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

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

Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains of &#945;1,4-linked glucose residues with branches introduced through the addition of &#945;1,6-linkages. Understanding how the synthesis and degradation of glycogen are regulated and how glycogen attains its characteristic branched structure requires the study of the enzymes of glycogen storage. However, the methods most commonly used to study these enzyme activities typically employ reagents or techniques that are not available to all investigators. Here, we discuss a battery of procedures that are technically simple, cost-effective, and yet still capable of providing valuable insight into the control of glycogen storage. The techniques require access to a spectrophotometer, operating in the range of 330 to 800 nm, and are described assuming that the users will employ disposable, plastic cuvettes. However, the procedures are readily scalable and can be modified for use in a microplate reader, allowing highly parallel analysis.

Techniques to measure the activity of key enzymes of glycogen metabolism are presented, using a simple spectrophotometer operating in the visible range.

用于研究真核糖原代谢的分光光度法

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains ...

科研

生物化学

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are influenced by its structure. Hence, a suitable extraction method that isolates representative samples of glycogen is crucial to the study of this macromolecule. Compared to other extraction methods, a method that employs a sucrose density gradient centrifugation step can minimize molecular damage. Based on this method, a recent publication describes how the density of the sucrose solution used during centrifugation was varied (30%, 50%, 72.5%) to find the most suitable concentration to extract glycogen particles of a wide variety of sizes, limiting the loss of smaller particles. A 10 min boiling step was introduced to test its ability to denature glycogen degrading enzymes, thus preserving glycogen. The lowest sucrose concentration (30%) and the addition of the boiling step were shown to extract the most representative samples of glycogen.

An optimal sucrose concentration was determined for the extraction of liver glycogen using sucrose density gradient centrifugation. The addition of a 10 min boiling step to inhibit glycogen-degrading enzymes proved beneficial.

肝糖原分子的提取用于糖原结构测定

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

使用透射电子显微镜定量骨骼肌纤维中的亚细胞糖原分布

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

糖原生化滴定<em&gt;在体外</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

生物学

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

聚丙烯酰胺凝胶电泳和银染色检测甘油糖

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

A Non-Degradative Extraction Method for Molecular Structure Characterization of Bacterial Glycogen Particles

Currently, there exist a variety of glycogen extraction methods, which either damage glycogen spatial structure or only partially extract glycogen, leading to the biased characterization of glycogen fine molecular structure. To understand the dynamic changes of glycogen structures and the versatile functions of glycogen particles in bacteria, it is essential to isolate glycogen with minimal degradation. In this study, a mild glycogen isolation method is demonstrated by using cold-water (CW) precipitation via sugar density gradient ultra-centrifugation (SDGU-CW). The traditional trichloroacetic acid (TCA) method and potassium hydroxide (KOH) method were also performed for comparison. A commonly used lab strain, Escherichia coli BL21(DE3), was used as a model organism in this study for demonstration purposes. After extracting glycogen particles using different methods, their structures were analyzed and compared through size exclusion chromatography (SEC) for particle size distribution and fluorophore-assisted capillary electrophoresis (FACE) for linear chain length distributions. The analysis confirmed that glycogen extracted via SDGU-CW had minimal degradation.

Bacterial glycogen structure is greatly impacted by extraction methods which may result in molecular degradation and/or biased sampling. It is essential to develop methods to minimize these problems. Here, four extraction methods have been compared using size distribution and chain length distribution as key criteria for minimizing extraction artifacts.

Currently, there exist a variety of glycogen extraction methods, which either damage glycogen spatial structure or only partially extract glycogen, ...

Periodic Acid-Schiff Staining of Cells: An In Vitro Technique to Detect Glycogen Levels in Peripheral Blood Mononuclear Cells

Source: Tabatabaei Shafiei, M., et al. <a href="https://www.jove.com/v/52199">Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining.</a> J. Vis. Exp. (2014).
This video demonstrates the periodic acid-Schiff staining technique for assessing glycogen levels of peripheral blood mononuclear cells in vitro. The stain imparts a magenta color to intracellular glycogen granules, rendering them visible under a microscope.

细胞高碘酸-希夫染色:一种检测外周血单核细胞中糖原水平的体外技术

Source: Tabatabaei Shafiei, M., et al. Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining. J. Vis. Exp. (2014).
...

JoVE Encyclopedia of Experiments

Biological Techniques

Staining Techniques

Cell staining

Transmission Electron Microscopy to Quantify Glycogen Distribution in Human Skeletal Muscles

Source: Jensen, R., et al. <a href="https://app.jove.com/v/63347">Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy.</a> J. Vis. Exp. (2022)
In this video, we demonstrate the sample preparation of human skeletal muscle tissue and its staining for transmission electron microscopy to visualize and quantify subcellular glycogen distribution.

透射电子显微镜量化人骨骼肌中糖原分布

Source: Jensen, R., et al. Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy. J. Vis. ...

Microscopy Techniques

Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array

Source: Olivera-Ardid, S. et al., <a href="https://app.jove.com/v/57662">Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates printed glycan array technology for analyzing circulating anti-glycan antibodies in mouse serum. The antibodies in the serum interact with specific glycans on the chip, which is confirmed by immunostaining followed by fluorescence scanning.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Glycochips Production

	...

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

Glycogen Branching Assay to Measure the Degree of Glycogen Branching

Source: Wilson, W. A., <a href="https://app.jove.com/v/63046">Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism.</a> J. Vis. Exp. (2021).
In this video, we describe a qualitative spectrophotometric assay to determine the extent of branching in a glycogen sample with an uncharacterized structure. Comparing the absorbance maxima of the uncharacterized sample with that of polysaccharides with known branched structures indicates the presence of branching in the uncharacterized sample.

糖原支化测定法，用于测量糖原支化的程度

Source: Wilson, W. A., Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism. J. Vis. Exp. (2021).
In this video, we describe a ...