻两个 FBS 的音标。将一个阿里报价添加到一瓶 500 毫升的 DMEM 中，制成 10% 的 FBS/DMEM 混合物。为此，添加 6 毫升的 Glut，并将由此产生的溶液储存在 4 °C。 此解决方案可以标记为"没有抗生素的 DMEM"。（德梅姆-阿贝）</li>
	<li>到第二瓶500毫升的DMEM中，加入其他50毫升的FBS。在此解决方案中，添加 6 毫升 PSG 和库存在 4 °C。 此解决方案可标记为"带抗生素的 DMEM"。（德梅姆+阿布）</li>
	<li>在层压罩中，取出 10 毫升 DMEM +Ab，放在 25 毫升组织培养瓶中。</li>
	<li>将烧瓶留在层压罩中，从液氮中取出冷冻的 H9c2 细胞，并迅速将管子置于 37 °C 的水浴中，直到培养物解冻。</li>
	<li>用70%的乙醇喷洒管子进行消毒，然后返回引擎盖。快速工作，以尽量减少在集中的DMSO的细胞时间，添加细胞的全部引用到10毫升的DMEM+Ab。 这充分稀释了 DMSO，使隔夜细胞的生存能力和粘性。</li>
	<li>将烧瓶放在组织培养孵化器中，在 37 °C、5% C02和 95% 湿度过夜。</li>
	<li>第二天早上，检查细胞层，以确保坚持。此时，细胞可能在 80-100% 汇流之间。</li>
	<li>在组织培养罩中，使用真空去除烧瓶内的介质，只留下烧瓶中的细胞。</li>
	<li>将约2毫升的0.25%的Trypsin-EDTA添加到细胞中，轻轻摇动约3秒。</li>
	<li>倒置烧瓶，使试剂溶液不再与单层接触，并通过真空去除试剂。</li>
	<li>在细胞中加入第二个 2 毫升的肌肽，并将烧瓶移动到倒置显微镜中。观察细胞，同时轻轻摇动烧瓶，以确保单层分散。</li>
	<li>单层分散后，立即返回组织培养罩，并在细胞悬架中加入 4 毫升 DMEM +Ab。</li>
	<li>取出细胞悬架的 2 毫升部分，并将其添加到包含 23 毫升 DMEM +Ab 的 50 毫升组织培养瓶中。 轻轻摇动烧瓶，然后在第 2.5 步所列条件下将其放置在组织培养孵化器中。</li>
	<li>每天检查细胞，直到它们达到约80%的汇合（约2.0-4.0 x 105 细胞每毫升）。细胞不要变得太结合是非常重要的，因为它们可能会分化成骨骼肌管时，长期饿伤的FBS14 和这种分化可以改变细菌入侵。 仔细监测细胞，并在培养成为汇流时从一个新的细胞管开始。</li>
	<li>一旦细胞达到80%的汇合，它们要么被传入另一个50毫升的烧瓶，如刚才描述，或准备入侵检测。</li>
</ol>3. 准备 H9c2 细胞进行入侵分析
<ol><li>在组织培养罩中，使用真空吸气将介质从 H9c2 细胞的 50 毫升烧瓶中取出，这些细胞的汇合率已达 80%。</li>
	<li>在单层中加入 4 毫升 0.25% 的 Trypsin-EDTA 解决方案，然后立即使用真空吸气消除。</li>
	<li>在烧瓶中再加入 4 毫升 0.25% 的 Trypsin-EDTA 部分，并将烧瓶移到倒显微镜中观察单层的分散。</li>
	<li>细胞分散后，返回引擎盖，并在细胞中加入4毫升DMEM-Ab。上下管道解决方案可确保从烧瓶底部完全去除细胞。</li>
	<li>单层悬念后，取出所有悬架并将其放置在 50 毫升圆锥管中。</li>
	<li>取出此悬架的 20 ul 部分，并使用血细胞计计算每毫升的细胞数量。</li>
	<li>确定溶液中细胞的浓度后，通过在新鲜 DMEM -Ab 中添加适量的细胞溶液，将浓度调整为2.25 x 10 4 细胞/毫升。调整后的溶液的总体积应为 25 毫升。</li>
	<li>在引擎盖中，将玻璃盖脂浸入 90% 乙醇中，并短暂地通过火焰短暂地传递每个盖片，从而对玻璃盖片进行消毒。在灭菌后，将每个盖片添加到 24 井组织培养处理板的单独井中。</li>
	<li>所有油井都有单独的盖片后，使用 25 毫升的管道将 1 毫升稀释的细胞溶液添加到板中的每口井中，然后用无菌针将每个盖片向下推。</li>
	<li>使用倒置显微镜检查每口井，以确保每口井中都有类似数量的细胞。将 24 个井板放在组织培养孵化器中过夜，湿度为 5% CO2 和 95%。</li>
	<li>第二天早上，查看每口井，以确保盖片有相当数量的附着肌母细胞，并且盖片不会漂浮在井中。</li>
</ol>4. 为入侵测定准备 L. 单细胞基因 应变
注:所有实验室工作均按照疾病预防控制中心生物安全2级指南进行。
<ol><li>准备24井板进行检测后，使用消毒循环去除应变的单一菌落，以检查细菌入侵，并接种每个到单独的14毫升管含有2毫升BHI汤。</li>
	<li>将接种的管子放入 37 °C 的孵化器中，在 45° 下倾斜，并在一夜之间静态地（不摇晃）孵化。</li>
	<li>第二天早上，从孵化器和漩涡中取出培养管，以确保细菌均匀地悬浮。</li>
	<li>使用 600 nm 波长的光谱仪测量培养的光学密度。在阅读光学密度之前，请务必使用无菌 BHI 汤将光谱仪归零。</li>
	<li>对于 L. 单细胞基因，光学密度与每毫升细菌的浓度直接相关，因此密度为 1.000 = 1.0 x 109 CFU 每毫升。</li>
	<li>计算感染 2.25 x 104 细胞所需的培养量，2.25 x 106（MOI = 100）。</li>
	<li>去除所需的培养量，并将其放置在1.5毫升离心管中。</li>
	<li>在 19，000 x g 下旋转 3 分钟，然后丢弃超自然人。将管子的开端对着纸巾，以去除粘在管子唇部的过量介质。</li>
	<li>将细菌重新吸收到1毫升的无菌PBS和漩涡中，以确保充分混合。这种混合物应包含大约 2.25 x 106 CFU 每 20 ul （1.125 x 108 CFU/毫升）.</li>
</ol>5. 执行入侵分析
<ol><li>入侵检测的前一天，准备第 3 节中描述的 24 井 H9c2 细胞板，并在第 4 节中描述的 李斯特菌 所需的菌株中接种无菌 BHI 汤。</li>
	<li>使用第 4 节中的协议，准备 PBS 恢复的菌株培养物，以评估入侵情况。注意:此时可以通过将每个样品的稀释电镀到固体介质上，并在夜间孵化后列举菌落来评估传染性滴定器。</li>
	<li>将至少 20 个 PBS 细菌溶液加载到 24 井板的单个井中。请注意，每个菌株至少需要三口井才能获得三联的结果，因此可以同时评估多达 8 个菌株。</li>
	<li>将油井装上细菌后，轻轻摇动油板，鼓励每个样品在井内均匀分布，并将24井板放回孵化器中，在37°C和95%的湿度下孵化45分钟，其中5%为二氧化碳。</li>
	<li>在此孵化过程中，只需取出 25 毫升 DMEM - Ab 并将其放入 50 毫升猎鹰管（或等效物），即可准备 DMEM - Ab （第 2 节）的别名报价。</li>
	<li>将根酰胺加入 25 毫升 DMEM - Ab 阿里报价，浓度为 15 微克/毫升（50 毫克/毫升库存溶液的 7.5 升）。在45分钟的孵化期间，将DMEM-Ab+Gent溶液置于37°C的水浴中。</li>
	<li>Aliquot 25 毫升 PBS 放入 50 毫升猎鹰管中，并在 45 分钟的孵化过程中放入 37 °C 的水浴中。</li>
	<li>45 分钟孵化完成后，取出 24 井板并将其放在引擎盖中。通过真空吸气从每口井中取出含有细菌的介质，在不同菌株之间更换玻璃管道尖端。</li>
	<li>在每个井中加入大约 1 毫升 PBS，以便从细胞表面清洗松散粘附的细菌，然后使用真空吸气去除 PBS。</li>
	<li>完全去除PBS后，在每口井中添加1毫升DMEM-Ab+根特。根酰胺素只会杀死那些留在细胞外的细菌，因此那些细胞内细菌将仍然存活。</li>
	<li>将 24 井板放回孵化器，并允许再孵化一小时。</li>
	<li>在长达一小时的孵化过程中，通过在每个管中加入 1 毫升无菌 ddH2 0来准备 14 毫升聚丙烯管。每个盖片需要一根管子，因此对于一个 24 井板，总共需要准备 24 根管子。</li>
	<li>经过一小时的孵化后，从孵化器中取出 24 井板，并将其与步骤 5.12 中准备的管子一起放在引擎盖中。</li>
	<li>使用无菌钳子，从各自的井中取出每个盖子，并立即将其放置在单独的管子中。请务必将钳子浸入乙醇中，并在每个盖子夹之间燃烧，以防止污染。</li>
	<li>在取下所有盖片并放入单独的管中后，丢弃 24 井板。</li>
	<li>返回到长凳上，每管漩涡5-10秒。</li>
	<li>从每个管中取出一部分，将每个样品放在 96 井板的单独井中，然后使用 1:10 稀释到 1:1，000 稀释连续稀释每个样品。</li>
	<li>使用预热和干燥的LB板，将稀释系列的每个点板点到 agar 板上。多通道管道可用于从每个样品的稀释系列中板 5-10 μl 点。</li>
	<li>此外，从每个未稀释的油井中去除 20 个 μl 样本，并将此量直接点到稀释系列中单独的板上的 LB agar 上。（此较大的体积可用于列举侵入性差的菌株）。</li>
	<li>所有样品都经过现场镀后，丢弃含有盖片的 14 毫升管。将 96 井板进行寄生膜，并在 -80 °C 的冰柜中放置，以在必要时重新镀板。</li>
	<li>让斑点完全干燥。通过将板放在引擎盖上并拆下盖子，这一过程大大加快了。</li>
	<li>斑点干燥后，将盘子放在37°C的孵化器中过夜。</li>
	<li>第二天早上，计算稀释系列中每个点的菌落数量，以便轻松评估殖民地数量（大约在 5 到 50 个殖民地之间）（图 1），并使用该系列计算评估的每株菌株的细菌数量。</li>
</ol>6. 为小鼠感染准备 L.单细胞基因 应变
<ol><li>感染前一天晚上，使用消毒循环去除所需的菌株的单菌落，并将每个菌株接种到含有2毫升BHI汤的14毫升管中。</li>
	<li>将接种的管子放入 37 °C 的孵化器中，在 45° 下倾斜，并在一夜之间静态地（不摇晃）孵化。</li>
	<li>第二天早上，从孵化器和漩涡中取出培养管，以确保细菌的均匀悬浮。</li>
	<li>使用 600 nm 波长的光谱仪测量培养的光学密度。在阅读光学密度之前，请务必使用无菌 BHI 汤将光谱仪归零。（对于 L. 单细胞基因，光学密度与每毫升细菌的浓度直接相关，因此密度为 1.000 = 1.0 x 109 CFU 每毫升）</li>
	<li>计算感染每种动物所需的培养量。注射通常含有 200 μl 的 PBS，其中含有 10，000 CFU 的单独菌株，这意味着所需的浓度为 5.00 x 104 CFU/ml。</li>
	<li>删除每个最终稀释的 5μl 别名，并直接将其铺在 LB agar 上。 在 37 °C 的夜间孵化，以确认用于接种的浓度。</li>
	<li>在稀释后 1 小时内注射每个 CFU 悬架（见下文）。</li>
</ol>7. 通过尾静脉注射和评估肝脏、脾脏和心脏内的细菌负担，将 L.单细胞基因 接种到小鼠体内
注:所有动物工作均按照疾病预防控制中心生物安全2级指南进行。 老鼠通常提前一周订购，每只笼子有5只动物被关在笼子里。在注射前，小鼠可以适应新的实验室环境四天。这些实验使用了6-8周大的瑞士-韦伯斯特雌性小鼠，这些老鼠被关在笼子里，在屏障环境中喂养非限制饮食。
<ol><li>通过取下盖子和食物/水箱，将笼子放在热灯下五分钟，一次准备一个老鼠笼。这个过程允许尾静脉扩张，使注射更容易执行。</li>
	<li>取出一只动物，并将其放在线束（或猎鹰管与结束切断），以抑制动物在注射期间。</li>
	<li>使用酒精垫清洁注射部位，让酒精蒸发。这不仅清洁了场地，还进一步扩张了尾静脉。</li>
	<li>使用装有 27.5 号针头的 1 毫升注射器，将需要培养的 200μl 轻轻注射到尾静脉中。</li>
	<li>使用纱布停止注射后可能出现的任何出血，并将动物放在一个新的笼子里。</li>
	<li>重复这个过程为剩余的动物和菌株。一定要给每个动物笼贴上他们收到的菌株的标签。</li>
	<li>每天检查动物，清除和牺牲任何似乎生病的动物。 如果没有动物表现出疾病的迹象，允许感染进行72小时，然后再牺牲所有动物。</li>
	<li>为了牺牲，使用瓶装源的二氧化碳 麻醉，直到所有呼吸停止，然后使用颈椎脱位，以确保动物死亡。</li>
	<li>牺牲后，将动物移到组织培养罩进行解剖。</li>
	<li>使用解剖块，用大尺针固定动物的每一条腿，并用70%的乙醇喷洒动物，直到它饱和。</li>
	<li>使用从阴道开口到 xiphoid 过程的 Y 形切口将腹部和胸腔的皮肤切回，然后进展到每只手臂的轴突。</li>
	<li>拉回皮肤，从每条腿上取出针头，以保持解剖打开。</li>
	<li>轻轻地切开腹膜，露出肠子、胃、肝脏和脾脏。</li>
	<li>首先切开肝脏顶部的肝静脉和冠状韧带，开始切除肝脏。肝脏下面还有其他韧带需要切除，将其连接到小肠的第一部分，以及背部的肌肉。</li>
	<li>将肝脏放在5毫升无菌ddH20的50毫升猎鹰管中。</li>
	<li>接下来，通过隔离来切除脾脏，并轻轻地切断血管和韧带。脾脏比肝脏更容易去除。将脾脏放在一个单独的猎鹰管中，内含 5 毫升无菌 ddH20。</li>
	<li>定位隔膜，沿着肋骨轻轻切割，以可视化胸腔。切开西福德过程和胸骨到颈部的水平，以打开胸腔，暴露心脏和肺部。</li>
	<li>使用钳子，轻轻地抓住心脏的顶点，并解除它，使主动脉和肺血管处于紧张状态。切这些血管来释放心脏。</li>
	<li>将心脏放入含有 5 毫升无菌 ddH2 0的 50 毫升猎鹰管中。</li>
	<li>丢弃小鼠尸体，并重复这个过程为每一个动物，使用干净的猎鹰管含有5毫升无菌ddH20为每个器官删除。</li>
	<li>所有器官被切除后，清洁工作站并返回工作台。</li>
	<li>准备一套5管，用于清洁和消毒每组器官的同质化剂从5只小鼠（即 一个5管设置5个肝脏，5管5个脾脏，5管5心脏）。每个管子中应填充 30 毫升无菌ddH 20，其中 1 个管应填充 30 毫升 90% EtOH。</li>
	<li>使用组织大师（或等效同质化剂），将同质化剂（同时运行）浸入步骤 7.21 中准备的管中，以清洁探头:</li>
	<li>管 1 （ddH2O） 5 秒在管 2 （ddh2O） 5 秒在地铁 3 （埃托赫） 5 秒在管 4 （ddh2O） 5 秒在管 5 （ddh2O）</li>
	<li>使用同质化器使一个肝脏同质化至少2分钟，或直到器官没有可见的部分保留。使用钳子清除探头中的任何大碎片。</li>
	<li>重复步骤 7.22 中描述的清洁过程</li>
	<li>重复其他肝脏的均质化过程，确保使用步骤 7.22 中描述的过程洗涤每个肝脏之间的探头。</li>
	<li>在所有的肝脏完全均质化后，丢弃肝脏的洗涤管（1-5）。</li>
	<li>重复脾脏和心脏的均质/清洁过程，确保为每个系列的器官使用一套新的洗涤管。如果洗涤管在任何时候变得浑浊，丢弃它们，代之以新的管子来完成该系列。</li>
	<li>在所有的器官都均质化后，对同质化器进行最后一次清洁循环，并将其干燥以储存。</li>
	<li>将每个器官的约 200μl 放入 96 井板的单独井中。</li>
	<li>通过稀释 1:10 系列，以 1:10，000 稀释（肝脏和脾脏）或高达 1:1，000 稀释（心脏），对器官样本进行连续稀释。</li>
	<li>将稀释系列点板放在预热的LB agar上。还将每个未稀释的器官和板的 20 个 ul 移到单独的 LB agar 板上，以便从殖民性较差的器官中列举 CFU。</li>
	<li>丢弃含有同质器官的猎鹰管，用半膜包裹含有稀释系列的96井板，并将其放在-80°C的冰柜盒中。</li>
	<li>让斑点干燥。通过将板放在引擎盖中并拆下盖子，可以加快这一过程。</li>
	<li>斑点干燥后，将盘子放在37°C的孵化器中过夜。</li>
	<li>计算第二天早上每个地点的菌落数量，并使用稀释系列计算每个器官的细菌总数。</li>
</ol>

<ol>
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作者报告没有相互竞争的经济利益。

L. 单细胞基因 是一种广泛且特征良好的人类病原体，能够引起多种不同的疾病表现。此前，该细菌曾被描述为能够跨越障碍物（如血脑屏障和胎盘-胎儿屏障）进行迁移，以便分别到达和殖民中枢神经系统并发育胎儿。生物体 的体内 殖民能力通常辅之以 体外 入侵构成目标器官的培养中具有代表性的细胞的能力。例如，胆囊丛中上皮细胞的入侵与生物体殖民CNS16</su...

李斯特菌单细胞基因是一种革兰阳性细胞内病原体，可在易感人群中引起严重疾病，包括老年人、孕妇、艾滋病毒/艾滋病感染者和接受化疗1的人。这些人群的感染往往是摄入受污染的食品造成的，大多数感染都与2，3的大规模食源性疫情有关。在人类和其他哺乳动物中，L.单细胞基因能够跨越小肠的上皮边界，然后被输送到肝脏4，5。动物模型表明，摄入的细菌在肠道内复制，并通过门户静脉传播到肝脏，或通过间质淋巴结扩散到血液中，导致血液向肝脏和脾脏传播6，7。在肝脏和脾脏中，细菌能够介入专业噬菌体和常驻帕丘奇细胞，并迅速在这些器官内建立感染。随着细菌负荷的增加，许多细菌被分散回血液中，在那里它们能够进一步殖民易感组织，包括中枢神经系统和胎盘（在目前）。这些部位的殖民化排除了人类李斯特菌病的最常见表现，包括脑膜炎、脑炎和胎儿流产2。
选定的L.单细胞基因亚种最近被证明具有增强的能力，入侵和复制心脏组织8。心脏介入的表现是多种多样的，范围从心内膜炎和心包炎，到完全由传导异常9-13完成的足部心肌炎。每年L.单细胞基因心脏病例总数较低，但估计可能不足，因为这种感染方面没有得到普遍认可。通过病原体殖民化心脏通常需要宿主倾向，如预先存在的血管损伤或人工心脏瓣膜。然而，已经发现的L.单细胞基因的分离物，是值得注意的能力殖民感染动物的心脏，没有任何心脏损伤和/或异常8。
本文介绍了 体外 和 体内 方法，用于评估受感染动物心脏组织中的细菌殖民化，使用组织培养和活体动物感染的入侵检测。这些方法不仅有助于识别有可能在受感染动物中殖民心脏组织的菌株，而且应该有助于识别细菌基因产品，这些基因产品有助于增强心脏细胞入侵和/或导致心脏病理学的变化。这些方法有助于比较多种菌株之间的心肌梗塞。对于此处描述的方法 ，L. 单细胞基因 10403S 用作非心电应变的经过充分研究的代表，临床隔离 07PF0776 用作心电应变的代表性示例。这两种菌株的选择为细菌入侵心脏细胞 体外 和殖民化受感染的小鼠的心脏 在体内提供了比较。分离07PF0776是一种临床隔离物，从导致艾滋病毒患者8致命心律失常的心室间脓肿中恢复过来。 L. 单细胞基因 分离物的毒性潜力可能有所不同，鉴于 李斯特菌 感染免疫抑制者和孕妇的倾向，这些人群中的人在评估不同的临床隔离物时应谨慎行事。

<table><tbody><tr><td>Difco</td><td>BHI Broth Base</td><td>237200</td><td>2 kg of Brain Heart Infusion powder (without agar)</td></tr><tr><td>Difco</td><td>Agar</td><td>214510</td><td>2 kg of Granulated Agar</td></tr><tr><td>Difco</td><td>BHI Agar</td><td>241810</td><td>2 kg of Brain Heart Infusion Powder with 7.5% Agar</td></tr><tr><td>in vitrogen</td><td>LB Broth Base</td><td>12975-084</td><td>2 kg of Luria Broth Base Powder (without agar)</td></tr><tr><td>Fisher</td><td>Glass Coverslips</td><td>12-545-80</td><td>12 mm glass coverslips</td></tr><tr><td>Falcon</td><td>24-well Tissue Culture Plate</td><td>35-3047</td><td>24 well, Plasma-treated Tissue Culture Plate</td></tr><tr><td>Falcon</td><td>14mL Polystyrene tube</td><td>352051</td><td>14 ml Polystyrene tubes</td></tr><tr><td>Falcon</td><td>96-well U-bottom plate</td><td>35-3077</td><td>Non-tissue culture treated 96-well plate</td></tr><tr><td>Corning</td><td>0.05% Trypsin, 0.53 mM EDTA (1X)</td><td>25-052-CI</td><td>Stock solution of Trypsin EDTA for tissue culture</td></tr><tr><td>Corning</td><td>DMEM (High glucose, high pyruvate)</td><td>15-013-CU</td><td>DMEM media without FBS, glutamine, or antibiotics</td></tr><tr><td>Corning</td><td>Pen/Strep/Glut Solution</td><td>30-009-CI</td><td>Stock mixture of penicillin, streptomycin, and L-glutamine for tissue culture</td></tr><tr><td>Corning</td><td>Gentamicin</td><td>30-005-CR</td><td>50 mg/ml Stock solution of Gentamicin for tissue culture</td></tr><tr><td>Cellgro (Corning)</td><td>L-Glutamine</td><td>25005197</td><td>Stock L-glutamine solution without antibiotics</td></tr><tr><td>Denville</td><td>75cm^2 Flask</td><td>T1225</td><td>75 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture treated flask</td></tr><tr><td>Denville</td><td>1.5mL microcentrifuge tubes</td><td>2013004</td><td>1.5 ml microcentrifuge tubes</td></tr><tr><td>ATCC</td><td>H9c2 Cardiac Myoblasts</td><td>CRL-1446</td><td>Rat cardiac myoblasts</td></tr><tr><td>Hyclone</td><td>Fetal Bovine Serum</td><td>SH30070.63</td><td>Fetal Bovine Serum</td></tr></tbody></table>

assessing bacterial invasion of cardiac cells in culture and heart colonization in infected mice using listeria monocytogenes

李斯特菌单细胞基因 是一种革兰氏阳性的细胞内病原体，能够引起免疫功能低下患者、老年人和孕妇的严重侵入性感染。 人类李斯特菌病最常见的表现包括脑膜炎、脑炎和胎儿流产。 侵入性 L.单细胞基因 感染的重要但记录较少的后遗症涉及心脏。 尽管治疗，心脏病的死亡率可能高达35%，但是对于心脏组织的 L.单细胞基因 殖民化及其由此产生的病理，人们所知甚少。 此外，最近很明显 ，L.单细胞基因 的亚群具有增强的能力，入侵和生长在心脏组织内。 该协议详细描述了 体外 和 体内 可用于评估 L.单细胞基因 分离心肌梗塞的方法。 介绍了在组织培养中感染H9c2大鼠心脏肌细胞的方法，以及确定受感染小鼠心脏的细菌殖民化。 这些方法不仅可用于识别有可能在受感染动物中殖民心脏组织的菌株，而且还可能促进细菌基因产品的识别，这些基因产品有助于增强心脏细胞入侵和/或推动心脏病理学的变化。 这些方法还规定了多个 L.单细胞基因 菌株之间的心电图谱的直接比较。

选定的 单细胞基因 分离物在细胞培养和小鼠感染模型中表现出对心脏细胞的增强入侵。 图1 显示了在阿加介质上当场电镀悬浮后细菌菌落可能如何出现的示例。此方法允许在样品中准确评估 CF，而无需使用大量 agar 介质板。 图2 显示了一个基于组织培养的检测示例，该分析将10403S菌株入侵心脏细胞的能力与菌株07PF0776的能力进行了比较。与感染10403S的细胞相比，?...

Watch this Scientific Journal Video about Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes at JoVE.com

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

李斯特菌单细胞基因 导致孕妇胎儿感染和易感人群脑膜炎。 细菌的亚种群可以殖民心脏组织，导致患者和实验室动物心肌炎。 在这里，我们提出了一个协议，描述了如何评估 L.单细胞基因 心脏细胞 入侵体外 和心脏殖民化感染动物。

使用李斯特菌单细胞基因评估受感染小鼠培养和心脏殖民化中心脏细胞的细菌入侵

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

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Immunology and Infection

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

5.4K 次观看.  College of Medicine, University of Illinois at Chicago. 李斯特菌单细胞基因 导致孕妇胎儿感染和易感人群脑膜炎。 细菌的亚种群可以殖民心脏组织，导致患者和实验室动物心肌炎。 在这里，我们提出了一个协议，描述了如何评估 L.单细胞基因 心脏细胞 入侵体外 和心脏殖民化感染动物。 

视频: Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

科研

免疫与感染

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

口头传播的<em&gt;单核细胞增生李斯特氏菌</em小鼠通过食入被污染的食物

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

成像InlC的分泌由细菌病原体调查感染蜂窝<em&gt;单核细胞增生李斯特氏菌</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

实验性感染<em&gt;李斯特菌</em&gt;作为研究主机干扰素γ响应模型

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled microscopic lesions (microlesions) that are remarkable due to the absence of infiltrating immune cells. Due to their release of cardiotoxic products, S. pneumoniae within microlesions are thought to contribute to the heart failure that is frequently observed during fulminate invasive pneumococcal disease in adults. Herein is demonstrated a protocol for experimental mouse infection that leads to reproducible cardiac microlesion formation within 30 hr. Instruction is provided on microlesion identification in hematoxylin &#38; eosin stained heart sections and the morphological distinctions between early and late microlesions are highlighted. Instruction is provided on a protocol for verification of S. pneumoniae within microlesions using antibodies against pneumococcal capsular polysaccharide and immunofluorescent microscopy. Last, a protocol for antibiotic intervention that rescues infected mice and for the detection and assessment of scar formation in the hearts of convalescent mice is provided. Together, these protocols will facilitate the investigation of the molecular mechanisms underlying pneumococcal cardiac invasion, cardiomyocyte death, cardiac remodeling as a result of exposure to S. pneumoniae, and the immune response to the pneumococci in the heart.

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Streptococcus pneumoniae forms discrete non-purulent microscopic lesions in the heart. Outlined is the protocol for a murine model of cardiac microlesion formation. Instruction is provided on microlesion visualization using microscopy, discrimination between early and late microlesions, and methods to detect cardiac remodeling in hearts of convalescent animals.

可视化<em&gt;肺炎链球菌</em&gt;在心脏Microlesions和随后的心脏重构

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled ...

医学

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.

李斯特菌脑部感染

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

细菌病原体在细胞和有机水平宿主反应探针中的应用

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

A Technique to Establish a Bacterial Infection Model in an Ex Vivo Organ Culture

Source: Rizzuto, G. A., et al. <a href="https://app.jove.com/v/54237">Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface.</a> J. Vis. Exp. (2016)
This video demonstrates a method to establish an infection model in placental tissues to study infection propagation. An ex vivo placental organ culture is infected with Listeria monocytogenes, a facultative intracellular parasite. The bacteria invade extravillous trophoblasts (EVTs) to enter the placental tissue and then propagate to other cells via cell-to-cell spread.

一种在离体器官培养中建立细菌感染模型的技术

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

In Vitro Models

Quantification of Bacterial Infection in Host Cells by the Colony-Forming Unit Assay

Source: Gayle, P. et al., <a href="https://app.jove.com/v/58775">Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses.</a>&#160;J. Vis. Exp.&#160; (2019).
This video demonstrates an in vitro quantification technique to determine Listeria monocytogenes infection of macrophages by the colony-forming unit assay. The bacterial colonies on the agar plate represent the intracellular bacterial population that escapes the host-defense mechanism and survives within the macrophages.

通过集落形成单位测定定量宿主细胞中的细菌感染

Source: Gayle, P. et al., Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses.&#160;J. Vis. Exp.&#160; (2019).
This video ...

Immune Response

Host-Pathogen Interaction

Assessing Bacterial Colonization in a Mouse Brain following Listeria monocytogenes Infection

Source: Ghosh, P., et al., <a href="https://app.jove.com/v/58723">Listeria monocytogenes Infection of the Brain.</a> J. Vis. Exp. (2018)
This video demonstrates the quantification of the infective bacterial load from the perfused mouse brain infected with Listeria monocytogenes.

评估单核细胞增生李斯特菌感染后小鼠大脑中的细菌定植

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.&#160;
1. Organ Harvesting and ...

使用李斯特菌单细胞基因评估受感染小鼠培养和心脏殖民化中心脏细胞的细菌入侵

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测量细菌感染小鼠和免疫反应李斯特菌

口头传播的

成像InlC的分泌由细菌病原体调查感染蜂窝

实验性感染

可视化

李斯特菌脑部感染

细菌病原体在细胞和有机水平宿主反应探针中的应用

一种在离体器官培养中建立细菌感染模型的技术

通过集落形成单位测定定量宿主细胞中的细菌感染

评估单核细胞增生李斯特菌感染后小鼠大脑中的细菌定植