This research was supported by a grant from the NSERC Discovery program grant number RGPIN 418522-2013. We thank R. Kilgour for helpful discussions, and Katelin Gresty and Dr. A. Berghdal for providing the mouse muscle sections.

研究与人类血液样本被批准Concordia大学伦理审查委员会，证书编号10000618.批准了康考迪亚大学伦理审查委员会，证书编号2010BERG对小鼠肌肉的工作。 从全血1 PBMC隔离注:执行此过程中使用无菌技术和制造商消毒设备生物安全柜。 <ol><li>小心倒入10-15毫升的肝素化（抗凝血剂）血液收集管的全血到50ml无菌锥形管中。对于轻微的血液外溢，使用清洁纸巾擦拭DDH 2 O的70％乙醇。 </li><li>稀释血液在锥形管以1:1的比例用磷酸盐缓冲盐水（PBS 1×）pH为7.4。确保最大总体积不超过30毫升</li><li>轻轻混匀，用血清学移液枪，避免气泡。 注意:不要颠倒离心管，以避免血液混合ccumulation在离心期间在盖和随后的渗流。 </li><li>添加13毫升菲可-帕克（见材料和设备表 ），以一个新的50毫升容量锥形管。保持直立管机架中。 </li><li>使用移液管，取稀释血液，触摸转印枪头靠近顶部管的内壁上。以缓慢而稳定的压力，转移的血液沿管上形成的蔗糖层的顶部上的血液层的内侧壁上。做到这一步几次，直到所有的血液已经转移。 </li><li>盖上管紧密，离心管在室温下放置30分钟，于700×g离心以摆动转子具有中等加速度设定为5，并减速置零。 </li><li>慢慢取出管，而不会干扰层，把它收回来的生物安全柜。 </li><li>仔细收集白膜层（细阴天白层），其中外周血单个核细胞的位置，放置在PBS /等离子之间的一次用Ficoll层的移液管。避免收集蔗糖层和不打扰红血细胞层。 </li><li>转移棕黄层到一个新的50毫升无菌锥形管中。它可能需要重复步骤1.8至收集所有的PBMC的几倍。 </li><li>加入PBS pH值7.4，与PBMC管，并填写到45毫升大关。彻底动摇管。不要旋涡。 </li><li>洗1:离心15分钟，在480×g离心既设置为9，观察在锥形管的底部形成的PBMC的颗粒的最大加速度和减速度。 </li><li>弃去PBS（上清液）到塑料烧杯中，用10ml漂白剂。 </li><li>轻轻"货架"对凹凸表面（ 即空架）松开细胞沉淀。不要旋涡。 </li><li>加25毫升新鲜的PBS pH值7.4到管。如果超过一个管正在处理，凝聚颗粒一起在这个步骤。 </li><li> 12分钟，在480 XG无线离心管:洗2TH均设置为9最大加速度和减速度。 </li><li>弃PBS（上清液）放入塑料烧杯中的漂白剂飞溅。 </li><li>轻轻的"机架"对凹凸表面（ 即空架）。不要旋涡。 </li><li>加25毫升新鲜的PBS到管。 <ol><li>取出50微升细胞，并转移到微量离心管中的生存力计数。 </li><li>加入台盼蓝（一生存能力染色）和吸管了等量（50微升），向下轻轻混匀。 </li></ol></li><li>取出10微升，并将其转移到血细胞计数检查每ml细胞的活力。记录的细胞/ ml的数量。 </li><li>取出细胞​​的所需量和离心反应管12分钟，在480×g离心既设置为9最大加速度和减速度。 </li><li>弃PBS（上清）与漂​​白剂的烧杯中。 </li><li>轻轻的"机架"对凹凸表面（ 即空架）。添加80微升PBS入管。 </li></ol> 2.制作的幻灯片PBMC <ol><li>放置80微升细胞到显微镜载玻片上。涂抹滴与另一滑动的帮助下或放置2滴各40微升在滑动的两端。 </li><li>离开幻灯片在生物安全柜干燥。标签上的磨砂面铅笔的幻灯片。 </li></ol> 3.固定在滑轨的样本<ol><li>通过混合0.5毫升37％甲醛与4.5毫升99％的乙醇制备的固定剂溶液。 </li><li>后的载玻片干燥，取出将2ml新鲜制备固定液和在滑动倒使得滑动的整个表面覆盖。 </li><li>离开在滑动1分钟的溶液中。冲洗幻灯片1分钟，用自来水，让它自然风干。 </li></ol> 4.制作的淀粉酶溶液为阴性对照<ol><li>就拿0.25克淀粉酶粉溶解在50毫升迪平息水。倒入一个干净的100毫升烧杯中的溶液中。 </li><li>沉浸在烧杯中的幻灯片，以便所述幻灯片的一半接收处理，而另一半保持未处理，然后孵育在室温下（ 图1C）15分钟。 </li><li>请记在其上滑​​动侧接受淀粉酶处理。绘制在幻灯片的背面表示治疗组和对照之间的边界线。 </li><li>与DDH 2 O的洗载玻片以除去淀粉酶溶液，并使滑动风干。 </li></ol> 5.执行碘酸雪夫（PAS）染色和成像注:PAS试剂是由吸入有毒和有腐蚀性的，所以步骤需要在化学通风橱中进行，且废产品必须妥善处置根据机构方针的。 <ol><li>放置在滑动在平坦表面上，倒在样品1.50-2.00毫升碘酸解的。 Incuba德5分钟，在室温下进行。 </li><li>冲洗载玻片在几个收费的蒸馏水。 </li><li>倾1.50-2.00毫升席夫试剂在滑动孵育它在室温下15分钟。 </li><li>洗用蒸馏水滑动5分钟并使其保持在空气中干燥。 </li><li>适用于10微升安装介质上的幻灯片，并盖上两个小盖玻片，或申请50微升，并使用一个大的盖玻片。 </li><li>应用透明指甲油盖玻片的边缘，让干燥过夜。 </li></ol> 6.获取图像与双目光学显​​微镜使用100X目标

<ol>
	<li>Rich, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+machinery+of+keilin's+respiratory+chain.">The molecular machinery of keilin's respiratory chain.</a> Biochem. Soc. Trans. 31 (Pt 6), 1095-1105 (2003).</li><li>Peter, J. B., Barnard, R. J., Edgerton, V. R., Gillespie, C. A., Stempel, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+profiles+of+three+fiber+types+of+skeletal+muscle+in+guinea+pigs+and+rabbits).">Metabolic profiles of three fiber types of skeletal muscle in guinea pigs and rabbits).</a> Biochemistry. 11 (14), 2627-2633 (1972).</li><li>Jones, R. V., Goffi, G. P., Hutt, M. S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+glycogen+content+in+various+disease.">Lymphocyte glycogen content in various disease.</a> J. Clin. Pathol. 15 (1), 36-39 (1962).</li><li>Scott, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+in+human+peripheral+blood+leukocytes.+I.+characteristics+of+the+synthesis+and+turnover+of+glycogen+in+vitro.">Glycogen in human peripheral blood leukocytes. I. characteristics of the synthesis and turnover of glycogen in vitro.</a> J. Clin. Invest. 47 (2), 344-352 (1968).</li><li>Fedele, 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=positive+index+of+lymphocytes+and+metabolic+control+in+insulin-treated+and+type+II+diabetes+mellitus.">positive index of lymphocytes and metabolic control in insulin-treated and type II diabetes mellitus.</a> Diabete Metab. 9 (3), 188-192 (1983).</li><li>Breli&#324;ska-Peczalska, R., Mackiewicz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochemical+studies+of+peripheral+blood+granulocytes+and+lymphocytes+in+patients+with+systemic+lupus+erythematosus.">Cytochemical studies of peripheral blood granulocytes and lymphocytes in patients with systemic lupus erythematosus.</a> Pol.Med.Sci.Hist.Bull. 15 (2), 231-234 (1976).</li><li>Yunis, A. A., Arimura, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymes+of+glycogen+metabolism+in+white+blood+cells.+I.+glycogen+phosphorylase+in+normal+and+leukemic+human+leukocytes.">Enzymes of glycogen metabolism in white blood cells. I. glycogen phosphorylase in normal and leukemic human leukocytes.</a> Cancer, Res. 24, 489-492 (1964).</li><li>Hagemans, M. 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=PAS-positive+lymphocyte+vacuoles+can+be+used+as+diagnostic+screening+test+for+pompe+disease.">PAS-positive lymphocyte vacuoles can be used as diagnostic screening test for pompe disease.</a> J. Inherit. Metab. Dis. 33 (2), 133-139 (2010).</li><li>Totsuka, Y., 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=Physical+performance+and+soleus+muscle+fiber+composition+in+wild-derived+and+laboratory+inbred+mouse+strains.">Physical performance and soleus muscle fiber composition in wild-derived and laboratory inbred mouse strains.</a> 95 (2), 720-727 (2003).</li><li>Murat, J. C., Serfaty, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+enzymatic+determination+of+polysaccharide+(glycogen)+content+of+animal+tissues.">Simple enzymatic determination of polysaccharide (glycogen) content of animal tissues.</a> Clin. Chem. 20 (12), 1576-1577 (1974).</li><li>Arrizabalaga, O., Lacerda, H. M., Zubiaga, A. M., Zugaza, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1+protein+regulates+glycogen+phosphorylase+activation+and+controls+interleukin+(IL)-2-dependent+T+cell+proliferation.">Rac1 protein regulates glycogen phosphorylase activation and controls interleukin (IL)-2-dependent T cell proliferation.</a> J. Biol. Chem. 287 (15), 11878-11890 (2012).</li><li>Pelletier, J., G, J., Mazure, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+titration+of+glycogen+in+vitro.">Biochemical titration of glycogen in vitro.</a> J.Vis.Exp. (81), (2013).</li><li>Roach, P. J., Depaoli-Roach, A. A., Hurley, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tagliabracci+V.S.+Glycogen+and+its+metabolism:+Some+new+developments+and+old+themes.">Tagliabracci V.S. Glycogen and its metabolism: Some new developments and old themes.</a> Biochem.J. 441 (3), 763-787 (2012).</li><li>Salmoral, E. M., Tolmasky, D. S., Krisman, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+presence+of+glycogen+in+rat+thymus.">Evidence for the presence of glycogen in rat thymus.</a> Cell Mol.Biol. 36 (2), 163-174 (1990).</li><li>Darlington, P. J., 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=Diminished+Th17+(not+Th1)+responses+underlie+multiple+sclerosis+disease+abrogation+after+hematopoietic+stem+cell+transplantation.">Diminished Th17 (not Th1) responses underlie multiple sclerosis disease abrogation after hematopoietic stem cell transplantation.</a> Ann.Neurol. 73 (3), 341-354 (2013).</li></ol>

The authors have nothing to disclose.

这个视频文章的关键步骤是在洗涤和淀粉酶处理的细胞。而洗涤载玻片，关键步骤是使用塑料挤压瓶洗涤并让水通过幻灯片上的样品轻轻地运行，并没有直接瞄准到样品。即使是最轻微的直接供水压力会导致细胞脱落的幻灯片。另一个关键步骤是使用相同的幻灯片±淀粉酶的条件。后的PBMC附着到滑动，滑动小心地放入烧杯所以只有一半的涂片的暴露于淀粉酶溶液。此步骤提供了一个强大的控制，因为血细胞是来自同一滑动，从而最大限度地减少因细微定时变化时可能出现的混淆变量。细胞粘附以及与不治疗，所以额外的成本和涉及用聚L-赖氨酸或聚乙二醇涂层例如时间不会被担保。 通过TRoubleshooting淀粉酶的最佳活性。对于肌肉切片，有人指出，在1小时温育的观察淀粉酶的最佳活性。在较长时间的肌肉部分会慢慢剥离滑动，而在较短的时间淀粉酶没有充分去除PAS-信号。对于淀粉酶孵育的PBMC幻灯片的定时必须减少相对于肌肉采样定时（为1小时）下降到仅15分钟。较长时间引起外周血单个核细胞脱落幻灯片，而较短的时间并不能有效影响PAS-信号。一个修改到PAS染色的标准协议被改变洗涤该幻灯片的方法。制造商的说明来表示用流动的自来水，从而导致细胞脱落的幻灯片洗幻灯片。的修改是用挤压洗瓶洗载玻片保存细胞。如在关键步骤部分中描述，它不是直接适用水很重要压力上的细胞。 有在该技术的一些限制。肌细胞膜用在肌肉细胞的细胞质中的点状颗粒染色沿。将颗粒通过淀粉酶处理，因此，很可能是糖原消除，而膜染色是不敏感的淀粉酶。的PAS阳性颗粒在细胞膜上的身份是未知的。它可能是肌肉地下室（肌束膜）膜。该膜围绕肌肉分册并由于其高含量的糖蛋白是已知的PAS阳性。 PAS染色的另一个限制是，糖原颗粒的直径必须为至少50纳米是通过常规的光学显微镜可见的。因此，更小的糖原颗粒可以存在于细胞中，但仍对的PAS测试寄存器阴性。所用的第二种方法通过提供检测糖原在一个裂解物克服了这一限制。在与标准曲线相结合这可以用来DETErmine糖原的确切数额。虽然这种技术是高度量化的，并且不通过的颗粒的尺寸的限制，但它本身具有一定的局限性。糖原颗粒与许多辅助蛋白和化学交联的复杂支链结构，使它不大可能水解酶充分溶解的整个糖原分子13。因此，酶检测（如PAS技术）可以下表示样品中糖原的实际数额。处理此限制的一种方法是用一种能解决即使是最小的糖原颗粒14电子显微镜。人们必须采用这些技术，PAS染色，酶消化，和电子显微镜相结合的糖原最全面的表征。 这篇文章和视频演示了适合于外周血单个核细胞使用碘酸雪夫（PAS）染色技术。本研究的意义被认为是在使用的选择外周血单个核细胞在血涂片，这使得它更可行枚举淋巴细胞。最初，一个典型的血涂片技术进行了测试，但是大部分细胞在滑动的是红血细胞和可能的嗜中性粒细胞（ 图4C）。以浓缩的淋巴细胞，将PBMC从血液利用标准的密度梯度技术纯化。使用类似于一个血涂片的技术，所述的PBMC容易附着的PAS程序，其中有许多有力的洗涤步骤的持续时间。该PAS技术已经使用了数十年，以确定在肌肉组织切片，其切成薄切片并粘附到载玻片糖原水平。 PAS染色被选择了其他碳水化合物的化学品染色，由于其高可靠性和在文献中预期的结果的存在。鼠标（ 小家鼠spretus）肌肉切片被用作阳性对照，发现，正如所料，该细胞的37％PAS阳性9。在成本和时间方面，制备PBMC是比血涂片更繁重的，但也有几个优点。首先，该制剂是在淋巴细胞，其是对于围绕自身免疫项目感兴趣的细胞更丰富。如果使用了血涂片，淋巴细胞将是少数，使其具有挑战性的找到感兴趣的细胞。人们必须准备和分析更为幻灯片以获得相同的号码，你会得到一个数PBMC幻灯片。研究人员将是在使用我们的新的优化技术在涉及1型糖尿病，多发性硬化症，狼疮，类风湿性关节炎等 ，其中T和B淋巴细胞和NK细胞中发挥作用的自身免疫项目很感兴趣。当然，对于其他应用场合的红细胞或粒细胞是感兴趣的血涂片将被推荐。另一优点是，PBMC中可用于研究人类淋巴细胞生物学体外 。 一项正在进行的研究正在调查糖原的外周血单个核细胞的来源。我n中的将来，计划来测量在多发性硬化症中，T淋巴细胞介导的自身免疫性疾病，全世界影响估计有230万人为2013 15,16的上下文糖原含量。什么是小的和大细胞PBMC样本？细胞在5微米与在其静止状态下的淋巴谱系一致。 T淋巴细胞，B淋巴细胞，自然杀伤细胞和其他次要子集在该尺寸范围内。较大的PBMC有可能由激活的淋巴细胞，它长得更大，因为它们接收来自免疫系统的炎性信号，并且单核细胞从骨髓谱系的。如荧光激活细胞分选或磁激活细胞分选先进的细胞分选技术都需要进一步缩小表达糖原的子集。 苏木反染色时使用染色的全血进行。这使我们能够区分红细胞单核细胞（图4C）。当染色上的PBMC进行，因为所有的细胞的单核，没有必要以对抗染色用苏木精，以确定细胞。还苏木被干扰细胞中的PAS信号。总之，我们已经证明一个适于PBMC中PAS染色过程。这种技术是对科学家和临床医生研究自身免疫，感染和过敏是有用的。

碘酸希夫（PAS）染色是一种免疫组织化学技术，被广泛用于肌肉研究和诊断。它也被用来作为对血样的诊断工具。该技术的工作原理是将高碘酸溶液的样品，其氧化，与无色希夫氏试剂，从而产生了深刻的品红产物反应的多糖产生醛基内单元。这个过程的步骤显示在图1的染色变为与多糖品红，包括糖原，糖蛋白，糖脂，粘蛋白，或其它分子与多糖部分任何东西。 PAS染色常常用于测量肌肉纤维糖原水平。肌肉组织切片是理想的，因为他们牢牢地固定在滑板和承受多次洗涤和染色步骤的技术。糖原是最存在于快肌II型肌纤维，它具有高的需求快速ATP生产要求糖原的最高性能1,2。糖原是葡萄糖的支链聚合物，可以通过对糖原磷酸酶的作用被分解成游离葡萄糖。在休息和营养自足倍，糖原通过糖原的过程中补充，而在营养不足或高能量需求的时间;糖原是由糖原分解分解成葡萄糖。从早在1950年的临床科学家们探索的血样PAS染色法来分析各种疾病3-7糖原含量。例如，在蓬珀病-一个真实糖原贮积无病白细胞积累大量糖原，从健康对照8显著不同的。 此视频文章演示了PAS染色的外周血单个核细胞（PBMC）从健康人的静脉血样使用改编版。 PBM铯包含大多淋巴细胞T淋巴细胞和B淋巴细胞的家庭，以及其他免疫细胞如自然杀伤细胞和单核细胞。第一纯化步骤除去红细胞，嗜中性粒细胞，和其它粒细胞。该技术提供了对淋巴细胞允许PAS阳性细胞的更健壮的枚举，相比使用全血涂片的浓缩比例的数据。 <img alt="图1" src="/files/ftp_upload/52199/52199fig1highres.jpg" /> 图1:第一步通过PBMC PAS染色步骤方法（一）首先，PBMC的隔离是通过聚蔗糖梯度实现的，左边的面板显示离心前的准备，右侧面板离心后显示它那里的棕黄色大衣含PBMC在管的中心观察到。（B）中分离的PBMC中使用福尔马林固定液乙醇SOLU固定到滑动化。滑动轻轻漂洗由塑料洗瓶蒸馏水。（C）的滑动，然后放置在填充有淀粉酶溶液的100毫升烧杯中的一半，这将溶解糖原。滑动轻轻漂洗。（D）将载玻片用高碘酸溶液中，其中糖的氧化发生处理。幻灯片轻轻冲洗;这将除去多余的高碘酸并停止氧化步骤。（E）当希夫试剂被添加到幻灯片，它将与在氧化步骤中创建的醛反应。然后，该无色试剂将导致深红色深红色产物。载玻片轻轻冲洗以除去过量的希夫试剂。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Periodic Acid Shiff Kit</td>
			<td>Sigma-Aldrich</td>
			<td>395B</td>
			<td>Bring to room temperature prior to use. Materials in this kit are toxic and harmful. Use caution&#160; 
			<a href="http://www.sigmaaldrich.com/catalog/product/sigma/395b?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/395b?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>&#945;-Amylase from porcine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>A3176</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a3176?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/a3176?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>Binocular Microscope&#160;</td>
			<td>Carl Zeiss Microscopy</td>
			<td>Axio Lab A0</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen Assay Kit</td>
			<td>Sigma-Aldrich</td>
			<td>MAK016</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/mak016?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/mak016?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>VWR, GE Healthcare</td>
			<td>17-1440-02</td>
			<td>Nonionic synthetic polymer of sucrose 
			<a href="https://us.vwr.com/store/catalog/product.jsp?product_id=4779441">https://us.vwr.com/store/catalog/product.jsp?product_id=4779441</a></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td>For PBMC isolation, swing buckets were used&#160;</td>
		</tr>
	</tbody>
</table>

detecting glycogen in peripheral blood mononuclear cells with periodic acid schiff staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

为了验证所用试剂和基本技术，PAS染色物根据制造商的对小鼠比目鱼肌节的指令进行的。染色做同一天的牺牲，并在染色的最后步骤，将切片固定二甲苯。比目鱼肌已知含有〜35％的糖原阳性细胞9。染色的肌肉细胞中显示两个不同的PAS阳性特性-点状颗粒的细胞内，并且连续线demarking细胞膜（ 图2A）。点状颗粒的存在是与糖原储存一致，并且被用来定义一个阳性肌细胞。用淀粉酶样品的处理除去 ​​的点状颗粒中是与它们被糖原（ 图2B）一致。 <img alt="图2" src="/files/ftp_upload/52199/52199fig2highres.jpg" width="700" /> 图2:PAS-<html>染小鼠肌肉切片用光学显微镜进行分析。鼠标比目鱼肌切片用PAS。一些样品被预处理以淀粉酶。（A）的 PAS阳性颗粒肌肉细胞内可见。（B）的更少的PAS阳性细胞时观察到的肌肉切片进行预处理用淀粉酶。周围的细胞膜染色保持淀粉酶处理（比例尺= 100微米）后， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52199/52199fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 即使经过淀粉酶处理的膜 - 染色信号仍然存在。正如所料，PAS阳性细胞在非淀粉酶肌节的比例为37％，而淀粉酶处理过的肌节有显著更少，约5％的PAS阳性细胞（ 图3）。 "&gt; <img alt="图3" src="/files/ftp_upload/52199/52199fig3highres.jpg" /> 图3:PAS阳性肌细胞的定量肌细胞是PAS阳性是从代表滑动计数而不或与淀粉酶预处理的比例。正如预期的那样，肌肉细胞的37％呈阳性反应，糖原。淀粉酶处理的PAS-信号显著降低至4％（* P &lt;0.001）。 接着，PAS-染色对PBMC使用改进的技术，如在协议部分所述进行从健康人受试者的静脉血，和图1所示。将PBMC粘附以及如果仔细进行洗涤步骤。该PAS染色外周血单个核细胞显示各种染色模式。小细胞（5微米）与颗粒很容易观察到。较大的细胞与弥漫染色图案的比例也观察到（ 图4A和插图A.1-6 </s仲&gt;）。用淀粉酶样品的处理除去 ​​颗粒信号并减少漫PAS-阳性（ 图4B）。 <img alt="图4" src="/files/ftp_upload/52199/52199fig4highres.jpg" width="700" /> 进行上的PBMC 碘 ​​酸-希夫（PAS）上的人PBMC染色（A）的 PAS染色:图4。两种类型的细胞进行观察。非淀粉酶处理过的载玻片（A.1-6）较小的细胞（5微米）显示品红粒子糖原一致。这些细胞可被静息细胞。非淀粉酶处理过的细胞的细胞较大（大于5微米）的漫PAS阳性染色。这些细胞可被活化的淋巴细胞。（B）中的PBMC用淀粉酶染色前，这削弱了的PAS信号处理15分钟。代表七个不同的健康人受试者。（℃）的PAS和hematoxy林染完成对全血的幻灯片。箭头显示了许多红细胞（比例尺= 10微米）包围的PBMC。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52199/52199fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 这是PAS阳性的比例为98％，为较小的细胞和40％的较大的细胞。淀粉酶处理消除了在小单元的PAS信号（P &lt;0.001）和显著减少的PAS-信号中的较大的单元，以7％（ 图5）。 <img alt="图5" src="/files/ftp_upload/52199/52199fig5highres.jpg" /> 图5:PAS阳性的PBMC定量那名PAS阳性是从代表滑动计数而不或与淀粉酶预处理的PBMC的比例。 98％的小型细胞呈阳性PAS。较大的细胞的40％的Positive的PAS。淀粉酶处理消除了在小单元的PAS信号（P &lt;0.001）和显著减少的PAS-信号中的较大的单元，以7％（P &lt;0.001）。 以确认的PBMC具有糖原的第二，独立的方法也提供了糖原的量的定量，使用10,11，和先前已发表 ​​在朱庇特的其它细胞类型12。的PBMC裂解在低渗缓冲液和糖原分离如在图6的说明简单说明。 <img alt="图6" src="/files/ftp_upload/52199/52199fig6highres.jpg" /> 图6:使用酶消化水解酶糖原的测量 。糖原是根据制造商的说明进行测量。简言之，该协议相伴的1×10 6个PBMC中低渗裂解，随后通过不溶物的造粒含有糖原。将沉淀洗涤并消化酶水解得到的葡萄糖，经分光光度法测量和比较标准曲线。将细胞裂解物稀释在含有水解缓冲指示比率。数据代表三次实验。在1检测糖原显著级别:1稀释（p值= 0.02），并且这个信号滴定出作为裂解物进一步稀释。 不溶糖原洗涤数次，然后消化用水解酶。用分光光度法测量和比较的标准曲线的葡萄糖从消化产生的量。一百万外周血单个核细胞有1.19微克的糖原。滴定下来作为细胞裂解物的糖原信号进一步稀释（ 图6）。

Watch this Scientific Journal Video about Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining at JoVE.com

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

detecting-glycogen-peripheral-blood-mononuclear-cells-with-periodic

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

21.1K 次观看.  Centre for Structural and Functional Genomics, PERFORM Centre, Concordia University. Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

视频: Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

小鼠腹膜腔细胞的分离

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

科研

免疫与感染

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

协同刺激信号封锁的同种抗原刺激诱导人外周血单个核细胞同种异体抗原特异性无能

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

扩展人外周血γδT细胞使用Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.