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，?...

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?...

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

Research

JoVE Journal

Immunology and Infection

21.0K 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

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 ...

科研

免疫与感染

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  &amp;#947;&amp;#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 ...

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 ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

传染性单核细胞增多症儿童外周血样本中免疫细胞亚群的分离

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

使用微泡从人外周血单核细胞样品中分离、活化和扩增基于浮选的 T 细胞

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

用于线粒体生物能量学分析的 PBMC 采集和低温储存

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

作者聚焦：冷冻保存后外周血单核细胞中生物能量参数的保存 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

人外周血单核细胞的冷冻保存与生物能量评价

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...