We would like to thank Henriette Mair-Meijers for invaluable attention to detail and hours devoted to processing the blood collections. We are grateful for the graphic design expertise of Natalie Jameson Kiesling. We also appreciate the approval of the manufacturers (Qiagen (Valencia, CA), BD Biosciences (San Jose, CA), Life Technologies (Grand Island, NY)) mentioned herein to publish the use of their products as described. Funding for this work was generously provided by the National Institutes of Health award numbers DA022720, RC1MH088283, and DA022720-05-S1.

底特律邻里健康研究进行了审查并批准了密歇根州的机构审查委员会的大学。所有参加者只要之前，他们在参与研究的知情同意书。 1.概述<ol><li>正确记录从招聘的各个阶段通过端点数据的分析。执行第1天同时协议，交换级和重叠时间允许。级的顺序被写入，以优化性能效率。 图1提供了整个过程的概述。 注：术语，“参与者”被用于整个来表示从个人同意的画去鉴定的血样。下面括号中的时间表示动手的时间。跟踪片和试样日志的实例可以在补充表S1和S2中找到，分别。样品通过在家庭抽血收集确定为快递同意参加由研究协调，并运送到实验室的个人（ 见补充信息（SI）1作进一步预处理的细节）的。 </li></ol> 2.第1天安装<ol><li>制备1×磷酸盐缓冲盐水（PBS）。 </li><li>预热加热块到56ºC。 </li><li>通过等分20微升到离心管当天的交付（每个参与者）相应的数字准备蛋白酶（见SI 2）。 </li><li>确保缓冲器AW1和AW2（在DNA提取试剂盒可提高DNA的纯度提供洗涤液），准备使用，增加100％的乙醇作为在标签上标明，如果必要的。 </li><li>降温冷冻离心机到4℃。 </li><li>替换在瓶子上的1×PBS的与所提供的橡胶隔帽盖和刺穿橡胶隔膜的转送穗，保持在转印的spi顶部的螺丝帽柯以防止蒸发。重复与瓶子的核糖核酸稳定溶液。 </li><li>下列制剂的缓冲剂，离心机和热块，记录对样品跟踪片的vacutainer交货的时间（见表SI）。 </li></ol> 3.第1天：处理全血样品的DNA，外周血单个核细胞和白细胞RNA（2小时） <ol><li>概观<ol><li>分配足够的时间，以尽量减少抽血和处理开始时刻之间的延迟。开始聚蔗糖含真空采血管的处理内2小时抽血，以避免增加红血细胞污染和在单核细胞回收的降低。 </li><li>假设集合2真空采血管含有聚蔗糖梯度凝胶，2，K 2乙二胺四乙酸（EDTA）真空采血管，以及每位成人与会者1血清真空采血管。交货时的真空采血管的实验室，有技术人员签字样本的跟踪表标志着验收。求与记录在每个真空采血管基础上，试件日志的开始时间立即处理。 </li></ol></li><li>血清的分离阶段1（1分钟） <ol><li>文件开始时间上的样本数（ 见表SI 2）。离心机（与制动和加速关）使用吊桶式转子与气雾剂帽7.0毫升血清（红色）的vacutainer（1每位与会者）（生物安全级别2; BSL2认证）20分钟，1300×g离心，4ºC。 </li></ol></li><li> DNA分离阶段1（15分钟） 注：有关此分离过程的详细信息，请参阅制造商的协议13。 <ol><li>文件开始时间。在一个BSL2细胞培养罩，转化含有聚蔗糖凝胶5倍然后添加200微升的全血从真空采血管中的一个的顶部到每个的蛋白酶的两个20微升等份（400微升总每位与会者）的真空采血管。离开蛋白酶+血的离心管在引擎盖，继续执行CENTRIfugation含有步骤3.4.1真空采血管的聚蔗糖的。 注意：使用真空采血管具有最大集合体积，牢记平衡离心机（也就是说，它可能有必要从每两个真空采血管的去除200微升）在步骤3.4.1纺丝真空采血管时的必要性。此外，为了确保蓝色真空采血管的处理期间适当分离，血液残留在真空容器的水平不应小于2.5英寸的聚蔗糖层上方。 </li></ol></li><li> PBMC的分离阶段1（1分钟） <ol><li>记录的开始时间（必须在2小时抽血，以避免增加红血细胞污染和在单核细胞回收减少）包含真空采血管的8-10倍.Invert聚蔗糖。离心机（带刹车和油门关闭），使用吊桶式转子气溶胶帽（BSL2认证）30分钟，1600 XG，22℃下的真空采血管（每个参与者）。 </li></ol></li><l我&gt; DNA分离阶段（20分钟） <ol><li>返回到引擎盖，并添加200微升缓冲AL每个蛋白酶+血的离心管（步骤3.3.1）的。盖，从发动机罩卸下，涡旋15秒，闪光灯旋转。 </li><li>孵育56ºC在热块中，10分钟。 </li><li>从加热块中取出试管。闪光旋转。返回到BSL2罩。加入200μl100％乙醇。盖，从发动机罩卸下，涡旋15秒，闪光灯旋转。其余的步骤可以在罩的外面完成。 </li><li>适用溶胞产物（内步骤3.5.3的30分钟），以标记的旋转柱（在2 ml收集管中）。关闭盖子通过气溶胶，以避免交叉污染。离心机6000 XG，1分钟。 </li><li>丢弃包含滤液收集管，并放置在旋转柱在新的2ml收集管中。 </li><li>加入500μlBuffer AW1于列，而不润燥的边缘，盖上，离心6000 XG，1分钟。 </li><li>丢弃包含FILTRA收集管德并将旋转柱在新的2ml收集管中。 </li><li>加入500μlBuffer AW2到列不润燥的边缘，盖上，离心20000 XG，3分钟。 </li><li>丢弃包含滤液收集管，并放置在旋转柱在一个新的1.5 ml收集管（未包括在试剂盒），和离心机20000×g离心，1分钟。 </li><li>丢弃包含滤液收集管，并放置在旋转柱在一个新的1.5 ml收集管（未包括在试剂盒）。 </li><li>加入200μl缓冲液AE或水到每个列和在室温下孵育5分钟。离心机6000 XG，1分钟。 </li><li>重复步骤3.5.11洗脱到同一个收集管。 </li><li>游泳池，距离每名参与者的2列洗脱的DNA。总产量〜800​​元参与者微升。 </li><li>使用分光光度计当时间允许定量样品。 </li><li>根据需要于2ml冷冻管，记录在specime每个浓度等分试样的DNA的n log。转移冻存管到一个cryobox和地点在-80℃长期储存。文档冰柜开始时间。 </li></ol></li><li>白细胞RNA分离阶段1（30分钟） 注意：请在生物安全2级认证的细胞培养罩。有关此隔离详见制造商的说明15。 <ol><li>皮尔斯K 2 EDTA真空采血管有转移尖峰的橡胶隔膜。保留外皮和螺丝帽在步骤3.6.11使用。继BSL2标准的做法，应注意避免接触血源性病原体。 </li><li>附加白滑连接器到转印穗的顶部。 </li><li>标记滤波器与相应参与者的ID则过滤器的入口（扩口端）连接到白滑连接器。 </li><li>附加的过滤器的护套25⅝ģ针到出口（锥形端）。准备过滤器组件交付前加快了程序，因此，附加的白滑连接器传输秒杀，加入过滤器，然后在护套针和设置的组件，培养管架，直至使用，值得推荐。 </li><li>以下的K 2 EDTA管系统的组装，安全地出鞘针（使用金属刮刀的末端）。刺针插入一个空10毫升真空采血管（血清接收管）和反转的K 2 EDTA真空采血管/过滤器/接收器管组件。继BSL2标准的做法，应注意避免接触血源性病原体。 </li><li>允许直到过滤器的楔形部分已清除血血液过滤通过。过滤需要约2分钟，在过滤期间可以放置在一个试管架上。 </li><li>取下组装过滤器。离开包含在滤液中的管针和丢弃整个组件在尖锐物容器。 </li><li>附加一个5毫升注射器向转印穗上的瓶的1×PBS的，在在用vert瓶，并撤销3毫升</li><li>附用PBS注射器到过滤器的入口和冲洗过滤器（3-5滴每秒）。收集流过的生物废料容器。分离来自过滤器的注射器，而不缩回柱塞。 </li><li>过滤后和一个PBS洗涤，使用新的5mL注射器，并且在步骤3.6.8中描述的方法撤回3毫升RNA稳定剂。冲洗过滤如在步骤3.6.9。在RNA稳定剂应保持在过滤器上。分离来自过滤器的注射器，而不缩回柱塞。 </li><li>密封该过滤器的入口和出口与护套和螺帽从转印道钉离开用RNA稳定剂的饱和的过滤器保留。该过滤器可以存储在这一点上。存储过滤在-80ºC到时间允许（〜2小时），完成步骤5.3至5.4.7。 注：储存在-80ºC长达一年采集后的过滤器没有增减已处理SE在RNA的质量。 </li></ol></li><li> PBMC的分离阶段2（30分钟） <ol><li>从离心机（步骤3.4.1）拆下菲含真空采血管，并继续在BSL2罩。真空采血管应显示分离如在图2中，如果不是， 见表 2。 </li><li>一旦真空容器被返回到BSL2罩，除去塞子和撤回1.5使用血清吸管顶端，淡黄，血浆层（图2）中的溶液没有得到接近单核（透明/白色）层。 （2真空采血管池 - - 1参与者）的等离子转移到5ml的离心管。记录收集的体积。请参阅存储说明的步骤3.7.6。 注意：最好分开和最伟大的外周血单个核细胞的产量来自于谁禁食至少12个小时，在采血前的参与者。 </li><li>转移剩余的血浆和白色，单核层（所有的凝胶层以上- 图2）U唱血清吸管，到15ml锥形管中，汇集从每两个聚蔗糖含有每参与者真空采血管的单个核层到一个锥形管中。 </li><li>加入1×PBS中，使在锥形管中的总体积至15ml。盖上盖子并翻转5倍。离心机（带刹车和油门关闭）15分钟，300 XG，22℃。 </li><li>返回在引擎盖的菲含真空采血管，并用5¾“巴斯德吸管周围流转，松开聚蔗糖凝胶层的外面，如果可能的话将其删除收集的红血细胞（红细胞）。使用血清吸管收集和红细胞（约为4.5毫升）转移至5ml的离心管。记录收集的体积。 </li><li>两个5 ml的冷冻管（等离子体在步骤3.7.2和红细胞在步骤3.7.5）转移到一个受控速率冷冻集装箱，并把在-80ºC至少24小时后，此时可以将它们转移至cryobox并返回到-80ºC用于长期存储。 </li><li>返回合作nical管罩，当在步骤3.7.4离心是完整的，并且吸出所有但〜500微升的PBS，而不会干扰沉淀。 PBMC收率大于如果〜200微升PBS留沉淀在这个阶段以上。 </li><li>添加新鲜的1×PBS使体积至10ml。轻轻悬浮颗粒。盖上盖子并翻转5倍。离心机（带刹车和油门关闭）10分钟，300 XG，22℃。 </li></ol></li><li>血清的分离阶段2（10分钟） 注意：请在BSL2罩。 <ol><li>从分装在真空采血管血清前血清层，离心后（步骤3.2.1），于2ml冻存管的期望。典型产量是2.5毫升（ 表1）。日志的卷。例如，使用4冷冻管等分200μl的冷冻管入1，1000微升到离心管2，然后除以剩余的冷冻管入3和4。 </li><li>转移到离心管一个cryobox和地点在-80ºC用于长期贮存。文件冰柜启动时间。 </li></ol></li><li> PBMC的分离阶段3（15分钟） <ol><li>返回到发动机罩，离心分离（步骤3.7.8），并吸出尽可能多的上清液/ PBS作为可能的后不干扰沉淀。悬浮颗粒吹打于2.5毫升外周血单个核细胞冻存液1（见SI 2）。 </li><li>添加2.5 ml的PBMC的冷冻培养基2（参照SI 2），以在步骤3.9.1细胞/培养基溶液。轻轻涡旋。 </li><li>分装10微升细胞溶液到0.65 ml离心管（进一步稀释可能是必要的）。加入10微升0.4％台盼蓝染色为0.65 ml离心管中，吹打混匀数次。更多详情请参见制造商的手册16。 </li><li>吸取10微升混合物的导入细胞计数室滑动件和地方滑进细胞计数器内混合3分钟。放大和聚焦的单元格。按“细胞计数”来获取外周血单个核细胞计数。 </li><li>等分试样中，如果可行的PBMC数目大于每毫升3百万细胞（MC / ml）的，根据需要进冷冻管并继续到步骤3.9.9。商店的PBMC中高达5的冷冻管在至少3 MC / ml的每种的浓度。 </li><li>如果可行的PBMC数量低于3 MC /毫升，通过乘以可行MC /毫升由5毫升计算细胞总数。除以该数乘以4，3，2或1毫升，以便将有至少一个管具有3 MC /毫升。 </li><li>离心锥形管包含细胞/冷冻介质5分钟，在300×g离心（制动和加速OFF）溶液。离心后，吸的冷冻介质（上清液）适当体积离开上述（4，3，2或1毫升）中计算出的体积。 </li><li>重悬沉淀，在剩余的上清液和等分至少3 MC /毫升到冷冻管（1-4）的适当数目以1毫升/冷冻管。最后冷冻介质是10％二甲基亚砜（DMSO）/ 20％胎牛血清（FBS）/ 70％罗斯韦尔园区纪念研究所（RPMI）1640。 </li><li>解决的文耳鼻喉科每个离心管的细胞计数。转移到离心管的受控速率冷冻集装箱，并把在-80ºC至少24小时之后将冷冻管可以被转移到一个cryobox并放入液氮罐（气相），用于长期存储。文档冰柜启动的时间。 </li></ol></li></ol> 4.第2天安装<ol><li>加热的不含核酸酶的0.1毫摩尔EDTA等分试样（每滤波器220微升）在一个无核酸管至80℃的热块。 </li><li>制备洗涤溶液1和2（见SI 2）。 </li></ol> 5.第2天：长期样品储存及白细胞过滤处理（3小时） <ol><li>概述。 <ol><li> 6个月的白细胞到过滤器的应用中执行此过程。 注意：总的来说，第2天的处理时间约3小时，虽然它被标记为“第2天”的关键要求是，滤波处理部应于一天的时被执行有在实验室中发生的任何1天的处理。 </li></ol></li><li>长期样品储存（15分钟） <ol><li>每天执行此过程中，交付新的真空采血管实验室进行1天的处理之前。传输该被储存的O / N的受控速率冷冻集装箱在第1天的冷冻管入适当标记cryoboxes，并将它们返回到-80ºC或液态氮（气相），用于长期存储。组织根据样品类型（例如，DNA，PBMC，红细胞等 ），以加速样品位置跟踪端点分析的冷冻管。 </li></ol></li><li>白细胞的RNA的过滤器处理中（45分钟）。 注：有关此过程的详细信息，请参阅生产商的说明17。 <ol><li>把过滤器RT（解冻约5分钟）。 </li><li>除去所述护套和螺丝帽从过滤器。缩回5毫升注射器的柱塞和其连接到过滤器的入口（扩口端），压下柱塞驱逐RNA稳定剂 从过滤口进入生物废料的容器。 </li><li>装入新的5mL注射器用4ml用于RNA分离的酚和异硫氰酸胍溶液，并将它附加到过滤器的入口，压下柱塞以冲洗通过过滤器的溶液，收集裂解物中的标记的15毫升锥形管（2％的参与者）。 </li><li>从过滤器上断开注射器，缩回柱塞，它重新连接到过滤器，并压下柱塞以排出收集在过滤器的磁盘残差采样到同一的15毫升锥形管中。丢弃过滤器和注射器。 </li><li>加入800微升BCP的锥形管，收管紧密，大力摇晃准备，持续30秒。 </li><li>在室温下孵育5分钟。离心机在2000×g下10分钟。 </li><li>水性（顶部）相转移到新鲜标记的15毫升锥形管（〜2.5毫升）中。 </li><li>加入的无核酸酶水的水相的0.5倍体积ð拌匀。再加入1.25倍的100％乙醇的含水量，并再次混合。 注意：对于2.5毫升的水的体积，增加1.25不含核酸酶的水毫升，混匀，再加入4.7毫升100％的乙醇（2.5毫升×0.5 = 1.25毫升无核酸酶的水，然后2.5毫升+ 1.25毫升等于3.75毫升新水容积THEN 3.75毫升×1.25 = 4.7 ml的100％乙醇）。这个步骤允许包含小RNA级分的总RNA分离。 A到忽略从隔离的小RNA的方法可以在手册17上找到。 </li><li>从5毫升注射器中取出柱塞和在原处插入旋转筒。附加盒/注射器组件到真空歧管。供到真空歧管更安全的连接，将墨盒/注射器组件内的1.5 ml离心管与底切断。 </li><li>从步骤5.3.8至纺丝筒慢慢地用在真空应用样品，小心地添加更多的样品，因为它是通过盒拉出。 注：如果没有真空现在，看到http://www.ambion.com/techlib/misc/leuko_iso.pdf离心法。 </li><li>自旋盒转移到1.5 ml离心管，并添加洗1.离心机750微升自旋盒/管组件5 - 10秒以12,000×克。 </li><li>从管丢弃滤液和自旋盒返回到相同的离心管中。 </li><li>加入洗净2离心机的750微升旋转筒/管5 - 10秒12,000×g的。弃滤液作为步骤5.3.12。返回自旋盒相同的离心管中。 </li><li>与其他750微升2洗净和离心时，重复步骤5.3.13。弃滤液。返回自旋盒相同的离心管中。离心纺丝筒/管以最大速度1分钟以干燥过滤器。 </li><li>旋转筒转移到一个新的，标记为1.5微升离心管中。 </li><li>加入200微升不含核酸酶的0.1毫摩尔EDTA（预热到80＆＃176℃）的旋筒式过滤器（每个参与者）的中心。在室温下孵育1分钟。 </li><li>离心1分钟，在12000×g离心，以洗脱RNA。保留滤液。丢弃旋筒。 </li><li>斯普利特各200微升滤液分为二，100微升等分到新的1.5 ml离心管中，标签和保持在冰上。在这一点上，从每个与会者2过滤器将得到每名参赛者RNA四个100微升等份。 </li></ol></li><li> DNase处理（1小时） 注：有关此处理的详细信息请参阅制造商的协议18。 <ol><li>通过将10微升的DNA酶我缓冲区和2微升rDNase我每等分+ 1（以弥补移液错误）创建一个主结构。 注意：对于四个样品，100微升等分使用50微升的DNA酶我缓冲区+ 10微升rDNase I. </li><li>分装12微升的预混步骤5.4.1到每个RNA等分的步骤5.3.18中，轻轻混匀。在37°下，在加热块30分钟。 </li><li>旋涡DNA酶失活试剂，并添加11.2微升到每个等份，混合均匀。在室温下孵育2分钟温育过程中涡旋的2-3倍。 </li><li>离心在10,000rpm xg离心1.5分钟。 </li><li>上清液（RNA）转移到一个新的1.5毫升管。从同一个参与者等分试样可以在这里汇集。 </li><li>运行在分光光度计每个样品得到的浓度。使用生物分析仪（见步骤5.5）建议在RNA的质量分析。 </li><li>分装成RNA作为冷冻管所需。如果一个生物分析仪分析将不会立即等分进行1.5微升样品放入离心管用于以后的分析。记录浓度和冷冻开始时间。样品储存在-80℃。 </li></ol></li><li>生物分析仪分析（1小时） <ol><li>按照制造商的协议19。当运行完成后，数据自动存储，但再次保存它瓦特第i个更小的，更容易识别的文件名。预期的结果（图3）：阶梯应 ​​具有6峰，样品应当具有3个峰（在44秒和50秒，分别并在25秒1早期标记物峰2的核糖体的峰）。 </li></ol></li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytomegalovirus+antibodies+as+a+marker+of+immunosenescence+in+the+Detroit+Neighborhood+Health+Study.">Cytomegalovirus antibodies as a marker of immunosenescence in the Detroit Neighborhood Health Study.</a> , (2010).</li><li>Bustamante, A. 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=.">.</a> Distinct gene expression profiles characterize lifetime PTSD and childhood maltreatment in the Detroit Neighborhood Health Study. , (2013).</li></ol>

作者宣称，他们有没有竞争的利益。

我们描述了一个精简的协议，已成功地应用于处理超过1600的全血样本底特律邻里健康研究进展。虽然许多这些技术可在现有的文献中，我们的一步一步汇编，包括每个步骤之间精确计时的改变，反映了成功地生产各种生物样本具有广泛的下游应用的优化，高效的协议，包括DNA甲基化，表达和免疫学分析。这些标本已经在各种实验，结果其中已发表 ​​在同行评审文献和/或提出了在国家会议11...

多项研究特征的基因表达，DNA甲基化和细胞亚群中的血液有和没有精神（或其它）毛病1-4个体间的差异。这些研究中，但是，已经从其中疾病相关的差异可能由于对哪个患者寻求治疗的疾病的一般更严重的自然放大临床环境获得。由于“组学”方法的进步，在过去十年的利息获得来自社区和/或流行病学设置5-7个生物样品，以提供疾病患病率人群为基础的估计爆炸和在一个更广阔的画面这些心理和/或身体疾病的环境因素。 这方面的一个主要挑战是对采集标本的快速处理的要求。单核细胞，关键免疫系统组件是frequentl降解ÿ用于评估个体的健康，立即开始在抽血用2小时收集8-10的后一个显著下降恢复。为了应对这一挑战，我们提出了一个优化的协议，其中全血多个组件同时从生活在大都市圈科目家庭获得的样品中分离。该协议是基于我们的汇编和当前技术，包括存储在事件未来技术允许进一步隔离所有的“额外”的级分的修饰/分析。而替代方法或试剂盒可以代替这里描述的各个方法，这些概述可以采用已被证明是用于在高通量的方式处理样品的可靠和有效的手段。高品质的级分（PBMC中，脱氧核糖核酸，血清，和RNA）的新鲜血液，可以在2小时收集的和所有的测定就绪标本制作可在2 天 （图1可）。 该协议的开发，使以社区为住宅，进行测试底特律邻里健康研究在底特律市的成年居民收集的样本有效地处理（DNHS; DA022720，RC1MH088283，DA022720-05-S1），以人口为基础研究创伤后应激障碍（PTSD）等精神疾病的社会和生物决定因素。创伤后应激障碍的底特律患病率超过全国平均数的两倍11,12。确定创伤后应激障碍的生物因素在这一人群可能有助于制定相应的药物和/或认知行为干预措施，以帮助那些来自错乱，无论是在这个城市的人口，而在其他高危人群 （如，返回退伍军人）。我们的实验室，以前位于韦恩州立大学在底特律，密歇根州，被选定为基于我们的专业知识在处理来自各种来源的新鲜组织标本处理来源，有必要开始2小时内收集处理的样品，并背靠集合地点。手头这次难得的机会，我们的目标是优化处理，从每个样本的DNA，RNA，血清和外周血单个核细胞（PBMC）中的最大收益率（总N = 1639的样品超过5浪标本采集）。这里所概述的程序，可以在非临床环境同时进行，由此产生用于多种下游应用，包括微阵列，后生，实时RT-PCR起始材料（见表1为平均产量），和流式细胞仪分析。 <img alt="图1" src="/files/ftp_upload/52227/52227fig1highres.jpg" width="700" /> 图1.整体工作流程。这里描述的整个过程包括取得血液样本的物流，从确定同意participants血液绘制自身。高品质的，级分（外周血单核细胞;外周血，脱氧核糖核酸，血清，和RNA）的新鲜全血可以在2小时收集的产生以及所有测定就绪标本可在2天内使用。而且，通过这种方法制得的级分都适合长期储存，如果样本不被立即测试。这里列出的整个时间线可以在一天内（〜5小时计）内完成。然而，这样的天将是极为劳动密集特别是对于具有与技术相当的经验的单个技术员。因此，我们建议分在第一天的过程至少有两名技术人员之间并完成第2天的RNA加工<a href="https://www.jove.com/files/ftp_upload/52227/52227fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>QIAamp DNA Blood Mini Kit</td>
			<td>Qiagen</td>
			<td>51104</td>
			<td>Day 1: DNA isolation</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>P5493-1L</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>5 &#190;&#8221; Pasteur pipets</td>
			<td>Fisher</td>
			<td>13-678-6A</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), heat inactivated</td>
			<td>Life Technologies</td>
			<td>10082147</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D8418-500ml</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>RPMI Medium 1640, liquid</td>
			<td>Invitrogen</td>
			<td>11875119</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>0.4% trypan blue stain&#160;</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>Countess Automated Cell Counter or cell counting device such as a microscope and hemocytometer</td>
			<td>Invitrogen</td>
			<td>C10281</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>LeukoLOCK Fractionation &#38; Stabilization Kit&#160;</td>
			<td>Ambion</td>
			<td>1933</td>
			<td>Day 1: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>25G x 5/8 in. needles</td>
			<td>Becton Dickinson</td>
			<td>305122</td>
			<td>Day 1: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>Syringes (5ml)</td>
			<td>Becton Dickinson</td>
			<td>309646</td>
			<td>Days 1 and 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>Denaturing Lysis Solution&#160;</td>
			<td>Ambion</td>
			<td>8540G</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>5M NaCl</td>
			<td>Life Technologies</td>
			<td>24740011</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>TRI Reagent&#160;</td>
			<td>Ambion</td>
			<td>9738</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>Bromo-3-chloro-propane (BCP)</td>
			<td>Sigma</td>
			<td>B-9673</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>spin cartridges&#160;</td>
			<td>Ambion</td>
			<td>10051G</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>0.1mM EDTA</td>
			<td>Ambion</td>
			<td>9912</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>DNA-free Kit</td>
			<td>Ambion</td>
			<td>AM1960</td>
			<td>Day 2: DNase treament</td>
		</tr>
		<tr>
			<td>RNA 6000 Ladder&#160;</td>
			<td>Agilent</td>
			<td>5067-1529</td>
			<td>Day 2: Bioanalyzer analysis</td>
		</tr>
		<tr>
			<td>RNA 6000 Nano Series II Kit&#160;</td>
			<td>Agilent</td>
			<td>5067-1511</td>
			<td>Day 2: Bioanalyzer analysis</td>
		</tr>
		<tr>
			<td>RNaseZAP</td>
			<td>Ambion</td>
			<td>AM8782</td>
			<td>Day 2: Bioanalyzer analysis</td>
		</tr>
		<tr>
			<td>Ethanol &#62;99%</td>
			<td>Sigma</td>
			<td>E7023-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol &#62;99%&#160;</td>
			<td>Sigma</td>
			<td>I9516-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free ultra pure water&#160;</td>
			<td>Invitrogen</td>
			<td>9938</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips (nuclease-free)</td>
			<td>Eppendorf</td>
			<td>22491253</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetter (serological)</td>
			<td>Eppendorf</td>
			<td>2223020-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetters (for volumes under 1ml)</td>
			<td>Eppendorf</td>
			<td>3120000-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes (serological)</td>
			<td>Fisher</td>
			<td>13-678-27E</td>
			<td></td>
		</tr>
		<tr>
			<td>Controlled rate freezing containers</td>
			<td>Nalgene</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryoboxes (to hold 2ml and 5ml cryovials and 1.5ml microcentrifuge tubes)</td>
			<td>Fisher</td>
			<td>03-395-464</td>
			<td></td>
		</tr>
		<tr>
			<td>Test tube rack</td>
			<td>Thermo Scientific</td>
			<td>14-804-134</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml polypropylene tubes&#160;</td>
			<td>Fisher</td>
			<td>14-959-49D</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml and 0.65 microcentrifuge tubes</td>
			<td>Fisher</td>
			<td>07-200-534 and 07-200-185</td>
			<td></td>
		</tr>
		<tr>
			<td>2ml and 5ml cryovials</td>
			<td>Fisher</td>
			<td>10-500-26 and 10-269-88F&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>8ml CPT vacutainer</td>
			<td>BD Biosciences</td>
			<td>362761</td>
			<td>2 tubes</td>
		</tr>
		<tr>
			<td>6ml K2 EDTA vacutainer</td>
			<td>BD Biosciences</td>
			<td>367863</td>
			<td>2 tubes</td>
		</tr>
		<tr>
			<td>8.5ml SST vacutainer</td>
			<td>BD Biosciences</td>
			<td>367988</td>
			<td>1 tube</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Fisher</td>
			<td>2215365</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry bath incubator with heating block for microcentrifuge tubes</td>
			<td>Fisher</td>
			<td>11-715-1250</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtration/vacuum system for use within the cell culture hood</td>
			<td>Fisher</td>
			<td>01-257-87</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed-angle rotor for microcentrifuge tubes with aerosol-tight lid</td>
			<td>Eppendorf</td>
			<td>22637002</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge with a swing-bucket rotor and aerosol-tight caps for 16 x 125mm vacutainers and 15ml polypropylene tubes&#160;</td>
			<td>Eppendorf</td>
			<td>22628157</td>
			<td>2, one does not need to be refrigerated</td>
		</tr>
		<tr>
			<td>Nanodrop 2000 (recommended for accuracy of small volumes) or other spectrophotometric device</td>
			<td>Fisher</td>
			<td>13-400-411</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2940CA</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen tank</td>
			<td>Thermo Scientific</td>
			<td>11-676-56</td>
			<td></td>
		</tr>
		<tr>
			<td>-80 &#730;C freezer</td>
			<td>Thermo Scientific</td>
			<td>992RAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharps container</td>
			<td>Fisher</td>
			<td>22-037-970</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological waste container</td>
			<td>Thermo Scientific</td>
			<td>1223P52</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Level 2&#160; certified cell culture hood</td>
			<td>Thermo Scientific</td>
			<td>13-261-315</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid fractionation and isolation of whole blood components in samples obtained from a community-based setting

采集和全血样本的处理在非临床环境提供了一个独特的机会来评价社区居住的人有和没有既往病史。这些样品的快速处理是必不可少的，以避免关键细胞成分的降解。这里包括用于同时外周血单核细胞（PBMC），DNA方法，RNA和从在跨首都圈同意参加者的家中进行的单一血液抽取血清隔离，与处理2小时内收集的启动。我们已经使用了这些技术来处理超过1600的血标本产生一致的，高品质的材料，后来被用在成功的DNA甲基化，基因分型，基因表达和流式细胞仪分析。一些采用的方法是标准;然而，当在所描述的方式相结合，它们使样品的高效的处理从与人口和/或社区的参与者基础的研究谁也不会在临床环境通常进行评估。因此，该协议具有以获得样本（以及随后的数据），该比较有代表性的普通人群的潜力。

至关重要的是，在整个过程生产出高品质的材料进行分析，在众多的下游应用，包括通过微阵列和RT-PCR分析基因表达，检测表观遗传修饰，和细胞亚群的变化。 表1表示的平均产量的材料和质量从每个过程。 图3提供的白细胞RNA分离和滤波处理方法的质量输出的一个例子。关于图3的左上方的图像是从毛细管电泳所得的凝胶图像。每个通道应该产生两个不同的?...

Watch this Scientific Journal Video about Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting at JoVE.com

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

快速分馏和全血的成分的分离样品中从社区为基础的设置获得

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

rapid-fractionation-isolation-whole-blood-components-samples-obtained

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JoVE Journal

Medicine

15.9K 次观看.  University of Illinois at Urbana-Champaign. We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

视频: Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

快速点护理依诺肝素的抗凝血剂功效的测定全血中

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

科研

医学

The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides1. Thus, identification of cell-free correlates that directly regulate the number of CD4+ T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates.
The number of stem cells that enter blood and are destined to become circulating CD4+ T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLECS) and the HLECS-reactive active &alpha;1proteinase inhibitor (&alpha;1PI, &alpha;1antitrypsin, SerpinA1)3. In HIV-1 disease, &alpha;1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active &alpha;1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 &mu;M, and to achieve normal levels during the symptomatic categories4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with &gt;220 CD4 cells/&mu;l, CD4 counts were correlated with serum levels of active &alpha;1PI (r2=0.93, p&lt;0.0001, n=26) and inactive &alpha;1PI (r2=0.91, p&lt;0.0001, n=26) 5. Administration of &alpha;1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting &alpha;1PI participates in regulating the number of CD4+ T cells in blood 3.
With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active &alpha;1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum &alpha;1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to &alpha;1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active &alpha;1PI in saliva. The resulting inhibition of PPE by active &alpha;1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/&mu;l), the concentration of &alpha;1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable7. Thus, active &alpha;1PI in saliva is calculated as a ratio to saliva protein content and is termed the &alpha;1PI Index. Results presented herein demonstrate that the &alpha;1PI Index provides an accurate and precise physiologic method for calculating CD4 counts.

The &amp;#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

A CD4 enumeration method, the &#x03B1;-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The &alpha;-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.

测试：α-细胞快速免费的CD4计数，使用全唾液

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

设置了一个<em&gt;体外</em大鼠血脑屏障&gt;模型（BBB）：在BBB抗渗性和受体介导的运输为中心

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

收集，分离，和人类宫颈样品的流式细胞仪分析

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

实时的细胞毒性分析在人全血

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

从整体组织和活检建立人类上皮Enteroids和Colonoids的

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.