作者要感谢Sanjiv甘比尔的fluc/mrfp/sr39tk报告基因的礼物。这项工作是由加拿大干细胞网络（SCN），和杰西之旅基金会。

1。 C2C12成肌细胞的维护和传播<ol><li>板C2C12细胞在75cm 2的烧瓶中，并在高葡萄糖Dulbecco改良的Eagle的血清（HG-DMEM）中至最终浓度为10％的牛胎儿血清（FBS）补充与维持细胞。不要让细胞变得汇合在任何时间，因为这会消耗成肌人口。应该改变每一天。 注：总是温暖的介质中，以37°C的水洗澡前使用。 </li><li>当成肌细胞成为约80％汇合时，到新的烧瓶中的传代细胞。吸液培养基中。 4-5毫升Hanks平衡盐溶液（HBSS）删除所有的痕迹，其中含有胰蛋白酶抑制血清的培养基洗涤细胞。简言之冲洗的细胞层与2-3毫升的0.25％（重量/体积）的胰蛋白酶-EDTA溶液，离解的粘合成肌细胞从烧瓶中。吸干胰蛋白酶和烧瓶中放入培养箱在37℃下5分钟。 </li><li>在此温浴TION，准备一个新的烧瓶9毫升HG-DMEM/10％FBS培养基。胰蛋白酶消化后，加入10毫升的完全培养基，细胞和吸液管的4-5倍，以确保在介质中的所有单元格的集合。的新烧瓶中加入1毫升的细胞悬浮液中，并在5％CO 2孵育在37℃下</li></ol> 2。 C2C12细胞转染<ol><li>一旦细胞达到50％汇合时，转染，根据制造商的说明从Invitrogen公司。简要地说，将90微升Lipofectamine 2000试剂，用4.5ml的OPTI-MEM培养基。在另一试管中，OPTI-MEM为4.5毫升36微克的CMV-trifusion记者基因DNA结合。滑动各管结合起来，并等待5分钟。结合两个管子的内容，轻轻混匀，在室温下培养了整整30分钟。 注：第二个烧瓶中也可以设置为未转染的细胞，只有收到脂质体和OPTI-MEM作为阴性对照。 </li><li>删除介质C2C12细胞，并添加15.5毫升新鲜HG-DMEM/10％FBS培养基中。转染培养基，将总体积为20毫升。 </li><li>让细胞转染过夜至少20小时，在37°C。 </li><li>第二天，吸转染培养基，并添加10毫升HG-DMEM/10％FBS。 </li><li>查看在倒置荧光显微镜下的细胞。拍摄时，可同时捕捉明亮的领域和红色荧光图像（使用TRITC过滤器的多维数据集的MRFP前/ EM：六百○七分之五百八十四纳米的）。除以总数下明场观察的视图来生成转染效率的多个字段的细胞的荧光下观察RFP-表达细胞数进行计数。 </li></ol> 3。细胞的生存能力/ MTT法评估<ol><li>前一天，转染，板1×10 5 C2C12细胞在24孔板，每孔。细胞应在500μl体积HG-DMEM/10％FBS的镀。 </li><li>第二天，转染的成肌细胞过夜如先前所述。按照制造商的建议转reagen吨量的24孔板。孵育一组只与Lipofectamine（不含DNA）作为对照井。查看细胞，荧光，以确保适当的转染的发生。 </li><li>将转染培养基，并培育成肌细胞与5毫克/毫升噻唑蓝四甲基偶氮唑蓝（MTT）HG-DMEM/10％FBS。加入D-荧光素转染井8，四个小时，并在37℃下孵育。 </li><li>从井中取出介质。溶解蓝色甲臜晶体通过加入180微升异丙醇，以各孔。摇动15分钟，在37℃下。 </li><li>避免的任何沉淀，吸移管溶液成96孔板，并读取在575 nm处的吸光度。 </li></ol> 4。制备成肌细胞进行移植<ol><li>删除介质，与HBSS洗成肌细胞，细胞中列出的1.1 trypsinize。 </li><li> 4 ml完全培养基中重新悬浮细胞。 </li><li>使用血球计数器，计数细胞产生含有10 6成肌细胞的体积。用移液管卷UME进入无菌1.5毫升微管。有了一个〜10％的转染效率，这些值表明，100000的荧光素酶表达的细胞是可以检测的移植后的的GE ExploreOptix扫描器上。 </li><li>达到最终的注射体积为15微升，含有10 6 C2C12细胞。如果有必要，在2,000 rpm离心微管一分钟。小心地用吸管吸出上清液。重新悬浮在15μl的HG-DMEM缺乏FBS的细胞。 </li></ol> 5。细胞移植<ol><li>麻醉，用2％异氟烷/ 2％O 2的鼠标。拨动头发从后肢背侧区。异氟醚/ 2％的O 2，在1.5％的维持麻醉。 </li><li>确保C2C12成肌细胞被暂停。用鼠标在俯卧位，延​​长后肢和使用胰岛素注射器直接注入细胞，成30°角腓肠肌外侧头。转染的成肌细胞注入右后肢和未转染的成肌细胞的诠释Ø肢体对侧（左）。 </li><li>执行基准的生物冷光扫描：快速将鼠标移动到舞台奠定了鼠标的光学扫描仪，在俯卧位。连接的麻醉行。为了确保的鼠标停留麻醉，第二个人应该是挂钩的麻醉线，而其他地方的动物在扫描仪。 </li><li>轻轻延长后肢，所以喷射的区域是可见的。带后肢的地方，一个温和的粘合剂，如医用胶带。 </li><li>关闭的腔室，并确保没有光可以访问的扫描仪的内部，因为这会增加检测到的背景信号。应设置扫描仪参数包括生物发光成像制造商的说明。具体而言，确保“无激光”的选择。 </li><li>周围画一个地区的利益（投资回报率）的弹拨区，并开始扫描。 </li></ol> 6。 mdx小鼠注射涨落基板，D-荧光素， &lt;OL&gt; <li>当鼠标麻醉腹腔内注射150毫克/公斤的萤火虫荧光素酶底物，D-荧光素（从40毫克/毫升原液，根据制造商的说明）。 </li><li>恢复前准备进行下一次扫描鼠标的鼠标，并允许15分钟的摄取。 </li></ol> 7。 BLI目标吕克表达的多用途储值卡的DMD小鼠模型植入<ol><li>摄取后期间，再次麻醉小鼠，如在5.1中描述。 </li><li>鼠标传输的光学扫描器，并作为背景扫描中相同的方式执行另一个生物发光扫描。 </li><li>虽然20分钟摄取期间应该提供最大的信号强度，随后的扫描可以被执行。 </li><li>图像采集完成后，牺牲的鼠标，根据实验动物伦理委员会和加拿大动物保护（CCAC）理事会的指导方针。我立刻隔离后肢肌肉和地方N 10％福尔马林固定的石蜡包埋。荧光素酶进行免疫组化染色证实成肌细胞肌肉注射。 </li></ol>

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	<li>Emery, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+muscular+dystrophies.">The muscular dystrophies.</a> Lancet. 359, 687-695 (2002).</li><li>Blake, D. 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=Function+and+genetics+of+dystrophin+and+dystrophin-related+proteins+in+muscle.">Function and genetics of dystrophin and dystrophin-related proteins in muscle.</a> Physiol. Rev. 82, 291-329 (2002).</li><li>Goldring, K., Partridge, T., Watt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells.">Muscle stem cells.</a> J. Pathol. 197, 457-467 (2002).</li><li>Partridge, T. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+validation+of+improved+triple+fusion+reporter+gene+vectors+for+molecular+imaging+of+living+subjects.">Construction and validation of improved triple fusion reporter gene vectors for molecular imaging of living subjects.</a> Cancer Res. 67 (7), 3085 (2007).</li><li>Ray, P., 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=Imaging+tri-fusion+multimodality+reporter+gene+expression+in+living+subjects.">Imaging tri-fusion multimodality reporter gene expression in living subjects.</a> Cancer Res. 64, 1323-1330 (2004).</li><li>Massoud, T. F., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+in+living+subjects:+seeing+fundamental+biological+processes+in+a+new+lights.">Molecular imaging in living subjects: seeing fundamental biological processes in a new lights.</a> Genes Dev. 17, 545-580 (2003).</li><li>Sicinski, P., 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=The+molecular+basis+of+muscular+dystrophy+in+the+mdx+mouse:+a+point+mutation.">The molecular basis of muscular dystrophy in the mdx mouse: a point mutation.</a> Science. 244, 1578-1580 (1989).</li><li>Mattson, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathogenesis of neurodegenerative disorders. In: Contemporary neuroscience. X, 294 (2001).</li><li>Andersson, J. E., Bressler, B. H., Ovalle, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+regeneration+in+hind+limb+skeletal+muscle+of+the+mdx+mouse.">Functional regeneration in hind limb skeletal muscle of the mdx mouse.</a> J. Muscle Res. Cell Motil. 9, 499-515 (1988).</li></ol>

作者没有透露。

在这项研究中，我们已经描述了一个快速和可靠的分子成像，记者基因的方法，以非侵入性的目标，成肌细胞/多用途储值卡的移植。虽然这项研究表明， 短期的本地化移植的多用途储值卡，其实很容易地应用于细胞移植的纵向评估，通过植入的细胞，细胞的方式 ，有针对性的通过bioluminescense成像（BLI），稳定表达报道基因。为此，本集团已怀有统一的报告基因的转基因小鼠品系。?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> C2C12成肌细胞</td><td> ATCC </td><td></td><td></td></tr><tr><td> Dulbecco改良的Eagle培养基</td><td> Life Technologies公司</td><td> 12800-017 </td><td></td></tr><tr><td>胎牛血清</td><td> Life Technologies公司</td><td> 12483020 </td><td></td></tr><tr><td> 0.25％胰蛋白酶-EDTA </td><td> Life Technologies公司</td><td> 25200-72 </td><td></td></tr><tr><td> Hanks平衡盐溶液</td><td> Sigma Aldrich公司</td><td> H6648 </td><td></td></tr><tr><td>脂质体2000 </td><td> Life Technologies公司</td><td> 11668-019 </td><td></td></tr><tr><td>尼康的Eclipse TE2000-5 </td><td>尼康仪器公司</td><td></td><td></td></tr><tr><td> xenolight D-荧光素</td><td>珀金埃尔默</td><td> 122796 </td><td>在PBS中的40毫克/毫升</td></tr></tbody></table>

molecular imaging to target transplanted muscle progenitor cells

杜氏进行性肌营养不良症（DMD）是一种严重的遗传性神经肌肉疾病，影响1，在3500名男生，其特点是渐进性肌肉变性1，2。患者，有能力的居民肌肉卫星细胞（SC）的再生受损的肌纤维变得越来越低效4。因此，移植的肌肉祖细胞器（MPC）/成肌细胞从健康到DMD是一种很有前途的治疗方法。利用干细胞治疗的主要限制因素，然而，植入的细胞，长期监测和评价其有效性是缺乏可靠的成像技术。在这里，我们描述了一种非侵入性的，实时的方法来评价成肌细胞移植的成功。该方法利用了一个统一的融合的基因组成的报告基因（萤火虫荧光素酶[ 涨落 ]，单体红色荧光蛋白[MRFP]和SR39胸苷激酶[sr39tk基因 ]），其expre裂变可以与不同的成像模式9，10成像。多种成像模式，包括正电子发射断层扫描（PET），单光子发射计算机断层显像（SPECT），磁共振成像（MRI），光学成像，和高频率的三维超声现已有售，每一个具有独特的优势和局限性11。生物发光成像（BLI）的研究，例如，具有的优点是相对低的成本和高吞吐量。这是因为这个原因，在本研究中，我们利用鼠标后的萤火虫荧光素酶（ 涨落 ）报告基因序列内包含为短期的本地化的可行的C2C12细胞的融合基因和生物发光成像（BLI）植入到模型的DMD（肌肉萎缩症的X染色体上[MDX]鼠标）12-14。更重要的是，BLI为我们提供了一个方法来检查标记的多用途储值卡植入后的动力学，将是有益的跟踪多次在T细胞IME和下面的迁移。我们的记者基因的方法还使我们能够合并在一个单一的生物体内的多种成像模式，断层性质，精细空间分辨能力扩展到较大 ​​的动物和人类的10,11，，PET将形成未来工作的基础上，我们建议可以方便快速的翻译方法在细胞的临床前模型和临床应用开发。

当50-60％汇合，C2C12细胞瞬时转染的与上述融合报告基因构建体组成的萤火虫荧光素酶[ 涨落 ]，单体红色荧光蛋白[MRFP]和SR39胸苷激酶[sr39tk基因 ]（ 图1A）。计算转染效率，通过荧光显微镜（ 图1B中的C），在我们的记者构造使得的MRFP序列使用。不影响细胞的生存能力，通过标记与BLI基板上，D-荧光素（ 图1D）。转染后，约100,000的MRFP?...

Watch this Scientific Journal Video about Molecular Imaging to Target Transplanted Muscle Progenitor Cells at JoVE.com

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

非侵入性的方法来评估成肌细胞移植的成功。该方法利用一个统一的融合的记者基因组成的基因的表达，可以被不同的成像方式成像。在这里，我们使用<em&gt;的波动</em&gt;报告基因序列到靶细胞通过生物发光成像。

目标移植的肌肉祖细胞的分子成像

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

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9.1K 次观看.  Lawson Health Research Institute. 非侵入性的方法来评估成肌细胞移植的成功。该方法利用一个统一的融合的记者基因组成的基因的表达，可以被不同的成像方式成像。在这里，我们使用的波动报告基因序列到靶细胞通过生物发光成像。

视频: Molecular Imaging to Target Transplanted Muscle Progenitor Cells

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

体内近红外荧光（NIRF）血管内分子成像炎性斑块，多式联运方针成像动脉粥样硬化

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

科研

医学

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes.
A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages 1. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo.
Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles 2-4. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images 5. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM-1 sec-1 strength4. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure 6. Our group has applied this agent in an &ldquo;off label&rdquo; use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.

We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.

Ferumoxytol，一个FDA批准的氧化铁纳米粒子标记干细胞

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

The Use of Thermal Infra-Red Imaging to Detect Delayed Onset Muscle Soreness

Delayed onset muscle soreness (DOMS), also known as exercise induced muscle damage (EIMD), is commonly experienced in individuals who have been physically inactive for prolonged periods of time, and begin with an unexpected bout of exercise1-4, but can also occur in athletes who exercise beyond their normal limits of training5. The symptoms associated with this painful phenomenon can range from slight muscle tenderness, to severe debilitating pain1,3,5. The intensity of these symptoms and the related discomfort increases within the first 24 hours following the termination of the exercise, and peaks between 24 to 72 hours post exercise1,3. For this reason, DOMS is one of the most common recurrent forms of sports injury that can affect an individual&#x2019;s performance, and become intimidating for many1,4.
For the last 3 decades, the DOMS phenomenon has gained a considerable amount of interest amongst researchers and specialists in exercise physiology, sports, and rehabilitation fields6. There has been a variety of published studies investigating this painful occurrence in regards to its underlying mechanisms, treatment interventions, and preventive strategies1-5,7-12. However, it is evident from the literature that DOMS is not an easy pathology to quantify, as there is a wide amount of variability between the measurement tools and methods used to quantify this condition6. It is obvious that no agreement has been made on one best evaluation measure for DOMS, which makes it difficult to verify whether a specific intervention really helps in decreasing the symptoms associated with this type of soreness or not. Thus, DOMS can be seen as somewhat ambiguous, because many studies depend on measuring soreness using a visual analog scale (VAS)10,13-15, which is a subjective rather than an objective measure. Even though needle biopsies of the muscle, and blood levels of myofibre proteins might be considered a gold standard to some6, large variations in some of these blood proteins have been documented 6,16, in addition to the high risks sometimes associated with invasive techniques. 
Therefore, in the current investigation, we tested a thermal infra-red (IR) imaging technique of the skin above the exercised muscle to detect the associated muscle soreness. Infra-red thermography has been used, and found to be successful in detecting different types of diseases and infections since the 1950&#x2019;s17. But surprisingly, near to nothing has been done on DOMS and changes in skin temperature. The main purpose of this investigation was to examine changes in DOMS using this safe and non-invasive technique.

The purpose of this investigation was to assess whether using an infra-red thermal camera is a valid tool for detecting and quantifying the muscle soreness after exercising.

热红外成像来检测迟发性肌肉酸痛

Delayed onset muscle soreness (DOMS), also known as exercise induced muscle damage (EIMD), is commonly experienced in individuals who have been physically ...

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

前列腺癌与小分子肽CLT1靶向造影剂的磁共振分子成像

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

顺序<em&gt;在体内</em&gt;成骨干/祖细胞在骨折修复的影像学

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

骨骼肌疾病的定量磁共振成像

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

主动脉钙化和炎症血管平滑肌细胞和成像的钙化

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

Contractility Measurements of Human Uterine Smooth Muscle to Aid Drug Development

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We describe methods to measure ex vivo myometrium contractility. This assay can be used to investigate factors and molecules involved in the modulation of myometrial contraction and to determine their excitatory or inhibitory actions, and hence their therapeutic potential in vivo. Biopsies are obtained from women undergoing cesarean section delivery with informed consent. Fine strips of myometrium are dissected, clipped and attached to a force transducer within 1 mL organ baths superfused with physiological saline solution at 37 &#176;C. Strips develop spontaneous contractions within 2&#8211;3 h under set tension and remain stable for many hours (&#62;6 h). Strips can also be stimulated to contract such as by the endogenous hormones, oxytocin and vasopressin, which cause concentration-dependent modulation of contraction frequency, force and duration, to more closely resemble contractions in labor. Hence, the effect of known and novel drug leads can be tested on spontaneous and agonist-induced contractions.
This protocol specifically details how this assay can be used to determine the potency of known and novel agents by measuring their effects on various parameters of human myometrial contraction. We use the oxytocin- and V1a receptor antagonists, atosiban and SR49059 as examples of known compounds which inhibit oxytocin- and vasopressin-induced contractions, and demonstrate how this method can be used to complement and validate pharmacological data obtained from cell-based assays to aid drug development. The effects of novel agonists in comparison to oxytocin and vasopressin can also be characterized. Whilst we use the example of the oxytocin/ vasopressin system, this method can also be used to study other receptors and ion channels that play a role in uterine contraction and relaxation to advance the understanding of human uterine physiology and pathophysiology.

This article describes experimental protocols to study ex vivo contractions of human myometrium and their application in drug discovery. This technique is used to improve the understanding of myometrial physiology and pathophysiology as well as to validate pharmacological data from novel research probes or drug leads.

人子宫平滑肌的收缩力测量对药物开发的帮助

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We ...