这项工作是由来自默多克儿童研究所和维多利​​亚州政府的基础设施运营支持项目资金支持。

上皮1。准备和细菌细胞<ol><li> CCL-23上皮细胞的解冻 <ol><li> Prewarm最小必需培养基（MEM）的含10％胎牛血清（FBS）在37℃水浴〜30分钟。 </li><li>消毒的组织培养批准的生物安全柜，用紫外灯使用前至少10分钟，然后擦拭手中的工作区用70％乙醇。请用70％的乙醇和地点在生物安全柜的温暖媒体瓶。 </li><li>用25毫升移液管转移14毫升的介质导入T-75烧瓶中。将烧瓶在37℃培养箱中培养，而细胞解冻（1.1.4节）。 </li><li>卸下液氮储存使用适当的安全预防措施，并在一个温暖的37℃水浴中，直到〜80％解冻CCL-23细胞的小瓶。 </li><li>返回在第1.1.3生物安全柜准备的烧瓶中。擦拭CCL-23细胞的小瓶的外表面用70％乙醇和传送内容（1毫升）到T-75瓶使用P1000的移液管。 </li><li>孵育在其一侧将烧瓶在37℃，5％CO 2，95％相对湿度组织培养箱中过夜（16-20小时）。 </li><li>检查在显微镜下的细胞，以确认一个健康的形态，去除介质，并更换15毫升新鲜，预热MEM +10％FBS。 </li></ol></li><li> CCL-23上皮细胞的维持 <ol><li>一旦细胞是〜80％汇合（1-2天），通过用25毫升移液管除去介质和轻轻地用8-10洗细胞两次毫升预热的磷酸盐缓冲盐水（PBS）中，用10毫升吸管。 </li><li>除去PBS中并加入1毫升无菌的0.25％胰蛋白酶-EDTA（0.25％（重量/体积），胰蛋白酶，0.1 mM EDTA）中。把烧瓶在其一侧，旋转，以确保胰蛋白酶涵盖细胞的汇合层，并培育烧瓶中2-3分钟在37℃，5％CO 2的培养箱中培养。 </li><li>回到瓶中的生物安全柜，加9毫升预热MEM +10％FBS，和吸管上下6 -8倍到重悬细胞，喷射液体向下烧瓶撞出细胞的一侧。 </li><li>分裂细胞1点15分（1毫升+14毫升MEM +10％FBS）或1点半（0.5毫升+14.5毫升MEM +10％FBS），弃去过量的细胞，并返回到烧瓶中，在37℃，5％CO 2培养箱中培养。 </li><li>通道的细胞至少两次在粘附试验后方可使用。典型的细胞分裂，每3-4天。 </li></ol></li><li> CCL-23上皮细胞接种 （前一天粘附试验，协议2） <ol><li>如在第1.2.1-1.2.3所述，重新悬浮在预热的MEM分裂汇合细胞+5％FBS（胎牛血清的浓度降低，以减少血清成分干涉检测的可能性）。去除20微升细胞，并使用台盼蓝染色和血细胞计数器计数。 </li><li>稀释细胞在预热的MEM + 5％FBS，以1.5×10 5细胞/在无菌的50ml管中ml的浓度。 </li><li>种子1毫升个细胞/孔在一个组织培养处理的24孔板中。 </li></ol></li><li> 细菌的制备 这个过程可以遵循为制备肺炎球菌和S的唾液 。细菌生长介质通常保持在37℃培养箱中过夜，以prewarm和测试不育（如果被污染一般会出现混浊）。 <ol><li>要坚持性对马血琼脂（HBA）板检测，连胜细菌和孵化，在35-37℃和5％CO 2的18-24小时之前两天。绵羊血琼脂可以作为一种替代方法。 </li><li>次日，通过吸取10毫升托德-Hewitt肉汤0.5％酵母提取物（THB + YE）介质进入无菌30毫升管制备的过夜培养每个细菌物种。用10微升环，接种约10-12很好的分离克隆。简要涡和孵化，在35-37℃和5％CO 2为14-16小时。需要注意的是肺炎球菌就会自溶如果离开太久的肉汤培养物。 </li></ol></LI&gt; </ol> 2坚持含量：加入细菌的上皮细胞单层<ol><li> 肺炎链球菌和S.中旬登录细菌培养物的制备唾液 <ol><li>移取10毫升预热泰铢+叶成每两个无菌30毫升管，一个标有肺炎球菌及其他S的唾液 。 </li><li>达到对数中期，肺炎球菌要求〜3小时和S.唾液需要〜2小时培养（参见图1）。同时启动两种文化。涡（3-5秒）过夜细菌培养，并加入100微升肺炎球菌和100微升S。唾液进入相应的管。 </li><li>简要涡和孵化培养，在37℃下为S。约2小时唾液和3小时的肺炎球菌。 </li></ol></li><li> CCL-23细胞粘附试验的制备 <ol><li>使用倒置显微镜，检查24孔板confluen吨单层与健康的细胞形态。单分子膜应&gt; 80％汇合，没有细胞聚集或致细胞病变效应如细胞变圆附着的或损失（参见图2）的证据。 </li><li>在每个用移液管好，倾斜板稍微以避免干扰细胞单层中取出介质。 </li><li>轻轻洗涤细胞，加入约500μL预热的PBS到每一个使用血清吸管好，用移液管将其删除，稍微倾斜板。弃PBS。重复洗涤步骤一次。 </li><li>加入500μl预热的MEM + 5％FBS在每孔中。返回的24孔板中的CO 2培养箱中，同时制备细菌培养物。 注意：为了确定感染复数（MOI）的多重性的确切CCL-23细胞的数目，两口井可以被胰蛋白酶消化并计数在感染当天。 </li></ol></li></ol> 3，坚持含量<ol><li> 添加到肺炎球菌在CCL-23细胞在MOI〜10:1。测试一式两份孔中在表1中列出的条件。 <ol><li>衡量的A 600（外径）1毫升S的光密度唾液文化。外径应为0.3-0.6之间。 </li><li>转帐5毫升S。唾液文化到10ml的试管和离心1,870×g离心4分钟。 </li><li>弃上清，并根据OD值在3.1.1节，重悬S。唾液在200-1,000微升0.85％的NaCl（终浓度为〜1.5×10 9 CFU /毫升）。作为一般指引，0.375的OD将悬浮在260微升。准备1:10和1:100稀释的S。用0.85％的NaCl 唾液 。 注：共有三种浓度的S.（代表高，中，低剂量）将唾液测定中进行测试。高剂量的〜10 S的比值唾液 ：1肺炎球菌，介质为〜1 S。唾液 ：1肺炎球菌，而低为〜1个S。唾液：10肺炎球菌。 </li><li>取出24孔板从保温箱。 </li><li>从肝素对照孔，取出40微升的媒体和100 U / ml的终浓度加入50微升肝素（1,000 U / ml的股票浓度）。 </li><li>加入10微升0.85％的NaCl只含CCL-23细胞和含CCL-23细胞和肺炎链球菌（S.无唾液 ）井井。 </li><li>加入10微升未稀释的1:10和1:100 S。唾液各自的水井。 注：S。唾液也可以在同一时间为肺炎双球菌（coaddition）或1小时，加入肺炎球菌（后加）后，研究益生菌给药时间的影响。 </li><li>离心24孔板中于114×g离心3分钟，以促进与细胞层的细菌接触并孵育1小时，在37℃，5％CO 2。 </li><li>执行S的连续稀释唾液股票在0.85％NaCl和平板上的重复的HBA板的活菌数。首先，加入50微升的现货，450微升0.85％的NaCl，使10 -1稀释，然后脉冲涡流（5秒），变更的提示，并加入50微升的10 -1稀释至450微升0.85％的NaCl作10 -2稀释度等等多达10 -7。 </li><li>板100微升的10 -5，10 -6和10 -7稀释液一式两份的HBA板和扩散用无菌吊具。在37℃，5％CO2倒过夜的HBA板。 </li><li>向1小时温育结束时，对肺炎球菌重复部分3.1.1-3.1.3。的OD应该是0.2-0.7之间。降速1毫升肺炎球菌培养和悬浮颗粒在300-2,000微升0.85％氯化钠（基于OD值）来的〜1.5×10 8 CFU /毫升的浓度。作为一般指引，0.3的OD将重新悬浮于500微升。 </li><li> 1小时后孵化与S。唾液 ，加入10μ;升未稀释的肺炎球菌对所有的井，除了阴性对照孔（含电池只）。 </li><li>离心24孔板中于114×g离心3分钟，并在37℃，5％CO 2中3小时。如第3.1.9和平板稀释10-4，10-5和10-6上的重复的HBA板的活菌数中描述的0.85％的NaCl进行肺炎球菌股票的连续稀释。 </li></ol></li><li> 3小时后，检查在显微镜下的细胞，保证细胞单层看起来很健康（ 见图2）。从各取出介质，并轻轻按2.2.3节中描述洗细胞3倍。使用不同的移液管的每个状态。这一步是必要的，以除去未粘附的肺炎球菌。单层不应该在洗涤过程中散开;这可以通过在显微镜下再次检查来确认。 </li></ol>注意：介质在该步骤中去除，可以储存在-20℃下进行另外的分析（&lt;em&gt;的例如细胞因子水平的测量）。 <ol><li>加入200μl0.1％毛地黄皂苷（温和的洗涤剂）的每个孔中，孵育7分钟，在37℃，5％CO 2。 </li><li>添加800微升THB的媒体的每个孔中。裂解细胞用移液器上下吹吸，并且，如果需要，刮井3至4倍的移液管尖，以确保细胞单层被完全除去。改变井之间的技巧。 </li><li>从每个使用P1000的移液管以及取出内容物并转移到标有微量离心管中。 </li></ol>贴壁肺炎球菌4。定量肺炎球菌粘附可通过测定活菌数（连续稀释和镀层在血琼脂）或通过定量实时PCR进行定量。 <ol><li> 活菌计数方法 必须坚持测定步骤3.5之后立即执行此方法。 <ol><li>对于每个分析孔，准备在收集样本的连续稀释通过加入50μl的样品到450微升0.85％NaCl中，脉冲涡流，并连续稀释如第3.1.9至10 -6稀释描述步骤3.5的粘附测定。 </li><li>板100微升的10 -3，10 -4，10 -5，和 10 -6稀释度一式两份在血琼脂，散布用无菌吊具，并于37℃过夜孵育平板倒置，5％的CO 2。注意，含有：CCL-23细胞的单独的孔中不应该包含任何细菌和可镀整齐（100微升未稀释）作为无菌控制。 </li><li>第二天，通过计算在坚持试验第3.1.13编写相应的稀释板（含30-300殖民地）的菌落确定肺炎球菌接种。计算CFU /毫升的用平均值由两个板块的接种物。同样，确定S。唾液接种计数编制板上的菌落在第3.1.10。 注：板应该只包含肺炎双球菌;其它物种的存在表明该测定法被污染和结果是无效的。 </li><li>要确定肺炎球菌附着水平，算上适当稀释平板（含30-300肺炎球菌菌落）在第4.1.1准备每个样品。需要注意的是S。唾液菌落将是小和白色，而肺炎链球菌将持平和绿色由于α-溶血，如示于图3。要计算的肺炎球菌粘附，从条件b正常化粘附肺炎链球菌的数量。 CCL-23细胞+肺炎链球菌为100％，和肺炎球菌粘附比较从其他条件这个条件。 </li></ol></li><li> 定量PCR方法 收集粘附试验样品步骤3.5可以储存在-20°C，直到DNA提取。使用商业试剂盒进行DNA提取和在各节中描述4.2.1-4.2.9和qPCR的作为说在第4.2.10-4.2.20。 <ol><li>制备酶促裂解缓冲液（含有50mM磷酸盐缓冲液pH 6.7 1毫克/毫升溶菌酶，0.075毫克/毫升的变溶菌素，和2毫克/毫升蛋白酶K）。以下几招可以用来打10毫升（不足50提取）。酶法裂解液应在使用前新鲜配制。 7.25毫升的50mM磷酸盐缓冲液（pH 6.7） 1.00毫升10的最终[1毫克/毫升]毫克/毫升溶菌酶股票 0.75 1毫克毫升/ ML变溶菌素股票作最后[0.075毫克/毫升] 1.00毫升20的最后[2毫克/毫升] mg / ml的蛋白酶K股票 </li><li>解冻在冰上或4℃下的样品涡和传输每个样品100μL分装到标有微量离心管中。返回原始样本放回-20℃冰箱。 </li><li>离心样品在6700×g离心10分钟。 </li><li>取出并弃去上清液。加入200μl酶裂解缓冲液的颗粒和涡旋重悬。 Incub吃的样品在56℃下进行30分钟。短暂离心（5秒），在6700×g离心在试管底部收集液体。 </li><li>加10微升20％的SDS，以完成细胞裂解，轻轻混匀，在室温下放置2分钟。 </li><li>加入4μLRNA酶A（100毫克/毫升的股票）中，轻轻混匀（在摇臂或反相管），在室温下2分钟。 </li><li>加入200μl缓冲液的Al（从工具包），涡旋15秒。孵育的样品在70℃下10分钟。短暂离心（5秒），在6700×g离心在试管底部收集液体。 </li><li>加入200μl100％乙醇，涡旋15秒。简要地旋转以收集液体在管的底部，则在不润湿的轮辋的混合物（包括任何沉淀物）转移到离心柱（在2 ml收集管中）。根据制造商的方案中遵循的洗涤步骤（此处未示出）。 </li><li>以洗脱DNA，转列到一个标有离心管中，加入50微升缓冲的AE（FR嗡试剂盒），并在室温下孵育2分钟。离心机在4300×g离心1分钟。重复一次，最后洗脱的100微升体积。 DNA可以贮存于-20℃直至使用。 </li><li>要执行定量PCR，消毒用紫外灯的罩15分钟前开始。准备一个96孔板地图工作表记录井位每个样品建议。 </li><li>使用的肺炎球菌的基因组DNA（从参考菌株ATCC 6305中提取）10纳克/微升库存制备标准曲线。 <ol><li>解冻肺炎球菌股票的DNA，并在台式离心机后短暂离心（5秒）。 </li><li>加入3微升的DNA，以27μL无核酸酶H 2 O，每个稀释度之间变化的技巧和涡旋执行1:10系列稀释液在6预先标记的离心管。最终的稀释系列由6个试管：1，0.1，0.01，0.001，0.0001和0.00001纳克/微升。 注意：这些可以被存储在4℃下长达一周。 </li></ol></li><li> PreparêLYTA mastermix在无菌的15毫升锥形管在无菌PCR罩如下。在冰上解冻并涡旋在使用前立即组件。 <table border="0"><tbody><tr><td> 元件 </td><td> 每个反应 </td><td> ×102（全板） </td></tr><tr><td> LYTA F </td><td> 0.25微升10微米股票</td><td> 25.5微升</td></tr><tr><td> LYTAř </td><td> 0.25微升10微米股票</td><td> 25.5微升</td></tr><tr><td> LYTA探头</td><td> 0.5微升10微米股票</td><td> 51微升</td></tr><tr><td> H 2 O的</td><td> 9.5微升</td><td> 969微升</td></tr><tr><td>预混</td><td> 12.5微升</td><td> 1,275微升</td></tr><tr><td>最终体积</td><td> 23微升</td><td> 2,346微升</td></tr></tbody></table&gt;注意：某些实时PCR机器需要使用一个参比染料，它被包含在一些商业mastermixes也可以单独添加的。这个协议被设计为在一个96孔板上运行25μl的反应，但可以调整，以适应不同的实时PCR机器。引物和探针序列由卡瓦略等人 15最初被发表的，并示于材料的列表。 </li><li>旋涡LYTA和负载23微升/孔混合到适当的水井。是指板图。 </li><li>交通运输板块的样品装载模板除了区域（在一个单独的房间最好是无菌柜）。 </li><li>使用P2移液管装入样品，改变提示每口井。所有样品一式两份运行。是指板图的位置。 </li><li>对于标准曲线的井，加入1μl/孔的适当稀释的标准曲线的DNA加1微升/孔H 2 O至使终体积为25微升。对于无模板对照孔，加2微升H 2 O </li><li>加入2μl/孔的样品DNA插入适当的孔。 </li><li>盖上使用的所有井。确保帽紧贴关闭。装载板进入实时PCR仪前快速旋转PCR板（5秒）。 </li><li>负载板进入PCR仪和运行的qPCR根据由机床制造商提供的说明。下面水解探针循环条件，建议： <table><tbody><tr><td>步骤1（1×）： </td><td> 3分钟，在95℃下</td></tr><tr><td>步骤2（40X）： </td><td> 20秒在95℃下</td></tr><tr><td></td><td> 20秒在60℃下</td></tr></tbody></table></li><li>收集数据，并使用标准曲线定量肺炎球菌的DNA。如前所述17，推定该1皮克基因组DNA的相当于447.4细胞，并报告为菌落形成单位/毫升（假设ö细菌负荷的计算每CFU NE基因组中，每个基因组LYTA的一个副本;参见图4）。对于一个成功的试验中，在R 2的值应≥0.98和标准曲线的范围内的所有阳性结果（Ct值小于为0.00001纳克获得/微升标准曲线点被认为是背景/ LYTA负）。 注意：某些实时PCR系统都带有可用于构建标准曲线，并计算未知分析软件。否则，这些计算可以在Excel或类似的程序来进行。一个例子的标准曲线示于图4。 </li><li>为了计算肺炎球菌坚持，从正常化条件b附着肺炎球菌的数量。 CCL-23细胞+肺炎球菌为100％，和肺炎球菌粘附比较从其他条件这个条件。 </li></ol></li></ol>

<ol>
	<li>O'Brien, K. L., 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=Burden+of+disease+caused+by+Streptococcus+pneumoniae+in+children+younger+than+5+years:+global+estimates.">Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates.</a> Lancet. 374, 893-902 (2009).</li><li>Bogaert, D., de Groot, R., Hermans, P. W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pneumoniae+colonisation:+the+key+to+pneumococcal+disease.">Streptococcus pneumoniae colonisation: the key to pneumococcal disease.</a> Lancet Infect. Dis. 4 (04), 144-154 (2004).</li><li>Weinberger, D. M., Malley, R., Lipsitch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotype+replacement+in+disease+after+pneumococcal+vaccination.">Serotype replacement in disease after pneumococcal vaccination.</a> Lancet. 378 (10), 1962-1973 (2011).</li><li>Jacoby, P., 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=Modelling+the+co-occurrence+of+Streptococcus+pneumoniae+with+other+bacterial+and+viral+pathogens+in+the+upper+respiratory+tract.">Modelling the co-occurrence of Streptococcus pneumoniae with other bacterial and viral pathogens in the upper respiratory tract.</a> Vaccine. 25, 2458-2464 (2007).</li><li>Kwambana, B., Barer, M., Bottomley, C., Adegbola, R., Antonio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+acquisition+and+high+nasopharyngeal+co-colonisation+by+Streptococcus+pneumoniae+and+three+respiratory+pathogens+amongst+Gambian+new-borns+and+infants.">Early acquisition and high nasopharyngeal co-colonisation by Streptococcus pneumoniae and three respiratory pathogens amongst Gambian new-borns and infants.</a> BMC Infect. Dis. 11, (2011).</li><li>Licciardi, P. V., 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=Protecting+against+pneumococcal+disease:+critical+interactions+between+probiotics+and+the+airway+microbiome.">Protecting against pneumococcal disease: critical interactions between probiotics and the airway microbiome.</a> PLoS Pathog. 8, (2012).</li><li>Popova, 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=Beneficial+effects+of+probiotics+in+upper+respiratory+tract+infections+and+their+mechanical+actions+to+antagonize+pathogens.">Beneficial effects of probiotics in upper respiratory tract infections and their mechanical actions to antagonize pathogens.</a> J. Appl. Microbiol. 113 (6), 1305-1318 (2012).</li><li>Pracht, 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=PavA+of+Streptococcus+pneumoniae+modulates+adherence,+invasion,+and+meningeal+inflammation.">PavA of Streptococcus pneumoniae modulates adherence, invasion, and meningeal inflammation.</a> Infect. Immun. 73, 2680-2689 (2005).</li><li>Adamou, J. E., Wizemann, T. M., Barren, P., Langermann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherence+of+Streptococcus+pneumoniae+to+human+bronchial+epithelial+cells+(BEAS-2B).">Adherence of Streptococcus pneumoniae to human bronchial epithelial cells (BEAS-2B).</a> Infect. Immun. 66, 820-822 (1998).</li><li>Wong, S. 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=Inhibition+of+Streptococcus+pneumoniae+adherence+to+human+epithelial+cells+in+vitro+by+the+probiotic+Lactobacillus+rhamnosus+GG.">Inhibition of Streptococcus pneumoniae adherence to human epithelial cells in vitro by the probiotic Lactobacillus rhamnosus GG.</a> BMC Res. Notes. 6 (1), 135 (2013).</li><li>Wescombe, P. A., Heng, N. C. K., Burton, J. P., Chilcott, C. N., Tagg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcal+bacteriocins+and+the+case+for+Streptococcus+salivarius+as+model+oral+probiotics.">Streptococcal bacteriocins and the case for Streptococcus salivarius as model oral probiotics.</a> Future Microbiol. 4, 819-835 (2009).</li><li>Di Pierro, F., Adami, T., Rapacioli, G., Giardini, N., Streitberger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+evaluation+of+the+oral+probiotic+Streptococcus+salivarius+K12+in+the+prevention+of+recurrent+pharyngitis+and/or+tonsillitis+caused+by+Streptococcus+pyogenes+in+adults.">Clinical evaluation of the oral probiotic Streptococcus salivarius K12 in the prevention of recurrent pharyngitis and/or tonsillitis caused by Streptococcus pyogenes in adults.</a> Expert. Opin. Biol. Ther. 13 (3), 339-343 (2013).</li><li>Fiedler, T., 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=Protective+mechanisms+of+respiratory+tract+streptococci+against+Streptococcus+pyogenes+biofilm+formation+and+epithelial+cell+infection.">Protective mechanisms of respiratory tract streptococci against Streptococcus pyogenes biofilm formation and epithelial cell infection.</a> Appl. Environ. Microbiol. 79, 1265-1276 (2013).</li><li>Slotved, H. C., Satzke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+growth+of+pneumococcal+isolates+representing+23+different+serotypes.">In vitro growth of pneumococcal isolates representing 23 different serotypes.</a> BMC Res. Notes. 6, 10-1186 (2013).</li><li>Carvalho, M. d. G. 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=Evaluation+and+improvement+of+real-time+PCR+assays+targeting+lytA,+ply,+and+psaA+genes+for+detection+of+pneumococcal+DNA.">Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA.</a> J. Clin. Microbiol. 45, 2460-2466 (2007).</li><li>Tonnaer, E. L. G. 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=Involvement+of+glycosaminoglycans+in+the+attachment+of+pneumococci+to+nasopharyngeal+epithelial+cells.">Involvement of glycosaminoglycans in the attachment of pneumococci to nasopharyngeal epithelial cells.</a> Microbes Infect. 8, 316-322 (2006).</li><li>Smith-Vaughan, H., 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=Measuring+nasal+bacterial+load+and+its+association+with+otitis+media.">Measuring nasal bacterial load and its association with otitis media.</a> BMC Ear Nose Throat Disord. 6, (2006).</li><li>Letourneau, J., Levesque, C., Berthiaume, F., Jacques, M., Mourez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+assay+of+bacterial+adhesion+onto+mammalian+epithelial+cells.">In vitro assay of bacterial adhesion onto mammalian epithelial cells.</a> J. Vis. Exp. , (2011).</li></ol>

该测定法的一个关键部分是加入S的适当浓度唾液和肺炎球菌在第3.1.7和3.1.12。浓度使用OD值估计，但确切的接种都没有确定，直到板计数第二天。出于这个原因，建议进行生长曲线测定OD和活菌数（CFU / ml）的随时间变化的测定中用于识别数中期，并协助由OD浓度估算所有的菌株。此外，肺炎球菌菌株可以在自己的坚持性方面都有很大差别。菌株具有良好的粘附性（&gt;接种3小时温育之...

肺炎链球菌 （肺炎球菌）是一种革兰氏阳性细菌，可引起感染，包括肺炎，中耳炎，脑膜炎。它是疾病的中低收入国家的儿童和负责估计有80万，每年死亡人数的1五岁以下儿童的主要病因。肺炎球菌是经常携带在幼儿的鼻咽部。虽然这定植通常被认为是无症状的，它先于肺炎球菌的感染，并作为贮存器为细菌在人群2。肺炎球菌结合疫苗接种有效地降低了运输包含在疫苗中的血清型。然而，有超过90肺炎球菌血清型，疫苗接种可导致血清型替换，从而消除疫苗血清型的后面是一个上升的马车和疾病引起的非疫苗血清型3。此外，在一些高危人群，肺炎球菌定植常发生在生命的早期，前第一剂疫苗4,5的管理。最近，使用益生菌已被提出作为一个额外的策略，以抑制肺炎球菌定植6,7。 体外粘附试验已被用于检查肺炎球菌粘附8,9。这些实验已经适应了调查益生菌鼠李糖乳杆菌 GG（LGG）对肺炎球菌坚持10的影响。 除了​​LGG和其他乳酸细菌， 唾液链球菌 ，口腔内的一种常见的居民，已被研究作为呼吸道一个潜在的益生菌，由于其潜在的定殖和抑制肺炎球菌和其它呼吸道病原体的体外能力11 - 13。这里介绍的协议描述用于调查S的影响的粘附试验唾液 K12对肺炎球菌坚持对人上皮细胞系CCL-23。在之前的实验使用，肺炎球菌菌株进行了评价粘附能力，生长和附着性可以有很大差异的菌株10,14之间。肺炎链球菌和S.的生长曲线唾液中执行通过光密度，以确定对数中期和估计浓度（菌落形成单位或CFU /毫升）（OD， 图1）。因此建议通过活菌数和OD检查生长的每个隔离在测定前使用。该测定可以在标准的组织培养设施和设备的任何实验室进行。在这个协议中，三个剂量的S的影响唾液给予1小时前对S的依从性增加肺炎球菌肺炎 PMP843，从鼻咽拭子派生的血清型19F车厢分离，进行检查。两种不同的方法来量化肺炎链球菌粘附呈现：电镀血琼脂阻止矿山活菌数，以及DNA提取和检测肺炎链球菌自溶素基因的qPCR 15。基本遵守试验方案可以很容易地修改，以测试不同剂量或益生菌的给药时间，也可以与其他的细菌菌株或物种中使用。

<table border="0" cellpadding="0" cellspacing="0" width="1318">
	<colgroup>
		<col />
		<col />
		<col />
		<col span="2" />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:264px;">Minimum Essential Media (MEM)</td>
			<td style="width:155px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">SH30244.01</td>
			<td style="width:377px;"> </td>
			<td style="width:377px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:264px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:155px;">Thermo Fisher Scientific</td>
			<td>SH30084.03</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">T-75 Flask</td>
			<td style="width:155px;">Nunc</td>
			<td>156472</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">CCL-23 cells</td>
			<td style="width:155px;">ATCC</td>
			<td style="width:147px;">CCL-23</td>
			<td style="width:377px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Trypsin</td>
			<td>Life Technologies</td>
			<td>15090046</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:264px;">24-well cell culture plate</td>
			<td style="width:155px;">Nunc</td>
			<td style="width:147px;">142475</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Horse blood agar (HBA) plates</td>
			<td style="width:155px;">Oxoid</td>
			<td style="width:147px;">PP2001</td>
			<td>Sheep blood agar plates also acceptable</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Todd-Hewitt Broth</td>
			<td style="width:155px;">Oxoid</td>
			<td style="width:147px;">CM0189</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Yeast Extract</td>
			<td style="width:155px;">Beckton Dickinson</td>
			<td style="width:147px;">212750</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Digitonin</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:147px;">D141-100MG</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Disposable cuvettes</td>
			<td style="width:155px;">Kartell</td>
			<td>1938</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Heparin</td>
			<td style="width:155px;">Pfizer</td>
			<td style="width:147px;">2112105</td>
			<td>comes in sterile ampules at 5,000IU/5mL </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">sterile spreader</td>
			<td style="width:155px;">Technoplas</td>
			<td style="width:147px;">S10805050</td>
			<td style="width:377px;">alternatively, a glass spreader can be dipped in 96% ethanol and flame sterilized before each use</td>
			<td style="width:377px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Lysozyme</td>
			<td>Sigma-Aldrich</td>
			<td style="width:147px;">L6876</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Mutanolysin</td>
			<td>Sigma-Aldrich</td>
			<td style="width:147px;">M9901</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Proteinase K</td>
			<td>Qiagen</td>
			<td>19133</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">SDS 20% solution</td>
			<td>Ambion </td>
			<td style="width:147px;">AM9820</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">RNase A</td>
			<td>Qiagen</td>
			<td style="width:147px;">19101</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">QIAamp DNA mini-kit (250)</td>
			<td>Qiagen</td>
			<td style="width:147px;">51306</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">LytA F primer</td>
			<td>Sigma-Aldrich</td>
			<td style="width:147px;">Custom made</td>
			<td>5&#39; -&#62; 3&#39; sequence ACGCAATCTAGCAGATGAAGCA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">LytA R primer</td>
			<td>Sigma-Aldrich</td>
			<td style="width:147px;">Custom made</td>
			<td>5&#39; -&#62; 3&#39; sequence TCGTGCGTTTTAATTCCAGCT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">LytA probe</td>
			<td>Eurogentec</td>
			<td style="width:147px;">Custom made</td>
			<td colspan="2">5&#39; -&#62; 3&#39; sequence Cy5-TGCCGAAAACGCTTGATACAGGGAG-BHQ3, alternative fluorophores can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Nuclease free water</td>
			<td>Ambion</td>
			<td style="width:147px;">AM9906</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Brilliant III Ultra Fast QPCR mastermix</td>
			<td>Agilent Technologies </td>
			<td style="width:147px;">600880</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Thermo-Fast 96 Detection plate </td>
			<td style="width:155px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">AB-1100</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:264px;">Ultra clear qPCR cap strips</td>
			<td style="width:155px;">Thermo Fisher Scientific</td>
			<td style="width:147px;">AB-0866</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Mx3005 QPCR System</td>
			<td style="width:155px;">Agilent Technologies </td>
			<td style="width:147px;">401449</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;"> </td>
			<td style="width:155px;"> </td>
			<td style="width:147px;"> </td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">* alternative sources are available for most/all products listed</td>
			<td style="width:147px;"> </td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating the effects of probiotics on pneumococcal colonization using an in vitro adherence assay

肺炎链球菌 （肺炎球菌）的坚持鼻咽上皮衬里可导致殖民化，被认为是肺炎球菌感染，如肺炎和中耳炎的先决条件。 体外粘附实验可以用来研究肺炎球菌的附着上皮细胞单层和调查潜在的干预措施，如使用益生菌，能抑制肺炎球菌定植。这里所描述的协议被用来研究对肺炎球菌的对人上皮细胞系CCL-23的粘附益生菌唾液链球菌的效果（有时也称为HEp-2细胞）。该法包括三个主要步骤：1）准备上皮细胞和细菌细胞，2）除细菌上​​皮细胞单层的，和3）检测肺炎链球菌粘附通过活菌数（连续稀释和电镀）或定量实时PCR（qPCR的）。这TEChnique是相对简单的，不需要专门的设备比组织培养的设置等。该法可用于测试其他益生菌种类和/或肺炎球菌定植的潜在抑制剂，并可以很容易地修改，以解决有关肺炎球菌粘附和侵袭等科学问题。

结果从有代表性的实验中，肺炎链球菌（ 肺炎链球菌 PMP843，一个定植19F分离物）加入到CCL-23细胞中加入S。后1小时唾液 ，和肺炎球菌粘附进行定量既活菌计数和LYTA的qPCR示于表2，结果是这两种方法对细菌的两个绝对数量（表示为菌落形成单位/毫升），％依从性，归一化到数之间是一致的的肺炎链球菌附着在仅含有肺炎球菌的水井。肝素被用作对照，因为?...

Watch this Scientific Journal Video about Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay at JoVE.com

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

体外坚持测定可用于研究肺炎链球菌的附着上皮细胞单层，并探讨潜在的干预措施，如使用益生菌抑制肺炎球菌定植。

使用一个调查益生素对肺炎球菌定植的影响<em&gt;体外</em&gt;粘附试验

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

investigating-effects-probiotics-on-pneumococcal-colonization-using

Research

JoVE Journal

Immunology and Infection

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

12.5K 次观看.  Murdoch Childrens Research Institute.  体外坚持测定可用于研究肺炎链球菌的附着上皮细胞单层，并探讨潜在的干预措施，如使用益生菌抑制肺炎球菌定植。 

视频: Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

在体外生物膜的形成以及8商会幻灯片

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

科研

免疫与感染

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

微孔板的检测，以评估RBL-2H3肥大细胞脱颗粒：三氯生的影响不使用有机溶剂的化学效应

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

荚膜血清型<em&gt;肺炎链球菌</em&gt;使用荚膜肿胀反应

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

荚膜血清型<em&gt;肺炎链球菌</em&gt;乳胶凝集

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

使用复分析定性测量细菌生产和敏感性，以肺炎球菌细菌素

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

一个<em&gt;体外</em&gt;血 - 脑屏障使用阻抗谱型号：对T细胞，内皮细胞的相互作用为中心

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

粒子结合Bet v 1的变应原PM10的流式细胞分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...