We thank our fellow Lee Lab colleagues, who provided valuable insight and expertise that greatly assisted this research. This work was supported by the Young Innovator Award from the National Institute of Health, awarded to S.W. Lee (NIH-1DP2OD008468-01). R.A. Flaherty is supported by the Albertus Magnus Fellowship provided by Dr. Robert C. Boguslaski, and a teaching assistantship through the Eck Institute for Global Health at the University of Notre Dame.

1.细菌培养<ol><li>产生等基因突变株用于感兴趣21的具体分泌因子的研究。 注意:这些实验包括使用的GASM1T1 5448株 WT 气"， 佐贺 Δ 猫 SLS缺陷的突变（简称文本为ΔSAGA）和SAGA补充株（简称文本为ΔSAGA SAGA +）21。 </li><li>制备感兴趣的细菌菌株的过夜液体培养物。生长气体M1T1在-10毫升的Todd-Hewitt肉汤的5448菌株在37℃下16-20小时感染人体细胞之前。 </li><li>隔夜离心细菌培养（2 400 RCF 10分钟），并悬浮在新鲜的细菌培养基。 注意:在相同的体积重悬的过夜培养物在（5-10毫升）生长通常产生期望初始光密度。 </li><li>规范化的细菌培养，以通过在600nm（OD 600）稀释在附加细菌培养基中再悬浮培养物，直至相同的光密度等于起始浓度获得用于所有菌株进行测试（OD的大约1.0 600被推荐为方便起见）。 注意:在这些研究中，固定相细菌培养物用于感染宿主细胞立即正常化以下。然而，如细菌出口固定相的一些菌株nonsynchronously它可能更合适，如果这被发现是对所关注的菌株的情况下，开始与对数期培养物宿主的感染。 </li><li>之前进一步分析，执行一个菌落计数测定以确定在起始培养物存在的每毫升菌落形成单位（CFU），使感染复数（MOI），或每宿主细胞的细菌的比率，可以被确定。 <ol><li>制备1毫升已归一化到所需的光学DENSI细菌培养物的系列稀释液在磷酸盐缓冲盐水（PBS）或适当的细菌生长培养基（的Todd-Hewitt肉汤在这些研究中使用）TY。制备稀释从10 -1至10 -6，以产生至少一组与可数菌落（30-300集落）琼脂平板。一式三份进行所有稀释。 </li><li>转移100μl的各系列稀释的到被分析含有用于细菌种类适当培养基的琼脂平板。 注意:在这些研究中，使用的Todd-休伊特琼脂。 </li><li>使用玻璃或塑料细菌吊具均匀分布的100微升等分试样在整个琼脂板的整个表面上，并且继续传播，直到液体已经被吸收到琼脂。使用无菌技术下进行的火焰。 </li><li>孵育在37℃的琼脂平板过夜，或直到可数的菌落出现。 </li><li>计数生长在各平板上的克隆和由以下formu计算在原始培养物中的CFU / ml的LA:菌落数×镀毫升连续稀释/培养体积成反比。 例如:（200个菌落点¯x1/10 -5稀释）/ 0.1毫升镀= 2.0×108 CFU /毫升</li></ol></li></ol> 2.宿主细胞培养<ol><li>获得用于所需的分析合适的宿主细胞系。 注:角质形成人体上皮角质月29日 ，从五Nizet一种礼物，被用于这些研究。 <ol><li>维持在100mM培养皿HaCaT细胞在Dulbecco改进的Eagle培养基（DMEM）和10％热灭活的胎牛血清（FBS）。孵育在37℃，5％的CO 2。 </li></ol></li></ol> 3.宿主细胞感染渗透膜插入系统<ol><li>板细胞在合适的组织培养皿中，让他们成长，直到他们达到90％汇合。 注意:在这些研究中通常使用2〜3天之内到达汇合的HaCaT细胞电镀后AYS。 <ol><li>对于溶胞产物收集（ 例如信令分析和三磷酸腺苷（ATP）的测定测定），板HaCaT细胞在6孔培养皿中以大约3×10 5个细胞的接种密度/孔。 </li><li>对于免疫荧光成像实验，板的细胞上在6孔培养皿中无菌盖玻片在约3×10 5个细胞的接种密度/孔。 </li><li>为乙锭同型二聚体膜通透性和乳酸脱氢酶（LDH）释放测定法，镀HaCaT细胞在24孔培养皿中以大约5×10 4个细胞的接种密度/孔。 </li></ol></li><li>紧接在治疗之前，洗涤细胞以1×无菌PBS。 </li><li>应用新鲜生长培养基细胞。对于这里所描述的条件下，应用每阱2毫升培养基为6孔板或每孔0.5毫升介质24孔板中。如果药物治疗正在测试中，拌入新鲜培养基，这期间STE申请页。 注意:有些试验可能需要替代的媒体。例如，如酚红和高血清浓度与LDH释放测定干扰，无酚红的DMEM补充有1％牛血清白蛋白（BSA）（重量/体积），2mM的L-谷氨酰胺和1mM丙酮酸钠是这些实验利用。 </li><li>新鲜培养基已经施加之后，小心地在每一个无菌0.4微米透过膜插入井。执行在层流罩此步骤中，并使用无菌镊子对渗透膜插入物转移到每孔中。 </li><li>根据制造商的说明适用新鲜的细胞培养基中的每个孔中的上腔室。对于这里所描述的条件下，应用每孔1ml培养基6孔板或每孔0.1毫升介质24孔板中。 </li><li>申请归细菌培养物（见第1）至渗透膜的插入系统的上部腔室的适当体积。使用细菌培养基对照感染醇治疗。对于这里所描述的结果，添加25微升每室的归一化培养物的24孔板中，并加入100微升每室中培养的6孔板中。 注意:在这些研究中，野生型或突变体气体施加到宿主细胞 在10 MOI的MOI基于最终的真核细胞数，计算（ 例如 2×10 6个细胞/孔），使得均在感染期的开始（ 例如 ，2×每宿主细胞中加入10倍的细菌每孔10 7 CFU）。适当的MOI将与不同的细菌种类，并与所希望的后续分析而变化。 注意:除了使用细菌培养基未感染的对照，对于本技术的某些应用可以包括热灭活的细菌或用于比较花费细菌介质其他有用的控制。 </li><li>孵育感染的细胞在37℃，5％的CO 2。 </li></ol> 4.样品采集 &lt;OL&gt; <li>收集细胞培养基，宿主细胞裂解物，或玻璃盖玻片与完整细胞的后续分析中，开始通过仔细地除去用无菌镊子渗透膜插入。 </li><li>对于分泌主机Protiens的评估: <ol><li>收集来自下部腔室中的介质进入1.5毫升管中。避免收集过程中的干扰单层。 </li><li>在4℃下14000 RCF离心10分钟离心样品以除去细胞碎片。 </li><li>在-20°C删除所有，但50微升上清到一个新的1.5 ml管和储存或立即使用。 </li></ol></li><li>对于宿主细胞Lystates的评估: <ol><li>轻轻吸宿主细胞的单层以上的介质。避免打扰单层，因为这可能会导致样品损失。 </li><li>用1X PBS冲洗细胞一次。 </li><li>轻轻吸PBS。立即应用冰冷的裂解缓冲液的适当体积，以获得所需的蛋白质浓度，并孵育在冰上15分钟的样品。 200微升和350微升每每6孔板的裂解缓冲液之间适用以实现0.5和1.5毫克/毫升之间蛋白浓度。 注意:在这些研究中所用的裂解缓冲液的组成为去离子水，1％（V / V）的Nonidet P40替代，磷酸酶抑制剂混合物，和蛋白酶抑制剂混合物。具体的试剂列在材料和设备部分。 </li><li>使用细胞刮棒从每个的板面分离细胞并在每个的全部内容吸取井到1.5毫升管。 </li><li>离心样品在4℃14000 RCF离心20分钟。 </li><li>为了评估水溶性成分溶解产物，去除上清到一个新的管并储存在-20℃或立即使用。 </li><li>评估核或其他不溶性裂解物组分，保留沉淀并贮存在-20℃或立即使用。 </li></ol></li><li>通过免疫荧光成像技术评价: <ol><li>如海盗中，洗1X冷PBS细胞。 </li><li>过夜在4％低聚甲醛（PFA）溶液固定细胞在PBS（重量/体积）。 注意:在这些研究中，将1ml的PFA每每6孔板的孔中加入。 </li></ol></li></ol> 5.建议应用<ol><li>主机信号转导分析SDS-PAGE和Western印迹<ol><li>收集宿主细胞裂解物如4.3节所述。 </li><li>确定由二辛可宁酸（BCA）测定法或等效使用蛋白质标准30各样品裂解物的蛋白质浓度。 </li><li>标准化样品之间蛋白浓度之前加载和运行在4-15％聚丙烯酰胺凝胶或合适的替代31样本。 <ol><li>样品体积-通过从每个样品（ 例如 20微克）吹打相同蛋白量成新的1.5毫升管并加入裂解缓冲液的可变体积（缓冲量=总体积正常化样品浓度加）到各管中，使总体积（ 例如 50微升）和最终蛋白质浓度相当于整个样品。 </li></ol></li><li>转移样品到一个聚偏二氟乙烯（PVDF）膜（或等效物）。对于这里描述的结果，增加电压至70伏为额外的45分钟，然后在25伏转移样品2小时。使用200mM的Tris碱，1.5M甘氨酸和去离子水的20％甲醇（体积/体积）组成的转移缓冲液。 </li><li>在5％BSA + 0.1％吐温20的Tris块膜缓冲盐水（TBS）。相应地调整根据制造商的建议，将要使用的抗体。 </li><li>孵育初级抗体的膜在4℃下过夜。在使用厂家推荐的稀释。 </li><li>洗膜在TBS + 0.1％吐温20 1.5小时;刷新缓冲每次10-15分钟。 </li><li>与辣根过氧化物酶（HRP）缀合的孵育二级抗体以1:1的稀释度:5000，在室温TEMPERAT 1.5小时URE。 </li><li>洗膜在TBS + 0.1％吐温20 1.5小时;刷新缓存每10 - 15分钟。 </li><li>根据制造商的说明在胶片显影前孵育化学发光检测试剂膜。如需要，可以使用其它的检测方法。 </li></ol></li><li>宿主细胞免疫荧光染色和成像<ol><li>如第4.4节所述的修复含有宿主细胞盖玻片。 </li><li>除去PFA溶液和洗涤含有处理的细胞两次在PBS盖玻片，洗涤之间进行抽吸。 注:PFA是剧毒的，必须适当处置。 </li><li>用1％阻断在PBS中室温下搅拌2小时的盖玻片（重量/体积）正常山羊血清，2％（V / V）的Triton X-100和0.5％（体积/体积）吐温20。 </li><li>用PBS洗涤盖玻片30分钟，刷新缓冲每10分钟。 </li><li>孵育初级抗体的盖玻片在1:50比率（或由制造商推荐的）在阻断溶胶ution过夜，在4℃。 </li><li>洗盖玻片在PBS 1.5小时，刷新缓冲区每隔30分钟。 </li><li>孵育盖玻片在二级抗体室温2小时用1（山羊抗兔IgG AlexaFluor488被用于这些研究）:抗体的阻断溶液200比。 </li><li>洗盖玻片1小时，刷新缓冲区每隔20分钟。 </li><li>应用所需的污渍。 注意:在封闭溶液1000:利用DAPI（核染色）中，在1比罗丹明 - 鬼笔环肽（肌动蛋白染色）这些研究。 </li><li>孵育核和肌动蛋白污渍盖玻片在室温下30分钟。 </li><li>洗盖玻片在PBS，在室温下30分钟，刷新缓冲每10分钟。 </li><li>安装在使用安装介质载玻片盖玻片。 </li><li>允许盖玻片前一夜密封和成像设置幻灯片。对于这里描述的结果，在一个荧光显微镜USI采集图像纳克一个标准的20倍的目标。使用ImageJ的和显微镜的成像软件来处理拍摄的图像。 </li></ol></li><li>乙锭同型二聚体细胞死亡含量<ol><li>从每个孔洗细胞两次用无菌PBS吸介质。 </li><li>在PBS申请4μM的乙锭同型二聚体-1。 </li><li>在室温下孵育细胞，在黑暗中30分钟。 </li><li>确定用酶标仪设定为528纳米激发和617与590纳米的截止值nm发射的荧光的程度。 </li><li>加0.1％（重量/体积）皂苷对每个孔之后的初始读数，并允许读出板的第二时间在相同的设置前板孵育在室温下（与摇动）一个额外的20分钟。 </li><li>由第二荧光读数（后皂素）除以初始荧光读数（后处理）单独确定百分之膜通透为每个孔中。 </li></ol></li><li> ATP含量测定<ol><li&gt;如上述第4.3节收集裂解液。 </li><li>通过BCA测定或类似30正常化的裂解物的蛋白质水平。 </li><li>确定使用基于发光的ATP的测定试剂盒，或根据制造商的说明等效细胞ATP水平。 </li><li>正常化处理值，以相应的未感染的对照样品。 </li></ol></li><li> LDH释放分析<ol><li>感染后，收集上清如4.3节所述。 </li><li>根据制造商的说明使用细胞毒性检测试剂盒为LDH释放评价LDH释放。 </li></ol></li></ol>

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The authors have nothing to disclose.

本文中描述了利用渗透膜基于插入物感染系统研究的在体外系统人类上皮角质化细胞的细菌毒素链球菌溶血素S的影响的方法。这个协议可以适用于其他细菌分泌的毒力因子的研究，以及替代宿主细胞类型。这家新近开发的系统提供了超过，利用纯化毒素或细菌过滤上清液1实验方法几个优点- 3,8,18 - 20。可渗透基于插入膜系统提供有关细菌毒素，因为它产生对宿主细胞，其允许维持最大毒素活性的保持恒定的剂量也实验之间增加的一致性。此外，该系统更紧密地通过允许分泌因子随时间累积的感染的进展和由ELIMI模拟生理条件内廷需要任意地选择具体的毒素浓度以适用于宿主细胞。此外，该系统也将提供装置，用于调查，以评估所关注的细菌因子是否被递送到宿主细胞在接触依赖方式。接触依赖性通常是通过在加入或通过使用该防止附着的试剂的缺陷等基因的细菌的突变体的产生进行测试。这里所描述的系统将提供一个简单的替代来补充或替代这些传统的方法。 除了在该协议的部分5中所述的测试的应用程序，其它应用到这种感染系统可以很容易地适应包括用于细胞因子阵列和ELISA测定的样品的集合。在这两种情况下，一个类似的协议，对于LDH释放测定法描述可以跟随。虽然这里未示出，该系统已被​​用于有效地收集宿主细胞样品是一个通过流式细胞仪nalyzed。在本申请中，渗透膜插入件是继感染期间除去，将细胞洗涤以除去细胞培养基，并胰蛋白酶被施加以使细胞进行分析的汇编。细菌从宿主细胞的物理分离是这种应用是特别有用的，因为它有助于防止由附着的细菌和宿主细胞中，可以以其他方式与精确细胞计数通过流式细胞仪干涉的大的聚集体的形成。 虽然比较与传统的直接感染研究渗透膜基于插入物感染的研究的结果时，很一致宿主反应已经观察到，在这些宿主反应的动力学一些显着的差异已经观察到21。例如，在宿主信令和膜 - 基于细胞毒性的变化在渗透膜基于插入物感染模型比心病发生取30-50％更长响应直接感染模型。因为渗透膜基于嵌件的系统需要介质要施加到每个上部和下部隔室以及，对于此处描述的研究开发的系统所需的总培养基体积增加了30％，每孔相比相应的先前使用的直接感染模型21。这种差异在总培养基体积，加上细菌与宿主细胞之间的距离增加的直接接触被禁止时，有可能会增加花费SLS通过介质扩散到达宿主细胞和引发所观察到的效果的时间。此外，可渗透基于插入膜系统删除许多气体毒力因子有可能向宿主细胞损害;没有在该系统中这些额外的因素也可能有助于在宿主细胞死亡和主机信号事件的开始相比直接感染21的延迟。这些应考虑的因素设计实验时，评估其他分泌的细菌成分。 找出最有意义的条件来测试对宿主细胞的分泌细菌因素的影响，检测的范围内的时间点的渗透膜以插入感染系统的每一个应用建议。在什么条件下感兴趣的特定致病因子最佳地生成（如对数期，静止期，响应于某些环境信号等 ），以便成功地观察其效果考虑是很重要的。在实验中重点是评估在宿主信号蛋白的变化，有必要选择，允许足够的时间用于SLS达到宿主细胞和以足够的量，以产生以诱导信令改变21的时间点。同时，在主机信令所需的更改的评估将之前SLS诱导膜通透性，因为这影响下进行，omplicates宿主细胞裂解物进行分析的集合。由SLS诱导的细胞死亡可在直接和渗透膜基于插入物感染模型被最佳观察到的报告信号事件21的开始后数小时。另外，在选择感兴趣的时间点，这也是重要的，以确保所研究的细菌是无法在研究期间穿透多孔插入物膜。在较长时间内生产某些细菌成分的可能破坏膜的完整性，并允许细菌的通道到下隔室。以确定这是否是在感兴趣的系统中的潜在问题，加以研究的细菌菌株可以在的范围内的时间点施加到可渗透的基于插入膜系统的上腔室。在每个时间点，插入件可以被小心地去除，并从底腔的介质可以被收集，并以标准菌落共同利用unting试验（参见1.5节）。如果没有菌落从在下部隔室中的细胞培养基形成，插入膜可以假定为有效地防止细菌的通道在所测试的时间点和细菌负荷。 另一个实验设计的组件，很可能需要优化的分泌细菌因子的有效的分析是感染（MOI）的多重性。的MOI是指每宿主细胞的细菌细胞的比率，并且由宿主细胞汇合感染的时间，由细菌菌落形成单位的（CFU）的数目，因此影响施加到细胞。在这些研究中，角质细胞细胞生长至90％汇合，这使得这些细胞形成用完整的紧密连接有凝聚力的单层。宿主细胞的生理组织的考虑周到进行研究是必要选择为感染实验的适当汇合。一旦appropr每孔被确定宿主细胞的莱特号码，合适的细菌的CFU可以计算基于该所需的MOI。在此处描述的研究，气体施加在10适当MOI的MOI对宿主细胞将与不同的细菌，并与所希望的后续分析而变化。低开始的MOI是典型地更生理学相关，并允许针对感兴趣的细菌因子积累足够慢在宿主细胞生存力的急剧变化之前捕获在宿主细胞中信号传导的细微变化是显而易见的。更高的MOI在许多研究中评估的细胞毒性使用的，但这种效果也可以通过用低的MOI开始并允许感染的时间更长的时间取得的进展。重要的是要确定一个准确的CFU至光密度推论为要测试的所有菌株，如同基因的突变体可以比野生型的细菌具有改变的生长速率，因此可能需要更高或更低的CFU每加到很好地确保菌株之间的宿主反应的一个合适的比较。在所研究的哺乳动物宿主细胞能够杀死细菌或抑制其生长，或者如果细菌菌株之间非同步生长被怀疑，它也可以是执行的研究在感染期的结束，以评估最终细菌负荷有用的情况。这可以通过收集可渗透插入件的内容，并执行类似于在该过程的第1.5节中所述的菌落计数测定来完成。 选择适当尺寸的孔中所需测定也用于获得最佳的结果很重要。可渗透膜插入在多种尺寸可用，但使用专为24孔和6孔组织培养板的插入，观察这里示出的研究中最一致的结果。这些刀片尺寸相对容易无菌镊子处理和易于操控的是预防的关键ING从上部隔室到井的下部隔室的细菌不需要的传输。对于涉及宿主细胞裂解物的收集实验中，6孔培养皿中提供每种条件对大多数分析适当的细胞数，并足够大以容纳样品收集细胞刮刀。对于细胞毒性试验，其中宿主细胞保持粘附，并贴有可在读板器（ 例如 ，乙锭同型二聚体测定法，台盼蓝排除法）测量荧光或比色染料，使用24孔板与相应的插入件是推荐的。对于其中细胞培养基将被收集和分析实验（ 如 LDH释放，细胞因子的研究）任一的24孔或6孔平板，可以使用，虽然小的孔一般为这些分析提供足够的样品体积，并尽量减少使用所需的试剂。总体而言，该方法是高度灵活的，并根据需要，制备SAMPL可适于ES众多后续的应用程序。

了解细菌毒力因子的作用在宿主细胞感染的背景是细菌发病机制研究的主要焦点。许多细菌病原体积极分泌毒素和其它可溶性因子在宿主组织的破坏，以帮助，以引发在宿主细胞中的信号的变化或感染1的过程中，操纵免疫系统反应- 10。虽然方法已经发展成功纯化和产生许多这些重要的致病因素的研究，某些细菌产物具有独特的结构或广泛的翻译后修饰，使对纯化方法他们顽抗候选人，因而不能孤立使用体外系统来研究。例如，A群链球菌，细菌病原体负责的感染范围从咽炎坏死性筋膜炎和中毒性休克综合征无数，产生分泌细菌毒素称为链球菌溶血素S（SLS）11 - 17。此核糖体产生的肽是由链球菌溶血素S相关基因（SAG）簇编码，且成熟的产品，估计为2.7 kDa的大小14 - 17。前毒素，通过SAGA编码，是由几个酶（SAGB，SagC和SAGD）翻译后修饰产生成熟的，功能性的形式15 - 17。在加上毒素的不寻常的氨基酸序列，这些翻译后修饰的复杂性已使该毒素与已尝试迄今15,17所有纯化和结构鉴定的努力不兼容。这些挑战复杂的努力，以确定这种毒素在宿主发病机制中的具体作用。 在的情况下的纯化的毒素或其他分泌的因子的制备或者是复杂的或不可能的，机制，以阐明这些产品的功能传统上一直通过过滤细菌的上清液，然后将其施加到宿主细胞18的制备研究- 20。有与该技术相关联的一些挑战。首先，存储和应用在稍后的时间对宿主细胞时许多这些分泌的因子，包括SLS，不保持最大或一致的活性。此外，当上清液在单一时间点被收集，然后应用到宿主细胞，它是难以确定的生理相关性，得出关于自然感染过程，其中分泌因子允许感染的过程中一个过程中积累的直接结论生理浓度。这第二个挑战不仅适用于使用细菌上清液的，而且要在宿主细胞中的研究1使用的纯化的毒素- 3,8。为了解决这些问题，可透膜樱雪基于RT-感染系统已经开发，以评估在维持最佳毒素活性和也消除细菌和宿主细胞之间的直接接触的可变的方式SLS对宿主细胞的影响。在这个系统中，人上皮细胞生长在一个单层中的两腔室的底部室中井，和细菌被引入到同一井的上部腔室。的多孔膜（0.4微米孔）分隔的上，下腔室，从而使两个室之间交换分泌因子但是防止细菌的通道。该系统允许的宿主反应是仅仅是由于持续分泌的细菌成分，同时消除了通过A组链球菌感染过程中直接接触任何可能发生的反应有效的评价。虽然除了SLS其他分泌细菌因素也可以通过多孔膜，使用的等基因突变型面板，包括野生型（WT）中，SLS-千牛ockout（ΔsagA）和SAGA补充株（ΔsagA+ SAGA）允许对宿主反应是严格SLS-21依赖性精确的评估。 虽然类似渗透膜插入系统已被用于参与病毒感染分泌的因子，肿瘤生物学和免疫细胞迁移22的研究- 26，一些研究涉及与宿主细胞的细菌的相互作用已经利用这种方法6,27,28。即使已采用这种系统，以探索细菌与宿主细胞之间的相互作用的研究主要集中于炎症细胞或细菌迁移通过镀上渗透膜插入上皮或内皮细胞单层。本文所述的可渗透基于插入物膜感染系统是通过一个多孔膜，以评估EFFE依赖于细菌和宿主细胞的分离的简单而有效的方法分泌的毒素，SLS，对宿主细胞膜的完整性，细胞活力，细胞信号转导和分泌宿主细胞因子的克拉。这种技术可以适用于其他分泌的毒力因子在多种哺乳动物宿主细胞类型的研究，调查特定细菌因子的过度的感染过程中的作用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Todd-Hewitt broth</td>
			<td>Acumedia</td>
			<td>7161A</td>
			<td>Use appropriate broth for bacterial species of interest.</td>
		</tr>
		<tr>
			<td>BioSpectrometer</td>
			<td>Eppendorf</td>
			<td>basic model</td>
			<td>Any UV-Vis spectrophotometer is suitable.&#160;</td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td>Other brands are suitable.</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma</td>
			<td>A1296-1kg</td>
			<td>Other brands are suitable.</td>
		</tr>
		<tr>
			<td>HaCaT human epithelial keratinocytes</td>
			<td></td>
			<td></td>
			<td>Gift from lab of Victor Nizet.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Life Technologies</td>
			<td>11995-073</td>
			<td>Use appropriate media for cell line of interest.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Biowest</td>
			<td>S162H</td>
			<td>Use appropriate media additives for cell line of interest.</td>
		</tr>
		<tr>
			<td>10cm tissue culture dishes</td>
			<td>Nunc</td>
			<td>150350</td>
			<td>Other brands are suitable.</td>
		</tr>
		<tr>
			<td>6 well tissue culture dishes</td>
			<td>CytoOne</td>
			<td>cc7682-7506</td>
			<td>Other brands are suitable but need to be compatible with the Corning insert.</td>
		</tr>
		<tr>
			<td>24 well tissue culture dishes</td>
			<td>CytoOne</td>
			<td>cc7682-7524</td>
			<td>Other brands are suitable but need to be compatible with the Corning insert.</td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Fisherbrand</td>
			<td>12-541-B</td>
			<td>These must be autoclaved prior to use for cell plating.</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red Free DMEM</td>
			<td>Life Technologies</td>
			<td>31053028</td>
			<td>Phenol Red Free media is only required for the LDH release assay.</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Fisher Scientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030149</td>
			<td>Only required to supplement cell culture media for LDH release assay.</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>BP356100</td>
			<td>Only required to supplement cell culture media for LDH release assay.</td>
		</tr>
		<tr>
			<td>Collagen-coated 0.4&#956;m Transwell inserts (6 well)</td>
			<td>Corning</td>
			<td>3540</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen-coated 0.4&#956;m Transwell inserts (24 well)</td>
			<td>Corning</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher</td>
			<td>3120018</td>
			<td>These must be sterilized with ethanol prior to handeling Transwell inserts.</td>
		</tr>
		<tr>
			<td>Cell scrapers</td>
			<td>Fisherbrand</td>
			<td>08-100-241</td>
			<td>These are only required for the collection of cell lysates.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>04042-500</td>
			<td>TOXIC.&#160; This is only required for immunofluorescence imaging.</td>
		</tr>
		<tr>
			<td>Bicinchoninic acid (BCA) assay kit</td>
			<td>Pierce</td>
			<td>23227</td>
			<td>Other protein quantification assays may be used.</td>
		</tr>
		<tr>
			<td>Tris-glycine 4-15% polyacrylamide gels</td>
			<td>BioRad</td>
			<td>4561083</td>
			<td>Buffer system and percent polyacrylamide should be selected based on proteins of interest.</td>
		</tr>
		<tr>
			<td>Electrophoresis cassette supplies</td>
			<td>BioRad</td>
			<td>1658063</td>
			<td>Only required for Western Blotting.</td>
		</tr>
		<tr>
			<td>Electrophoresis power supply</td>
			<td>BioRad</td>
			<td>1645050</td>
			<td>Only required for Western Blotting.</td>
		</tr>
		<tr>
			<td>Western blot transfer cassette</td>
			<td>BioRad</td>
			<td></td>
			<td>Only required for Western Blotting.</td>
		</tr>
		<tr>
			<td>Polyvinylidene (PVDF) Membrane</td>
			<td>EMD Millipore</td>
			<td>IPVH00010</td>
			<td>Only required for Western Blotting.</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P1379-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit IgG-HRP secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc2030</td>
			<td>The secondary antibody should be determined based on the selected method of detection.&#160;</td>
		</tr>
		<tr>
			<td>Mouse IgG-HRP secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc2031</td>
			<td>The secondary antibody should be determined based on the selected method of detection.&#160;</td>
		</tr>
		<tr>
			<td>Phospho-p38 (T180+T182) MAPK antibody</td>
			<td>Cell Signaling</td>
			<td>4511</td>
			<td>Select appropriate primary antibodies based on proteins of interest.</td>
		</tr>
		<tr>
			<td>Total p38 MAPK antibody</td>
			<td>Cell Signaling</td>
			<td>8690</td>
			<td>Select appropriate primary antibodies based on proteins of interest.</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG Alexafluor488</td>
			<td>Molecular Probes</td>
			<td>A-11034</td>
			<td>Other secondary antibodies may be used for immunofluoresence imaging detection.</td>
		</tr>
		<tr>
			<td>LumiGLO Detection Reagent</td>
			<td>KPL</td>
			<td>54-61-00</td>
			<td>Only required for Western Blotting with detection on film.</td>
		</tr>
		<tr>
			<td>Detection film</td>
			<td>Biodot</td>
			<td>BDB57</td>
			<td>Only required for Western Blotting with detection on film.</td>
		</tr>
		<tr>
			<td>DeltaVision Nikon 90i fluoresence microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Other fluorescence microscopes are suitable for these analyses.</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Thermo Scientific</td>
			<td>31873</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T9284-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI nuclear stain</td>
			<td>Cell Signaling</td>
			<td>4083</td>
			<td>Select stains based on specific immunofluorescence applications of interest.</td>
		</tr>
		<tr>
			<td>Rhodamine-phalloidin actin stain</td>
			<td>Molecular Probes</td>
			<td>R415</td>
			<td>Select stains based on specific immunofluorescence applications of interest.</td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>Only required for immunofluorescence imaging.</td>
		</tr>
		<tr>
			<td>Ethidium homodimer 1</td>
			<td>Molecular Probes</td>
			<td>E1169</td>
			<td>Only required for membrane permeabilization cytotoxicity assay.</td>
		</tr>
		<tr>
			<td>Spectramax M5 Microplate Reader</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Other microplate readers capable of detecting UV-vis, fluorescence and luminescence may be suitable.</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>47036-50G-F</td>
			<td>Only required for membrane permeabilization cytotoxicity assay.</td>
		</tr>
		<tr>
			<td>Molecular Probes ATP Determination Kit</td>
			<td>Life Technologies</td>
			<td>A22066</td>
			<td>Other kits are likely to be suitable.</td>
		</tr>
		<tr>
			<td>Cytotoxicity Detection Kit for LDH release</td>
			<td>Roche</td>
			<td>11644793001</td>
			<td>Other kits are likely to be suitable.</td>
		</tr>
		<tr>
			<td>Nonidet P40 Substitute</td>
			<td>Sigma</td>
			<td>74385-1L</td>
			<td>Other suppliers are suitable.</td>
		</tr>
		<tr>
			<td>HALT Phosphatase Inhibitor Cocktail</td>
			<td>Thermo Scientific</td>
			<td>78420B</td>
			<td>Other cocktails are likely to be suitable.</td>
		</tr>
		<tr>
			<td>SIGMAFAST Protease Inhibitor Cocktail Tablets, EDTA-free</td>
			<td>Sigma</td>
			<td>S8830-2TAB</td>
			<td>Other cocktails are likely to be suitable.</td>
		</tr>
	</tbody>
</table>

implementation of a permeable membrane insert-based infection system to study the effects of secreted bacterial toxins on mammalian host cells

许多细菌病原体分泌强效的毒素在宿主组织的破坏援，以引发在宿主细胞中的信号的变化或感染的过程中操纵免疫系统的反应。虽然方法已经发展成功纯化和产生许多这些重要的毒力因子，但仍有许多细菌毒素，其独特的结构或广泛的翻译后修饰使它们难以纯化并在体外系统学习。此外，即使当能够得到纯的毒素，存在与学习毒素相关的生理条件下的具体影响相关的许多挑战。大多数在旨在评估分泌细菌毒素对宿主细胞的作用的体外细胞培养模型涉及孵育宿主细胞毒素的一次性剂量。感染，其中的毒素不断所产生过程中的这种方法不好大致与宿主细胞的实际经验细菌细胞，并允许感染的过程中逐渐累积。这个协议描述了基于插入渗透膜细菌感染系统的设计研究链球菌溶血素S，由A群链球菌产生的强效的毒素，对人上皮角质细胞的影响。这个系统的感染比，其中纯毒素或细菌上清液直接施加到宿主细胞的方法中更紧密地模仿自然的生理环境。重要的是，这种方法也消除了宿主反应是由于直接在细菌和宿主细胞之间的接触的偏压。该系统已被​​用来有效地评估链球菌溶血素S（SLS）的对宿主膜完整性，细胞活力和细胞信号的响应的影响。这种技术可以容易地应用于其他分泌的毒力因子的研究在多种哺乳动物宿主细胞的类型，以调查分泌细菌系数d的具体作用uring感染过程。

对于分泌的细菌因子的研究开发的渗透膜基于插入物感染系统协议是图1该系统通过一个多孔膜依赖于细菌和宿主细胞的分离，以评估分泌细菌因素的影响的详细，在这种情况链球菌溶血素S（SLS），对宿主反应如主机膜的完整性，细胞生存力，细胞信号转导，和分泌的宿主细胞因子。 图2提供代表蛋白质印迹数据表明，该系统可被用于评估在接触的活化变化 - 独立主机信号蛋白。具体地，该代表数据显示显著增强p38蛋白的活化在SLS产气菌的存在。该系统还可以应用于显现对宿主蛋白质定位分泌细菌因素通过免疫​​荧光显微镜的影响（&lt;STRONG&gt;图3）。数据显示的关键炎症介质核因子κB（NFκB），其从细胞质在激活21转移到细胞核的依赖性SLS-活化。在图2和3的结果表明，这两种SLS依赖性炎性信号传导的响应不需要的细菌与宿主细胞之间的直接接触。如前面的实验已经表明SLS依赖性p38和NFκB活化的直接感染模型21，在渗透膜基于镶嵌系统三种气体株中的p38或NFκB活化的类似水平将已表明所需的响应之间的直接接触细菌和宿主细胞。 图4表明，该感染系统可以应用于评估通过乙锭同型二聚体和LDH释放测定中在宿主细胞毒性毒素依赖性变化。这是由显著增加我证明Ñ​​既膜通透性和乳酸脱氢酶的从暴露于含有SLS相比暴露于SLS缺失菌株未感染的细胞或细胞GAS菌株的宿主细胞的释放。这些变化在细胞毒性不直接的，如显著效果并不明显，直到12小时后感染膜透的情况下和在LDH释放的情况下，16小时。代表性数据示出选择合适的时间点和感染的条件对这些宿主反应的评价的重要性。除了 ​​允许对宿主信令变化和细胞毒性的评估中，渗透膜基于插入感染系统，适用于代谢变化如准确确定主机ATP水平的研究通过防止宿主细胞裂解物的细菌污染（ 图5） 。这些数据表明响应于GAS感染16小时角化细胞的ATP的显著损失感染后，具有增强的ATP损耗我ñSLS的存在。这些结果与在宿主应激响应信令和细胞毒性所观察到的毒素依赖性增加是一致的。 <img alt="图1" src="/files/ftp_upload/54406/54406fig1.jpg" /> 图1:基于插入渗透的膜示意图感染系统协议，以评估对宿主细胞分泌的细菌因素的影响人角质形成细胞铺在底层舱和成长至90％汇合。用0.4微米孔的胶原涂覆的隔膜分隔的上，下腔室，和细菌被添加到所需感染期间的渗透膜插入系统的上腔室。细胞培养上清和宿主细胞可以被收集后的感染期和各种分析利用。 <a href="https://www.jove.com/files/ftp_upload/54406/54406fig1large.jpg" target="_blank">请点击此处查看大图</a>这一数字。 <img alt="图2" src="/files/ftp_upload/54406/54406fig2.jpg" /> 图2:在基于插入渗透膜感染系统可以通过SDS-PAGE和Western印迹被用于宿主细胞裂解物分析中的代表性数据表明SLS增强在感染角化细胞的p38蛋白激酶途径的活化。 （A）中HaCaTs分别经由可渗透膜插入物感染系统感染气体为7小时（MOI = 10）和裂解物进行了评估的p38的活化。 （B）的来自三个独立印迹光密度进行定量的p38的相对激活响应于GAS&#39;infection。从三个生物学重复的平均值被示出，与表示标准偏差误差线。对p38的相对活化被表示为磷酸化/总蛋白水平。相比于被确定统计学意义未受感染的细胞。整体p值是通过ANOVA（p值= 0.0063）测定。邓尼特的测试进行事后比较每个条件对应的未感染控制的意思。 *，P = 0.01〜0.05; **，p值= 0.001-0.01; ***，P = 0.0001-0.001; ****，P &lt;0.0001。这个数字已经从弗莱厄蒂等。2015年21修改。 <a href="https://www.jove.com/files/ftp_upload/54406/54406fig2large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54406/54406fig3.jpg" /> 图 3: 基于插入渗透膜感染系统允许通过免疫荧光显微镜主机信号的变化的可视化的具有代表性的数据表明，链球菌溶血素s至NFκB的活化增强了促炎症信号。将HaCaT人角质形成细胞感染了气体的M<html>使用可渗透基于插入物膜感染系统8小时10 OI。 NFκB的（A和B）核定位免疫荧光成像评估。核局部化的细胞的百分数通过计数在NFκB已经从细胞质易位到细胞核对于给定的字段细胞的数量除以该号码由细胞同场的总数。刻度条表示100μm左右。 （A）的三次生物学重复的平均值被表示为与表示标准偏差误差棒各条件。整体p值是通过ANOVA确定; P &lt;0.0001。进行Dunnett的检验以各条件比较相应未感染的对照条件。 *，P = 0.01〜0.05; **，p值= 0.001-0.01; ***，P = 0.0001-0.001; ****，P &lt;0.0001。这个数字已经从弗莱厄蒂等。2015年21修改。/ftp_upload/54406/54406fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54406/54406fig4.jpg" /> 图 4: 基于插入渗透膜感染系统允许细菌介导膜通透性和细胞毒性的细菌和宿主细胞之间的直接接触缺位的决心代表性的数据表明，角质形成细胞的活力活跃SLS毒素的存在减少。 GAS -诱导的细胞死亡是在角质形成细胞WT存在评估，SLS缺陷或补充佐贺角质形成气用渗透膜基于插入感染系统暴露GAS 8-16小时，在10的MOI（。 A）存活率，乙啶同型二聚体检测或（B）LDH释放法检测。在两个面板，3重plicates被平均及误差棒表示标准偏差。对于每个时间点显着性通过ANOVA（A）的8小时，p值= 0.0241确定; 12小时，P &lt;0.0001（B）8小时，P = 0.0287; 12小时，P = 0.1977; 16小时，P = 0.0031。进行Dunnett的检验从每个状态到野生型感染的相应的时间点进行比较的装置。 *，P = 0.01〜0.05; **，p值= 0.001-0.01; ***，P = 0.0001-0.001; ****，P &lt;0.0001。这个数字已经从弗莱厄蒂等。2015年21修改。 <a href="https://www.jove.com/files/ftp_upload/54406/54406fig4large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54406/54406fig5.jpg" /> 图5: 通过防止BACT对主机ATP水平的准确测定基于插入渗透膜感染系统允许宿主细胞的erial污染裂解液，代表数据表明A组链球菌感染时的ATP依赖的SLS损失。 HaCaT细胞感染了气体使用可渗透基于插入物膜感染系统8-16小时。技术重复（N = 3）从一个代表性生物复制（每个样品2×10 6个细胞）的平均值为每个条件，与表示标准偏差误差线。整体p值分别通过ANOVA确定（12小时，P &lt;0.0001; 16小时，P &lt;0.0001）。进行Dunnett的检验以比较野生型感染每个条件为相应的时间点。 *，P = 0.01〜0.05; **，p值= 0.001-0.01; ***，P = 0.0001-0.001; ****，P &lt;0.0001。这个数字已经从弗莱厄蒂等。2015年21修改。 <a href="https://www.jove.com/files/ftp_upload/54406/54406fig5large.jpg" target="_blank">请点击此处查看本图的放大版本。</a>

Watch this Scientific Journal Video about Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells at JoVE.com

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

这里，说明使用可透膜基于插入物感染系统研究链球菌溶血素S，由A群链球菌产生的分泌毒素的效果的方法中，在角质形成细胞。这个系统可以很容易地应用到各种宿主细胞类型的其他分泌的细菌蛋白的感染过程中的研究。

基于插入渗透膜感染系统的实现研究分泌细菌病毒对哺乳动物宿主细胞的作用

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

implementation-permeable-membrane-insert-based-infection-system-to

Research

JoVE Journal

Immunology and Infection

10.9K 次观看.  University of Notre Dame. 这里，说明使用可透膜基于插入物感染系统研究链球菌溶血素S，由A群链球菌产生的分泌毒素的效果的方法中，在角质形成细胞。这个系统可以很容易地应用到各种宿主细胞类型的其他分泌的细菌蛋白的感染过程中的研究。 

视频: Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

到哺乳动物的上皮细胞在体外细菌粘附的含量

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

科研

免疫与感染

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

监视分泌细菌毒力因子的组装使用站点特定的交联

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

电功能性细菌效应器到哺乳动物细胞

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

对白细胞从流程的分析招聘基质细胞的影响

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

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 ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...