“ 作者感谢提供正常的脑组织样本的人的大脑和脊髓液资源中心（洛杉矶，CA）。这项研究是支持由美国国家卫生研究院（NIH）R01NS062787，的克雅氏病基金会，联盟生物安全，以及在大学中心老化和健康的支持麦格雷戈基金会和总统的全权委托基金（Case Western Reserve大学） 。

1。脑匀浆的制备和洗涤剂可溶性（S2）和不溶性的（P2）的馏分<ol><li>取100毫克冷冻人脑组织中，并加入100μl裂解缓冲液中（10mM Tris，150mM的NaCl，0.5％NONIDET P-40（NP-40），0.5％EDTA，pH7.4）中。 </li><li>均质在冰上，用杵由无绳马达驱动，在干冰中冷冻它为5分钟，并再次均质它使用杵先用手驱动，然后由马达，并加入300μl或800微升的裂解缓冲液（这是20 ％或10％总脑匀浆，分别）。 </li><li>以1,000 xg离心10分钟，在4℃下，收集上清液（S1），用台式离心机离心20％或10％的总的脑匀浆。 </li><li>超速离心机在4℃下1小时，在35,000转（100,000 xg离心）在SW55转子（Beckman Coulter公司，富勒顿，加利福尼亚州）的10％S1 （S2）的上清液转移到一个干净的试管中包含的洗涤剂可溶部分。重悬含有洗涤剂不溶性的颗粒馏分（P2）在100μl或200μl的裂解缓冲液中，使10 - 或5倍浓缩的制剂。 </li><li> PK消化，与相同量的PK孵育样品在50微克/毫升，在37℃下为1小时，S2和P2馏分。苯甲基磺酰氟添加至终浓度为5 mM的和相等体积的SDS样品缓冲液（3％SDS，2mM EDTA的，4％β-巯基乙醇，10％甘油，50mM的Tris，pH值6.8）终止的PK消化。煮沸10分钟的样品，并在室温下冷却5分钟。他们已经准备好为西方墨点法。 </li></ol> 2。在蔗糖密度梯度的速度沉降<ol><li>孵育450微升20％S1，用等体积的在冰上30分钟，在H 2 O 2％肌氨酸。 </li><li>加入0.5毫升的60％，30％，25％，20％，15％，和10％的蔗糖成具有最大的5毫升容量的Beckman管。 </li><li>将0.4毫升S1到10-60％的蔗糖密度梯度的顶部。 </li><li>离心48000转（200,000 XG，SW55转子）1小时后，在4°C。 </li><li>收集283微升馏分，各从管的顶部和获得总共12馏分。 </li><li> 20μl的每个级分取到新的Eppendorf管中，加20μl样品缓冲液中，煮沸10分钟。 </li><li>酷样品遮光罩4分钟，1分钟和涡他们在1,000 xg下离心。装入样品到预铸凝胶。 </li></ol> 3。尺寸排阻色谱法<ol><li>使用Superdex 200 HR珠（Pharmacia公司，Uppsala，瑞典）中的1×30厘米柱，以确定的PrP分子的寡聚状态。 </li><li>七分子量标记物（Sigma公司，圣路易斯，MI），包括葡聚糖蓝（2,000 kDa的），甲状腺球蛋白（669 kDa的），脱铁铁蛋白（443 kDa的），β-淀粉酶（200 kDa的），乙醇脱氢酶（150 kDa的），白蛋白（ 66 kDa的），碳酸酐酶（29 kDa）的独立装载的浓度由Sigma 200微升样品量的建议。 </li><li>蓝色DEXT的洗脱体积然是用来确定的空隙体积（V 0 = 8.45毫升），总体积（ 的 V t = 24毫升）中所提供的产品的指令。峰的洗脱体积（V E）计算出的色谱图和分数保证金。 K表AV的分配系数（定义样的行为），计算公式：K AV =（V E - V-0）/（V T - V 0）。的校准曲线来确定通过绘制针对日志兆瓦的标准8的蛋白质标准的K AV。 </li><li>分子量（MW）是在不同的FPLC馏分回收的各种的PrP物种根据与各种标准的凝胶过滤生成的校准曲线进行评价。 </li><li>注入200μl的裂解缓冲液中含有1％肌氨酸成的列的每个运行中的S1。 </li><li>在FPLC系统（GE Healthcare公司）的流速为0.25毫升/分钟和馏分进行色谱s的0.25毫升收集的一小部分收集器（Amersham Biosciences公司，RediFrac）。 </li><li>孵育0.125毫升各馏分用0.5毫升预冷的甲醇在-20℃下2小时。 </li><li>离心样品在13,000 xg离心30分钟，在4℃下在台式的离心。弃去上清液，将沉淀重悬于在20μl如上述的SDS样品缓冲液，煮沸10分钟，冷却至室温，将它们加载到一个预制凝胶。 </li></ol> 4。捕获的PrP G5p以<ol><li> G5p以分子（100微克）（请提供由英国朴次茅斯大学的博士。杰夫Kneale和约翰·McGeehan）是共轭7×10 8对甲苯磺酰基活化磁珠孵化100的微克g5p和中加入350μlg5p珠1毫升的磷酸盐缓冲盐水（PBS），在37℃下20小时。 Eppendorf管的侧壁外部的磁力吸引G5p以珠，然后删除所有的解决方案。洗净珠，用1ml的PBS中含有0.1％牛血清白蛋白（BSA）为三次。 </li><li>孵育g5p共轭珠在1毫升的0.2M Tris-盐酸，pH为7.4，含有0.1％BSA，37℃下5小时，以阻断非特异性结合，然后洗珠，用1ml的PBS中含有0.1％BSA的三次，如上所述。移除该溶液，并加入1毫升的PBS成珠。所制备的g5p珠为至少3个月是稳定的，在4℃下</li><li>捕获的PrP G5p以进行孵育100μl的S1馏分或P2与60微升g5p共轭珠（10微克蛋白质/ 6×10 7珠）在1毫升的结合缓冲液（3％的Tween-20，3％NP 40在PBS中，pH值7.5）。 </li><li>孵育后，在室温下的3小时的恒定的旋转，含有的PrP G5p以珠被吸引到Eppendorf管的侧壁上，通过外部的磁力，允许所有未结合的分子在溶液中容易地取出。 </li><li>继洗涤三次在洗涤缓冲液（2％Tween-20和2％NP-40在PBS中，pH值为70.5），G5p以珠被收集，并加热，在95℃下为5分钟在SDS样品缓冲液（3％SDS，2mM的EDTA，10％甘油，50 mM Tris-盐酸，pH为6.8）。 </li><li>样品冷却后，在室温下5分钟，将样品离心分离以1,000 xg离心5分钟，在室温下。上清液准备Western印迹。 </li></ol> 5。蛋白质印迹<ol><li>负载样品在SDS样品缓冲液中煮沸15％的Tris-HCl判据的预制凝胶（Bio-Rad公司）上，在150 V〜80分钟。 </li><li> 2小时，将凝胶上的蛋白转移到PVDF 70V。 </li><li>封闭后的膜在5％牛奶的Tris盐缓冲液含有0.01％Tween-20（TBS-T），将膜孵育2小时，在室温下用初级单克隆抗体或多克隆抗体3F4（1:40,000），1E4（1 1000），抗-N 8（1:6,000），或抗-C 8（1:6,000）进行探测的PrP分子。 </li><li>与HRP标记的羊抗小鼠IgG温育后在1:3000，或驴抗兔IgG在1:3,000的PrP频带柯达膜用ECL Plus可视化，在根据制造商的协议。 </li></ol> 6。代表性的成果偶发性CJD的样品相比，在P2馏分检测IPRPÇ少量的在正常的大脑，虽然大多数的PrPÇ在S2的部分（ 图1）回收。正如前面所指出的8，IPRP帐户的约5-25％的总的PrP包括全长度和N-端截短的物种。 分析用蔗糖的步骤梯度沉淀透露，而大部分的PrPÇ非克雅二氏症的大脑中回收的顶端部分1-3，少量的PrP也被检测到的底部部分9-11，通常含有大量聚集（ 图2）。 各种的PrP Sc的物种范围ING从单体，小的低聚物更大的聚集体进行分离，凝胶过滤在大脑中与克雅氏病（ 图3A）。然而，正常的大脑的不溶组分（ 图3C）中也检测到少量的较大的聚集体的分子量大于2000 kDa的。此外，二聚物和四聚物的PrPÇ不仅检测不溶组分，而且在可溶性级 ​​分（ 图3B和3C）。 PK和PNGase治疗后，G5p以所捕获的PrP与1E4抗体检测对PrP97-105 8。三PK性的核心片段，称为PrP的朊病 ​​毒* 19，和PrP * 20，* 7〜20 kDa的，〜19 kDa的〜7 kDa的，分别为（ 如图4所示 ，左图）进行了检测，迁移。然而，没有被检测到的PrP时，1E4抗体预孵育的合成肽具有序列中身份ICAL 1E4的抗原表位（ 图4中 ，中间面板），表明检测1E4的频带是PrP的片段。此外，抗-C抗体揭示了两种不同的朊蛋白片段〜18 kDa的迁移（PRP * 18）〜12-13 kDa的“（PrP-CTF12/13），除了对朊病毒* 20（ 图4，右图）。 <img alt="图1" src="/files/ftp_upload/3788/3788fig1.jpg" /> 图1。IPRP C和IPRP SC的检测。 PNGase（糖肽酶）F在反应总体积的1/10，在37℃下1小时的处理后，以除去蛋白，全长或N-端截短PrP的物种中的可溶性和不溶性组分（S2和P2）孤立聚糖对PrP23-40（中间面板），正常对照组（CTL）和零星的克雅氏病（SCJD）的脑组织标本中检测到的超速离心3F4对PrP106-112（左），抗-N，和对朊蛋白的抗-C220-231（右图）。在CTL的样品中，有少量的PrP P2中被检测到，而大量存在在S2中。相反，更多的PrP P2中检测比S2 SCJD样品。 <img alt="图2" src="/files/ftp_upload/3788/3788fig2.jpg" /> 图2。蔗糖密度梯度离心沉淀的PrP。 PRP在个人分数从1到11的非CJD脑样品S1检测Western印迹3F4的。虽然大多数的PrPÇ中的顶级馏分1-3检测到，少量的PrP也观察到了在底部馏分9-11。此外，从顶部和底部的带型的PrP是不同的朊蛋白的顶部部分恢复上带的主导，而PrP的恢复的底部分数的一个占主导地位较低的带。一个PK处理的PrP Sc为加载控制右侧的印迹。 <i毫克ALT =“图3”SRC =“/ files/ftp_upload/3788/3788fig3.jpg”/&gt; 图3。检测中的可溶性和不溶性的PrPÇ低聚物。通过超速离心法分离的可溶性和不溶性蛋白C从正常人类大脑，然后进行凝胶过滤，分别。通过运行的基团的分子质量标记测定各个级分的分子大小。 （A）的PrP Sc的物种SCJD脑组织标本。两个种群的PrP物种进行检测凝胶过滤组分49 - 65含有单体和小的低聚物，而部分27-33含有大量聚集。的PrPÇ种从可溶组分（S2），（B）和不溶部分（P2）的（C）的正常对照组进行检测。朊病毒用的3F4抗体。二聚体（馏分55）和四（馏分51）的PrP检测不仅在P2中，而且，在S2中正常脑SAMPL上课（B和 C）。大的聚集体，只在P2的正常样本（C）的检测。 <img alt="图4" src="/files/ftp_upload/3788/3788fig4.jpg" /> 图4。G5p以富硒准备从正常人类大脑的各种抗PK-IPRP片段的检测。 G5p以富集样品与PK和PNGase F处理之前，Western印迹探测1E4（左面板），1E4预孵育有1E4抗原表位（中间面板）相同的序列的合成肽，和抗-C（右图）。 1E4检测到的三个PK抗性的PrP片段称为朊病毒* 20的PrP * 19，和PrP * 7（左面板）。朊病毒水库阻断1E4与肽后，没有进行检测（中间面板），表明检测到的频带与1E4 PrP的片段。抗-C发现除了PK抗朊蛋白片段称为PrP的18和PrP-CTF12的/13，除了朊病毒* 20。

<ol>
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没有利益冲突的声明。

报告相结合的办法，隔离始终不溶性的朊蛋白的Ç聚集和可溶性PrP的Ç从正常的人脑的低聚物。超速离心法是一个典型的方法，该方法已被广泛用于从可溶的PrP C 14的分离的不溶性的PrP Sc的一小时以100,000 xg离心。虽然它是有效的，其中之一的注意事项是拉离心分离后的上清液（S2）过程中，以避免污染。 IPRPÇ是不同的朊蛋白凝胶调，这是不可能的朊蛋白检?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>苯甲基磺酰氟</td><td> Sigma-Aldrich公司</td><td> P7626 </td><td> 72 mM的2 - 丙醇中</td></tr><tr><td>蛋白酶K </td><td> Sigma-Aldrich公司</td><td> P2308 </td><td>在H 2 O 2毫克/毫升</td></tr><tr><td> PNG酶F </td><td> New England Biolabs公司</td><td> MWGF200 </td><td></td></tr><tr><td>超速离心机LE-80K，SW55转子</td><td> Beckman Coulter公司</td><td>料号：365668，356860 </td><td></td></tr><tr><td> Superdex 200 HR珠</td><td> GE医疗集团</td><td> 17-1088-01 </td><td></td></tr><tr><td> AKTA FPLC系统</td><td> GE医疗集团</td><td> 18-1900-26 </td><td></td></tr><tr><td>分子量标记</td><td> Sigma-Aldrich公司</td><td> MWGF200 </td><td>多变</td></tr><tr><td>的Dynabeads M-280磁珠</td><td> Invitrogen公司</td><td> 143-01 </td><td></td></tr><tr><td> 3F4抗体</td><td>科文斯</td><td> SIG-39600 </td><td></td></tr><tr><td> 1E4抗体</td><td>细胞科学</td><td> M1840 </td><td></td></tr><tr><td>就绪凝胶15％的Tris-HCl预制胶</td><td> Bio-Rad公司</td><td> 345-0020 </td><td></td></tr></tbody></table>

isolation of soluble and insoluble prp oligomers in the normal human brain

朊病毒疾病的发病机制中的中心事件涉及的主机编码朊蛋白PrP的Ç转换成其致病亚型的PrP Sc的1。朊蛋白C是水溶性清洁剂和敏感的蛋白酶K（PK）消化的PrP Sc的 ，而形成不溶于洗涤剂的聚集和部分耐PK 2-6。朊病毒Ç的PrP Sc的转换是已知涉及的构象的蛋白的β-折叠片结构的α-螺旋结构的过渡。然而， 在体内的途径仍然是知之甚少。暂定内源性的PrP Sc的 ，中间的PrP *或“沉默的朊病 ​​毒”，在未受感染的大脑还没有确定。 使用相结合的生物物理和生物化学的方法，我们发现不溶性的朊蛋白的Ç聚集（IPRP C）未感染的哺乳动物的大脑和培养的神经元单元8，9。在这里，我们将详细介绍这些方法的程序，其中包括超速离心法在洗涤缓冲液，蔗糖步骤梯度沉降，尺寸排阻色谱法，IPRP富集基因5蛋白（g5p）特异性结合的结构改变的PrP表格10，和PK-处理。这些方法的组合隔离：不仅不溶性的PrP Sc和朊病毒Ç聚集体，但也可溶性蛋白Ç从正常人类大脑的低聚物。由于这里描述的协议已被用于从受感染的大脑和未感染的大脑IPRP C的隔离双方的PrP Sc的 ，他们为我们提供了一个机会，比较两种异构体之间的物理化学特性，神经毒性，传染性。这样的研究将大大提高我们了解蛋白质病原体的感染。生理学和病理生理学IPRPÇ目前还不清楚。值得注意的是，在一个新的-id的可变entified人类朊病毒疾病被称为蛋白酶敏感的prionopathy，我们发现了一个新的PrP Sc的共享的免疫反应行为IPRP C 11，12和碎片。此外，我们最近表明，IPRP C是主要品种，与β-淀粉样蛋白在阿尔茨海默病13互动。在相同的研究中，这些方法被用来隔离Abeta聚合物和低聚物，在阿尔茨海默氏病13，提示它们的其他神经退行性疾病中涉及的非朊病毒蛋白聚集体中的应用。

Watch this Scientific Journal Video about Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain at JoVE.com

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

朊蛋白（PrP的一新种<sup&gt; C</sup&gt;）最近被鉴定使用这里描述的方法在未感染的人脑。这些方法可以用来隔离不同的朊蛋白物质，而其中一些也可在其他错误折叠的蛋白质聚集体从人类的大脑隔离开来。

在正常人脑分离的可溶性和不溶性朊蛋白低聚物

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

isolation-soluble-insoluble-prp-oligomers-normal-human

Research

JoVE Journal

Medicine

10.7K 次观看.  Case Western Reserve University School of Medicine. 朊蛋白（PrP的一新种 C）最近被鉴定使用这里描述的方法在未感染的人脑。这些方法可以用来隔离不同的朊蛋白物质，而其中一些也可在其他错误折叠的蛋白质聚集体从人类的大脑隔离开来。

视频: Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

慢分裂的细胞在人脑胶质瘤羧基荧光素琥珀酰亚胺酯（CFSE）的分离与鉴定

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

科研

医学

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

利用人类纤维原细胞线粒体表型的监测领域的帕金森氏病

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer&#039;s Disease and Normal Elderly

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

病变资源管理器：一个视频引导，标准化为老年痴呆症准确，可靠的MRI衍生Volumetrics和正常老年人协议

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

人类癫痫术后皮层组织的多电极阵列录音

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

分离，冻存和文化的人羊膜上皮细胞的临床应用

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

使用Saccadometry与深部脑刺激来研究正常和病理脑功能

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Mechanical and Controlled PRP Injections in Patients Affected by Androgenetic Alopecia

23 patients (18 male and 5 female) aged 21-70 years who displayed male pattern hair loss (MPHL) in Stage 1 to Stage 5 as determined by the Norwood-Hamilton classification scale, and female pattern hair loss (FPHL) in Stage 1 to Stage 2 as determined by the Ludwig classification scale, were treated with non-activated autologous platelet-rich plasma (A-PRP). Autologous blood (55 mL) was harvested using sodium citrate as an anticoagulant. A-PRP (23 mL) was produced for all cases using a closed system according to the transfusion service protocol. Following centrifugation (260 x g for 10 min) the A-PRP was inserted in a laser light selector device, and after the centrifugation, 9 mL of A-PRP was collected.
The scalp of the patients affected by androgenetic alopecia (AGA) was divided into four areas (frontal, parietal, vertex, and occipital); local anesthesia was not performed. Interfollicular A-PRP injections (0.2 mL x cm2) were performed by controlled and mechanical injections scheduled at a depth of 5 mm using a medical injector gun. Treatment sessions were performed with a 30-day interval. For each patient, three treatment sessions were performed.
PRP was injected in the androgen-related areas of scalp affected by hair loss. Placebo (normal saline solution) was loaded in another syringe (10 mL) and injected on the adjacent side in a similar fashion.

This paper aims to describe the interfollicular autologous platelet-rich plasma injections (0.20 mL x cm2) in the scalp of 23 patients affected by androgenetic alopecia at a depth of 5 mm using a medical injector gun equipped with a 30 gauge needle, in three sessions.