我们要感谢马蒂亚斯S.迪克，罗兰F.德雷尔和塞巴斯蒂安鲁尔讨论。这项工作是由一个SNSF​​教授PP00P3_139120 / 1和巴塞尔大学的项目赠款ID2153162到PB支持

1.毛地黄皂苷测定与mCherry表达沙门氏菌 （基于FACS分析） <ol><li>准备细菌 注意： 沙门氏菌野生型SL1344（链霉素抗性）从质粒（PFPV编码mCherry下rpsM启动子）表达mCherry用于该测定。细菌吞噬作用和增殖，其中通过mCherry的组成型表达没有改变（数据未示出）16。 <ol><li>在3ml LB培养基，在37℃的CO / N的一摇床补充有链霉素（90微克/ ml）和氨苄青霉素（100微克/毫升，mCherry表达载体）感染前接种菌株1天。 </li></ol></li><li>电镀细胞感染。 <ol><li>在24孔板/孔在1ml初级巨噬细胞培养基中的种子的野生型BMDMs在1.25×10 5个细胞的密度（DMEM补充有10％胎牛血清（FCS，热灭活，56℃，30分钟），1％HEPES，1％非必需氨基酸（NEAA）中，1％L-谷氨酰胺和10％的L929上清液（来自巨噬细胞集落刺激因子（M-CSF的条件培养基）的制造的L929细胞）。根据表1种子井占个体条件。加盖玻片用作对照（ 表1）的孔中。 </li><li>过夜孵育细胞在培养箱中在37℃和5％的CO 2。 </li></ol></li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td></td><td></td><td colspan="2">透</td><td colspan="3">染色</td></tr><tr><td></td><td>玻璃盖玻片</td><td>沙门氏菌</td><td>洋地黄</td><td>皂素</td><td>抗沙门氏菌</td><td>抗钙联接蛋白</td><td>抗PDI </td></tr><tr><td> A1（样品） </td><td> - / + * </td><td> + </td><td> + </td><td></td><td> + </td><td></td><td></td></tr><tr><td> A2（样品） </td><td> - / + * </td><td> + </td&gt; <td> + </td><td></td><td> + </td><td></td><td></td></tr><tr><td> A3（样品） </td><td> - / + * </td><td> + </td><td> + </td><td></td><td> + </td><td></td><td></td></tr><tr><td> A4（不透控制） </td><td> - / + * </td><td> + </td><td></td><td></td><td> + </td><td></td><td></td></tr><tr><td> A5（完全通透控制） </td><td> - / + * </td><td> + </td><td></td><td> + </td><td> + </td><td></td><td></td></tr><tr><td> B1（染色钙联接） </td><td> + </td><td></td><td></td><td></td><td></td><td> + </td><td></td></tr><tr><td> B2（染色钙联接） </td><td> + </td><td></td><td> + </td><td></td><td></td><td> + </td><td></td></tr><tr><td> B3（染色钙联接） </td><td> + </td><td></td><td></td><td> + </td><td></td><td> + </td><td></td></tr><tr><td> C1（染色对于PDI） </td><td> + </td><td></td><td></td><td></td><td></td><td></td><td> + </td></tr><tr><td> C2（染色的PDI） </td><td> + </td><td></td><td> + </td><td></td><td></td><td></td><td> + </td></tr><tr><td> C3（染色的PDI） </td><td> + </td><td></td><td></td><td> + </td><td></td><td></td><td> + </td></tr></tbody></table> 表1：在24孔格式进行后续感染沙门氏菌，透化和染色骨髓来源的巨噬细胞（BMDMs）电镀方案 *根据方案1或方案2是否完成。 <ol start="3"><li>准备细菌感染。 <ol><li>第二天，通过在PBS中稀释的培养1:10，测量OD 600针对PBS仅控制确定沙门氏菌的过夜培养的浓度。这确保了OD 600测量在升inear范围分光光度计。 </li><li>计算每毫升沙门氏菌的浓度。 1-3的OD 600相当于1×10 9个细菌。 </li><li>稀的细菌在巨噬细胞培养基中以达到1.25×10 6个细胞/ ml的沙门氏菌浓度。 </li></ol></li><li>感染细胞与沙门氏菌 。 <ol><li>从BMDMs移除介质。加入1毫升的巨噬细胞培养基中，以未感染的对照孔（B1〜B3，C1〜C3）。加入1毫升沙门氏菌悬浮液至孔A1 - A5至达到每个细胞10细菌感染（MOI）的多重性。 </li><li>离心板15分钟，300×g离心，在37℃以同步感染。传送板在培养箱中在37℃和5％的CO 2。 </li></ol></li><li>杀死沙门氏菌外与庆大霉素。 <ol><li>在1小时感染后，从孵化器中取出板，并添加0.1毫升巨噬细胞培养基中含有0.1毫克/毫升庆大霉素杀死细菌细胞外。添加庆大霉素到所有孔中，甚至对未感染的对照，以确保所有的细胞相同的方式处理。传送板在培养箱中在37℃和5％的CO 2。 </li></ol></li><li>洗涤细胞。 <ol><li>在2小时后感染，从孵化器中取出板和洗涤各孔2×1毫升纯DMEM中（预热至37℃），并将其与巨噬细胞的培养基更换（预热至37℃）含有10微克/毫升&#160;庆大霉素防止任何剩余胞外细菌的生长。传送板在培养箱中在37℃和5％的CO 2。 </li></ol></li><li>准备新鲜的缓冲液用清洁剂和抗体。 <ol><li>刚分析的所需时间点之前，准备毛地黄皂苷的新鲜储备液以1mg / ml，在KHM缓冲液（110毫乙酸钾，20mM的HEPES，2mM的MgCl 2的，pH为7.3）。筛选股票，并在稀释缓冲KHM到工作浓度的毛地黄皂苷的50微克/毫升。另外，制备0.1％皂苷中KHM缓冲液，抗沙门氏菌抗体偶联到FITC（CSA-1-FITC）在1/500，抗钙联接蛋白在1/100或抗PDI在1/100 KHM溶液缓冲补充有3％BSA封闭。 </li></ol></li><li>细胞透。 <ol><li>从培养箱中取出盘子并洗孔3×用0.5ml KHM缓冲液（预热至37℃）。除去KHM缓冲器和与毛地黄皂苷添加0.25毫升平原KHM缓冲区到井A4和B1 / C1，KHM缓冲区到井A1 - A3和B2 / C2或KHM缓冲器，具有皂素孔A5和B3 / C3（根据表1）。孵育正好1分钟，RT和立即清洗各孔3×用0.5ml KHM缓冲液（预热至37℃）。 </li></ol></li><li>初级抗体染色。 <ol><li>除去KHM缓冲器和添加第一抗体溶液（山羊抗沙门氏菌 -FITC，250微升/孔，0.1微克/毫升），以适当的孔（ 图1 </st荣&gt;， 表1）。孵育板15分钟在培养箱中在37℃和5％的CO 2。 </li><li>洗涤细胞1X用1毫升的PBS。 </li></ol></li><li>感染的细胞（井A1 - A5）：FACS分析<ol><li>洗涤细胞3倍，用PBS，并添加0.5毫升冰冷PBS含有0.1％的Triton。转移到5毫升FACS管细胞滤网盖，打破了细胞聚集。不断的冰样本。 </li><li>分析流式细胞仪的样本。使用完全透控件，设置为基于前向总细菌和侧向散射（FSC / SSC）第一栅极，而所述mCherry信号（610毫微米±20nm的过滤器）。 </li><li>接着，分析在细菌总人口使用完全透和不透的控制来设置门，用于胞浆菌素（FITC + mCherry +）和液泡菌（FITC-，mCherry +）（ 图3）的FITC信号。 </li><li>与门设置，分析样品，并确定胞质对的百分比根据制造商的协议使用的FlowJo软件液泡细菌。 </li></ol></li><li>未感染的通透性控制（井B1 - B3，C1 - C3）：镜<ol><li>固定术<ol><li>除去PBS中并在PBS中加入250微升的PFA 4％。孵育10分钟，在37℃。 </li><li>洗孔2×1毫升PBS中，并加入250微升的0.1M甘氨酸的PBS 10分钟以淬灭固定液。洗井2×1毫升的PBS。 </li></ol></li><li>二级抗体染色。 <ol><li> 1小时在室温下与连接到荧光团在PBS中适当的二级抗体染色透控制补充有0.1％皂苷和3％的BSA，充分透细胞。 </li><li>洗涤盖玻片3次用1毫升的PBS并装入盖玻片分析在安装介质用DAPI（1.5微克/毫升）。 </li></ol></li><li>显微镜分析。 <ol><li>使用共聚焦FLUO在40X或63X放大率分析与对照的盖玻片rescence显微镜。如果透工作过，应该没有染色用于非渗透细胞，钙联接蛋白染色为digitonin-和皂苷透化细胞，并只PDI染色为皂苷透化细胞（ 图4）。 </li></ol></li></ol></li></ol> 2.毛地黄皂苷含量与未标记沙门氏菌（显微镜为基础的分析） <ol><li>准备细菌O / N培养。 <ol><li>接种未标记沙门氏菌野生型SL1344（链霉素抗性）1天在3ml LB培养基，在37℃的CO / N的一摇床补充有链霉素（90微克/毫升）的感染之前。 </li><li>电镀细胞感染。 <ol><li>在24孔板含有无菌盖玻片在1.25×10 5个细胞的密度种子BMDMs /孔在1ml巨噬细胞培养基（DMEM补充有10％胎牛血清（FCS，去补充，56℃，30分钟），1％HEPES，1％L-非必需氨基酸（NEAA），1％L-谷氨酰胺和10％的L929上清液）。根据表1种子井占个体条件。 </li><li>孵育细胞O / N中的培养箱中在37℃和5％的CO 2。 </li></ol></li><li>准备细菌感染。 <ol><li>第二天，通过在PBS中稀释的培养1:10并测量OD 600针对PBS仅控制确定在O / N培养沙门氏菌的浓度。这确保了OD 600测量在分光光度计线性范围。 </li><li>确定每毫升沙门氏菌的浓度。的1的OD 600相当于1×10 9个细菌。 </li><li>稀的细菌在巨噬细胞培养基中以达到1.25×10 6个细胞/ ml的沙门氏菌浓度。 </li></ol></li><li>感染细胞与沙门氏菌 。 <o升&gt; <li>从所有孔中取出。加入1毫升纯巨噬细胞中到未感染对照孔（B1 - B3，C1 - C3）。加入1毫升沙门氏菌悬浮液至孔A1 - A5至达到每个细胞10细菌感染（MOI）的多重性。 </li><li>离心板15分钟，300×g离心，在37℃以同步感染。传送板在培养箱中在37℃和5％的CO 2。 </li></ol></li><li>杀死沙门氏菌外与庆大霉素。 <ol><li>在1小时感染后，从孵化器中取出板，并添加0.1毫升巨噬细胞培养基中含有1mg / ml的庆大霉素，以杀死胞外细菌到所有孔中。传送板在培养箱中在37℃和5％的CO 2。 </li></ol></li><li>洗涤细胞。 <ol><li>在2小时后感染，从孵化器中取出板和洗各孔3次用1毫升含有10微克/毫升新鲜的巨噬细胞培养基 庆大霉素防止生长的任何剩余的胞外细菌。传送板在培养箱中在37℃和5％的CO 2。 </li></ol></li><li>准备新鲜的缓冲液用清洁剂和抗体。 <ol><li>刚分析的所需时间点之前，准备毛地黄皂苷的新鲜储备液以1mg / ml，在KHM缓冲液（110毫乙酸钾，20mM的HEPES，2mM的MgCl 2的，pH为7.3）。过滤库存和KHM缓冲稀释至50微克/ ml的工作浓度 毛地黄的。 </li><li>另外，制备0.1％皂苷中KHM缓冲的溶液中，抗沙门氏菌 -FITC（CSA-1，0.1微克/毫升），在KHM缓冲器抗钙联接蛋白在1/100或抗PDI在1/100补充有的3％BSA（见材料表用于抗体浓度）。 </li></ol></li><li>细胞透。 <ol><li>从培养箱中取出盘子并洗孔3×用0.5ml KHM缓冲液（预热至37℃）。删除KHM缓冲区，并添加0.25毫升平原KHM缓冲区井A4和B1 / C1，KHM缓冲器，具有毛地黄皂苷井A1 - A3和B2 / C2或KHM缓冲器，具有皂素井A5和B3 / C3（根据表1）。孵育正好1分钟，RT和立即清洗各孔3×用0.5ml KHM缓冲液（预热至37℃）。 </li></ol></li><li>初级抗体染色。 <ol><li>除去KHM缓冲器和添加第一抗体溶液（山羊抗沙门氏菌 CSA1-FITC，250微升/孔，0.1微克/毫升），以适当的孔（ 图2，表1）。孵育板15分钟在培养箱中在37℃和5％的CO 2。洗3次用1毫升的PBS。 </li></ol></li><li>固定。 <ol><li>除去PBS中并在PBS中加入250微升的PFA 4％。孵育10分钟，在37℃。 </li><li>洗孔2×1毫升PBS中，并加入250微升的0.1M甘氨酸的PBS 10分钟以淬灭固定液。洗井2×1毫升的PBS。 </li></ol></li><li>总受感染的样本沙门氏菌污染。 <ol><li>洗涤盖玻片3次用PBS用0.1％皂苷/ 3％BSA和孵育的样品与抗CSA1抗体（山羊抗CSA1，0.1微克/毫升，小鼠抗LPS作品以及0.1微克/毫升）1小时在室温在PBS 0.1％皂苷/ 3％BSA封闭。 </li><li>洗3次在PBS与0.1％皂苷/ 3％BSA封闭。孵育的样品与第二抗山羊抗体耦合到所选择的荧光团在PBS与0.1％皂苷/ 3％BSA在室温30分钟在黑暗中。 </li><li>洗涤盖玻片3次用1毫升的PBS并装入盖玻片分析在安装介质用DAPI（1.5微克/毫升）。 </li></ol></li><li>显微镜分析。 <ol><li>通过计数胞质沙门氏菌 （抗沙门氏菌 -FITC阳性）和总沙门氏菌 （双阳性）（ 图5）获得了共焦显微镜的数据。通透的控制应该显示：无染色无渗透细胞，钙联接s泰宁为毛地黄皂苷透化细胞和PDI染色皂苷透化细胞（ 图4）。韦尔斯A4和A5将作为内部控制的沙门氏菌污染。 </li></ol></li></ol></li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+Toxoplasma+gondii+parasitophorous+vacuole+by+IFNgamma-inducible+immunity-related+GTPases+(IRG+proteins)+triggers+necrotic+cell+death.">Disruption of the Toxoplasma gondii parasitophorous vacuole by IFNgamma-inducible immunity-related GTPases (IRG proteins) triggers necrotic cell death.</a> PLoS Pathog. 5, e1000288 (2009).</li><li>Meunier, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-11+activation+requires+lysis+of+pathogen-containing+vacuoles+by+IFN-induced+GTPases.">Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases.</a> Nature. 509, 366-370 (2014).</li><li>Yamamoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cluster+of+interferon-gamma-inducible+p65+GTPases+plays+a+critical+role+in+host+defense+against+Toxoplasma+gondii.">A cluster of interferon-gamma-inducible p65 GTPases plays a critical role in host defense against Toxoplasma gondii.</a> Immunity. 37, 302-313 (2012).</li><li>Degrandi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+guanylate+binding+protein+2+(mGBP2)+controls+Toxoplasma+gondii+replication.">Murine guanylate binding protein 2 (mGBP2) controls Toxoplasma gondii replication.</a> Proc Natl Acad Sci U S A. 110, 294-299 (2013).</li><li>Mariathasan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+activation+of+the+inflammasome+by+caspase-1+adaptors+ASC+and+Ipaf.">Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf.</a> Nature. 430, 213-218 (2004).</li><li>Knodler, L. A., Nair, V., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+cytosolic+Salmonella+in+epithelial+cells.">Quantitative assessment of cytosolic Salmonella in epithelial cells.</a> PLoS One. 9, e84681 (2014).</li><li>Ng, H., Smith, D. J., Nagley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+flow+cytometry+to+determine+differential+redistribution+of+cytochrome+c+and+Smac/DIABLO+from+mitochondria+during+cell+death+signaling.">Application of flow cytometry to determine differential redistribution of cytochrome c and Smac/DIABLO from mitochondria during cell death signaling.</a> PLoS One. 7, e42298 (2012).</li><li>Krawczyk, E., Suprynowicz, F. A., Sudarshan, S. R., Schlegel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+orientation+of+the+human+papillomavirus+type+16+E5+oncoprotein.">Membrane orientation of the human papillomavirus type 16 E5 oncoprotein.</a> J Virol. 84, 1696-1703 (2010).</li><li>Keller, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+measurements+of+vacuolar+rupture+caused+by+intracellular+pathogens.">Single cell measurements of vacuolar rupture caused by intracellular pathogens.</a> J Vis Exp. , e50116 (2013).</li></ol>

We have nothing to disclose.

差透是一个简单而可靠的方法来分析和量化的液泡车厢和细胞质的细菌性病原体的亚细胞分布。相同的测定已被成功地用于与细菌，如弗朗西斯novicida 2,4和志贺氏菌 12。然而，由于许多细胞内病原体改变或修改主机内膜结构，毛地黄皂苷透化的鲁棒性将必须在个体基础上确定。在对于所用可能是必要的病原体或细胞系微调该测定的。该测定不仅限于感染生物学，但也...

细胞内细菌病原体复制或者直接在宿主细胞胞质溶胶，或者在专门的液泡区室1。在感染的初始阶段大多数病原体获得通过吞噬​​作用由专门的细胞（如巨噬细胞），或通过积极推动自身摄取进入非吞噬细胞内化两种。在吞噬细胞，吞噬体通常与溶酶体融合，以形成降解隔室，其中所述吞噬颗粒被消化。专门胞浆菌，如弗朗西斯novicida或志贺氏菌 ，逃避吞噬体降解通过诱导吞噬体的破裂和随后逃逸进入细胞质2,3。这需要毒力相关的机制，如弗朗西斯致病岛（FPI）或痢疾杆菌 T3SS，其中注入效应蛋白促进液泡破裂2-4。宿主细胞细胞质的功能一些保守模式识别受体，通常承认的病原体的存在和诱导先 ​​天免疫信号5。此外，xenophagy，自噬的抗微生物形式能降解该进入细胞质细菌。胞浆菌通常很好地适应生硬或采用不同的策略规避这些反应。例如， 弗朗西斯修改其LPS以避免主机识别和志贺防止自噬招聘使用分泌效应蛋白6,7。 另一种策略逃脱溶酶体降解由模型液泡病原体沙门伤寒采用，此后简称为鼠伤寒沙门氏菌 。 沙门氏菌使用T3SS注入该重塑的初始吞噬体进入细胞内利基效应， 沙门氏菌含液泡（SCV） 8,9。连续操纵宿主的途径是要保持这种空泡招募脂类等营养物质到液泡。事实上， 沙门氏菌的SIFA突变体不能维持液泡稳定，并进入宿主细胞后数小时内细胞质感染后，这导致活化先天免疫途径和自噬招募8。 沙门氏菌的液泡逃逸取决于细胞类型即研究，一些报告显示，在上皮细胞中甚至WT 沙门氏菌能逃脱SCV和hyperreplicate在细胞质10。最近的报告表明，先天免疫检测的病原体空泡也取决于主机的认可和破坏含病原体的空泡（PCVs）11-14能力。 鉴于这两个主机或细菌基因型可影响空泡和细胞内室之间的胞内细菌的分布，有必要进行量化空泡对的数目胞浆菌。因为，在大多数实验设置宿主细胞感染有一个以上的细菌，并随后可以在不同的亚细胞区室怀有几个细菌，一种技术是必要的，它允许量化在单细菌的水平。通透性差（也称为吞噬体保护法）提供了这一决议2,4,10,12。该测定法是基于宿主细胞质膜与洗涤剂毛地黄皂苷，这让液泡膜完好的选择性透和因此允许胞浆菌的抗体的选择性染色。在这里，我们提供了两种协议的使用差分通透性胞浆空泡和鼠伤寒沙门氏菌的定量。这个方法的原理在图1中被描述：骨髓衍生的巨噬细胞（BMDMs）感染固定相mCherry表达沙门氏菌 。固定相沙门氏菌需要牛逼ø被使用，因为它们下调的SPI-1 T3SS，其活性将另外由NLRC4炎性被识别，诱导BMDMs 15的快速细胞死亡（pyroptosis）的表达。以下洗涤细胞并用毛地黄皂苷50μg/ ml的1分钟处理过的感染。将细胞立即再次洗涤和温育与耦合到FITC与访问到细胞质标记细菌的抗沙门氏菌抗体。后的附加洗涤步骤细胞裂解和mCherry +（液泡）和FITC + / mCherry +（细胞质）的细菌的百分比是通过FACS测定。我们还报告这种方法，可用于在没有荧光蛋白表达菌株是可用的（ 图2）的适应。附加步骤的FITC标记，其中将细胞固定并完全透后进行了介绍。此后所有的细菌都沾满抗沙门氏菌抗体和相应的二次抗体。 DET挠度然后通过显微镜代替FACS分析进行。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Digitonin</td>
			<td>Sigma</td>
			<td>D5628</td>
			<td>50ug/mL</td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>mpbio</td>
			<td>219998380</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Life Technologies</td>
			<td>15630</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Acetate</td>
			<td>Sigma</td>
			<td>791733</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Salmonella CSA-1-FITC</td>
			<td>KPL</td>
			<td>01-91-99-MG</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>anti-Salmonella CSA-1</td>
			<td>KPL</td>
			<td>02-91-99-MG</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>anti-Calnexin</td>
			<td>Enzo Lifesciences</td>
			<td>SPA-860D</td>
			<td>1/100</td>
		</tr>
		<tr>
			<td>anti-PDI</td>
			<td>Enzo Lifesciences</td>
			<td>SPA-890</td>
			<td>1/100</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vectorlabs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti Rabbit antibody-488</td>
			<td>Molecular Probes</td>
			<td>A-11070</td>
			<td>1/500</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Life Technologies</td>
			<td>15710-49</td>
			<td>100ug/mL and 10ug/mL</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Promega</td>
			<td>H5141</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>20012-019</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Amimed</td>
			<td>5-13K00-H</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>imaging done at 63x</td>
		</tr>
		<tr>
			<td>FACS Fortessa</td>
			<td>BD technologies</td>
			<td></td>
			<td>detection mCherry 610 nm</td>
		</tr>
		<tr>
			<td>FACS Fortessa</td>
			<td>BD technologies</td>
			<td></td>
			<td>detection FITC 530 nm</td>
		</tr>
	</tbody>
</table>

quantification of cytosolic vs. vacuolar salmonella in primary macrophages by differential permeabilization

细胞内细菌病原体可在细胞质或专门含病原体的空泡（PCVs）复制。到达细胞质，像痢疾杆菌和弗朗西斯novicida细菌需要诱导吞噬体破裂。与此相反， 鼠伤寒沙门氏菌复制在液泡室，被称为沙门氏菌含液泡（SCV）。 沙门氏菌然而某些突变体不能维持SCV完整性并因此释放到胞质溶胶中。胞质与单菌的水平液泡细菌的百分比可以通过差通透性进行测量，也被称为吞噬体的保护测定法。该方法利用洗涤剂毛地黄皂苷的财产选择性结合胆固醇。由于质膜包含比其它细胞膜更胆固醇，毛地黄皂苷可用于选择性地透质膜同时留下细胞内膜完好。简言之，感染后表达的荧光标记蛋白（ 例如 mCherry等等）的病原体，宿主细胞的质膜透化与含有缓冲液短暂孵育毛地黄皂苷。然后将细胞洗涤并孵育一抗（偶联至所选择的荧光团）针对所选择的细菌（ 例如抗沙门氏菌 -FITC），并再次洗涤。如果未标记的细菌时，额外的步骤可以做到的，其中所有的膜透化和所有细菌染色与相应的抗体。以下染色，空泡和细胞内细菌的百分比可以通过FACS或显微镜来量化通过计数单或双阳性事件。在这里，我们提供了实验细节使用这种技术与细菌鼠伤寒沙门氏菌 。该测定的优点在于，相对于其他测定法，它提供了一个量化单bacte的水平河口，如果通过显微镜分析提供胞质和液泡细菌在给定的细胞的精确数量。

图1和图2显示的毛地黄皂苷测定在方案1和方案2示出在协议的关键步骤和所获得的结果中描述的原理图; 图3示出了典型的FACS结果。阳性和阴性对照用于设置的闸门的mCherry + / FITC-菌（液泡沙门氏菌 ）和mCherry + / FITC +菌（细胞质沙门氏菌 ）。基于这些门胞质和液泡细菌的百分比可以在实验样本中确定。在这个例子中，我们比较了野生型和ΔsifA 沙门氏菌 。 ...

Watch this Scientific Journal Video about Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization at JoVE.com

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 ...

quantification-cytosolic-vs-vacuolar-salmonella-primary-macrophages

Research

JoVE Journal

Immunology and Infection

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

10.8K 次观看.  University of Basel. We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

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

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

胞内钙的测量 2 +</sup在隔离的收缩淋巴管

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

科研

免疫与感染

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

单细胞测量液泡破裂引起细胞内病原体

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

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 ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

人THP-1巨噬细胞通过核转高效转染

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

巨噬细胞通过二维电泳蛋白组学分析

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

吞噬体偏移和速度与HIV-1感染的活小学的人巨噬细胞测量

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

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. ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

小鼠巨噬细胞吞噬的延时3D 成像

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

原代人细胞中寨卡病毒抗体依赖性增强的定量研究

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...