1。 GIMM结肠组织的准备工作<ol><li>为了准备肠胃蠕动监视器（GIMM），首先在冰冷的Krebs液隔离冒号（121毫米氯化钠，氯化钾5.9 mM的，2.5毫米氯化钙 2，MgCl 2的1.2毫米，25毫米碳酸氢钠 3远端结肠段1.2毫米的NaH 2 PO 4，和8毫米葡萄糖，95％O 2 / 5％CO2充气） 。从外壁清理余下的肠系膜和一个小切口在口腔端，所以它可以区分放置到器官浴时。注：组织可能在冰Krebs液保持长达2个小时前，实验。 </li><li>下一步，位置在器官浴的流入和流出的渠道，使他们的相机领域，以防止干扰与图像采集外。连续灌注与器官水浴预热（37℃）含氧（95％，5％CO 2）的Kreb在10毫升/分钟的流速的解决方案。 </li><li>跟踪的口腔与肛门两端，在器官浴的两端引脚上的远端结肠段（至少5厘米），允许一个松弛的小程度，使该段可以自由移动在中间1厘米。口头到底应该定位走向，便于放置的粪便颗粒研究员。应固定结肠段内的每一个给定的实验的实验研究员以同样的方式，因为段的长度和紧张会影响推进运动的速度， 减少过境（迪克森等率与纵向舒展。 ，2007）。 </li><li>允许至少30分钟的准备，以平衡。 </li></ol> 2。设置GIMM和数据采集<ol><li>在GIMM系统中，在灌注室的结肠段是从下面照亮。数码摄像机与计算机相连的是定位上面室。确保光照明光源和GIMM软件打开。 </li><li>设立GIMM软件应用程序中的一个新的实验后，开始插入环氧树脂涂层的粪便颗粒口服结肠段启动肠蠕动的一审判决。相机上的计算机上的切换开关，点击打开相机和点击记录按钮开始录制。颗粒在肛门方向的运动是由摄像机和记录以供日后分析的PC上存储的数字电影。当颗粒已经达到了结肠段结束后，单击“记录”按钮来停止录制。 </li><li>为了获得控制推进速度值，开始从健康动物的结肠段，并没有申请药物。 3-5试验，在一个单一的准备行为，与彼此之间运行5分钟的恢复时期。 </li><li>要确定一定的条件或药物对结肠运动的影响，每个实验条件下执行3-5试验/准备。此外，在执行上的每个实验至少五年至少有五种不同动物的不同冒号。 </li><li>在数字电影的分析，粪便颗粒推进率的计算公式为弹丸穿越x厘米的结肠段时间。 </li></ol> 3。建筑的时空地图<ol><li>从个人GIMM运行取得的数字视频可转​​换为使用自定义的GIMM软件时空地图。 </li><li>横轴上，结肠直径的变化是随着时间的推移绘制转换结肠的形象，在每个视频帧的一个剪影，并计算和转换沿整个长​​度为灰度的直径。最终的结果是，沉淀和放松的地方显示为黑色，而收缩的区域显示为白色。 </li><li>颗粒穿过结肠段的距离，垂直轴代表。 </li></ol> 4。具有代表性的时空地图<ol><li>这里展示的是具有代表性的颗粒运动的时空地图显示在各种实验条件下，在结肠段。 y（垂直​​）轴代表颗粒的位置，随着时间的推移，而X轴（横向）代表的距离，通过结肠段的颗粒进展。 <img src="/files/ftp_upload/2435/2435fig1.jpg" alt="图1" /> </li><li>在从控制冒号（未经处理的，正常）的时空地图，颗粒会进步的线性〜2毫米/秒的速度。相比之下，管理，对某些药物或在结肠的炎症可以导致如停止蠕动和蠕动受阻中断蠕动模式。 </li><li> GIMM结果也可以作为图形显示，Y轴代表在粪便颗粒，X轴代表时间，以秒前往毫米的距离。例如，管理，DAMGO（D - 助理法律顾问2，N - ME - Phe4，甘氨酸 - ol5）鸦片类μ-受体激动剂，导致减少在结肠推进运动。 </li></ol>

<ol>
	<li>Bayliss, W. M., Starling, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+movements+and+innervation+of+the+small+intestine.">The movements and innervation of the small intestine.</a> J of Physiol (Lond). 24, 99-143 .</li><li>Dickson, E. J., Spencer, N. J., Hennig, G. W., Bayguinov, P. O., Ren, J., Heredia, D. J., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enteric+occult+reflex+underlies+accommodation+and+slow+transit+in+the+distal+large+bowel.">An enteric occult reflex underlies accommodation and slow transit in the distal large bowel.</a> Gastroenterology. 132, 1912-1924 (2007).</li><li>Foxx-Orenstein, A. E., Jin, J. G., Grider, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-HT4+receptor+agonists+and+delta-opioid+receptor+antagonists+act+synergistically+to+stimulate+colonic+propulsion.">5-HT4 receptor agonists and delta-opioid receptor antagonists act synergistically to stimulate colonic propulsion.</a> Am J Physiol. 275, 979-983 (1998).</li><li>Grider, J. R., Foxx-Orenstein, A. E., Jin, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Hydroxytryptamine4+receptor+agonists+initiate+the+peristaltic+reflex+in+human,+rat,+and+guinea+pig+intestine.">5-Hydroxytryptamine4 receptor agonists initiate the peristaltic reflex in human, rat, and guinea pig intestine.</a> Gastroenterology. 115, 370-380 (1998).</li><li>Kadowaki, M., Wade, P. R., Gershon, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Participation+of+5-HT3,+5-HT4,+and+nicotinic+receptors+in+the+peristaltic+reflex+of+guinea+pig+distal+colon.">Participation of 5-HT3, 5-HT4, and nicotinic receptors in the peristaltic reflex of guinea pig distal colon.</a> Am J Physiol. 271, 849-857 (1996).</li><li>Krauter, E. M., Strong, D. S., Brooks, E. M., Linden, D. R., Sharkey, K. A., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+colonic+motility+and+the+electrophysiological+properties+of+myenteric+neurons+persist+following+recovery+from+trinitrobenzene+sulfonic+acid+colitis+in+the+guinea+pig.">Changes in colonic motility and the electrophysiological properties of myenteric neurons persist following recovery from trinitrobenzene sulfonic acid colitis in the guinea pig.</a> Neurogastroenterol Motil. 19, 990-1000 (2007).</li><li>Langley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Autonomic Nervous System. , (1921).</li><li>Linden, D. R., Sharkey, K. A., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+excitability+of+myenteric+AH+neurones+in+the+inflamed+guinea-pig+distal+colon.">Enhanced excitability of myenteric AH neurones in the inflamed guinea-pig distal colon.</a> J Physiol. 547, 589-601 (2003).</li><li>Linden, D. R., Sharkey, K. A., Ho, W., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclooxygenase-2+contributes+to+dysmotility+and+enhanced+excitability+of+myenteric+AH+neurones+in+the+inflamed+guinea+pig+distal+colon.">Cyclooxygenase-2 contributes to dysmotility and enhanced excitability of myenteric AH neurones in the inflamed guinea pig distal colon.</a> J Physiol. 557, 191-205 (2004).</li><li>Linden, D. R., Chen, J. X., Gershon, M. D., Sharkey, K. A., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+availability+is+increased+in+mucosa+of+guinea+pigs+with+TNBS-induced+colitis.">Serotonin availability is increased in mucosa of guinea pigs with TNBS-induced colitis.</a> Am J Physiol Gastrointest Liver Physiol. 285, 207-216 (2003).</li><li>Linden, D. R., Couvrette, J. M., Ciolino, A., McQuoid, C., Blaszyk, H., Sharkey, K. A., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indiscriminate+loss+of+myenteric+neurones+in+the+TNBS-inflamed+guinea-pig+distal+colon.">Indiscriminate loss of myenteric neurones in the TNBS-inflamed guinea-pig distal colon.</a> Neurogastroenterol Motil. 17, 751-760 (2005).</li><li>Lomax, A. E., Mawe, G. M., Sharkey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+facilitation+and+enhanced+neuronal+excitability+in+the+submucosal+plexus+during+experimental+colitis+in+guinea-pig.">Synaptic facilitation and enhanced neuronal excitability in the submucosal plexus during experimental colitis in guinea-pig.</a> J Physiol. 564, 863-875 (2005).</li><li>Lomax, A. E., O'Hara, J. R., Hyland, N. P., Mawe, G. M., Sharkey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+alterations+to+enteric+neural+signaling+in+the+guinea+pig+colon+following+the+resolution+of+colitis.">Persistent alterations to enteric neural signaling in the guinea pig colon following the resolution of colitis.</a> Am J Physiol Gastrointest Liver Physiol. 292, 482-491 (2007).</li><li>Strong, D. S., Cornbrooks, C. F., Roberts, J. A., Hoffman, J. M., Sharkey, K. A., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+neuromuscular+transmission+is+selectively+attenuated+in+ulcerated+regions+of+inflamed+guinea+pig+distal+colon.">Purinergic neuromuscular transmission is selectively attenuated in ulcerated regions of inflamed guinea pig distal colon.</a> J Physiol. 588, 847-859 (2010).</li><li>Wade, P. R., Chen, J., Jaffe, B., Kassem, I. S., Blakely, R. D., Gershon, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+and+function+of+a+5-HT+transporter+in+crypt+epithelia+of+the+gastrointestinal+tract.">Localization and function of a 5-HT transporter in crypt epithelia of the gastrointestinal tract.</a> J Neurosci. 16, 2352-2364 (1996).</li><li>Wood, M. J., Hyman, N. H., Mawe, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effects+of+Daikenchuto+(DKT)+on+Propulsive+Motility+in+the+Colon.">The Effects of Daikenchuto (DKT) on Propulsive Motility in the Colon.</a> J Surg Res. , (2009).</li></ol>

生产此视频赞助野猫的研究和开发，公司，生产在这篇文章中使用的仪器。

在胃肠道推进运动神经介导的首次描述了超过一个世纪前由贝里斯和椋鸟（贝里斯和椋鸟，1899年）。这一观察结果导致肠道神经肠神经系统（ENS），自主神经系统的不同分工（兰利，1921年）的指定。神经源性肠道蠕动，涉及到在一个给定的点和反射介导的收缩以上，或口头，在aboral方向上的刺激和放松的水平拉伸和/或粘膜刺激。其结果是产生一个压力梯度，推动在一个口头aboral方向上的管腔内容。在各种物种的小肠，并在大鼠大肠收缩蠕动波可以被激活进入肠段管腔输液。在豚鼠远端结肠，天然或人工粪粒可以插入口服结肠，沿结肠段的进展可以很容易地监控。因此，豚鼠远端结肠提供了一个简单而有用的实验调查在肠道推进运动。 蠕动是一个复杂的神经反射，涉及的neuroactive化合物和受体的数量。因此，影响神经递质的ENS的化合物，影响推进运动的速度。羟色胺，主要在肠黏膜肠嗜铬细胞释放，参与发起的蠕动反射。利用豚鼠远端结肠，Grider和同事证明，推进运动是增强5 -羟色胺4受体激动剂（福克斯，Orenstein 等人 ，1998年。; Grider 等 ，1998）的intralumenal管理，同时应用浴5 -羟色胺和5 -羟色胺受体拮抗剂降低推进率（门胁等 ​​，1996;。菩提树等 ，2003年b）。此外，抑制羟色胺转运与选择性血清素再摄取抑制剂（SSRIs），在低浓度增加肠蠕动，但跌幅在较高浓度的蠕动，可能是由于5 -羟色胺受体（门胁等 ​​人 ，1996年的脱敏;。韦德等人， 1996年）。其他豚鼠推进运动的信号分子，包括乙酰胆碱尼古丁受体，这是在肠内interneuronal信号的主要调停。封锁与尼古丁受体拮抗剂六烃的突触传递，减少在高浓度颗粒推进，而较小的浓度不影响推进率（门胁等 ​​人，1996年）。阿片类药物，阿片受体激动剂，它长期以来一直被肠胃蠕动有抑制作用，减少颗粒在豚鼠推进运动模式的推进（福克斯，Orenstein等，1998;。Wood等，2009） 。有趣的是，日本的传统中草药，临床上用于治疗呕吐，腹痛，和无序运动，daikenchuto，跌幅颗粒推进的速度与阿片拮抗剂纳洛酮管理，单独给药时，逆蠕动的结果（ Wood等。 2009年）。这些发现说明豚鼠推进运动模型，以评估各种化合物和结肠运动模式及其受体的贡献的实用性。 大量研究表明，炎症导致结肠神经元“（2005 年林登等人，2003年。;洛马克斯等，）的电气和突触的变化，这些变化可以坚持周炎症恢复后（Krauter 等 ，2007;洛马克斯等，2007）。豚鼠远端使用GIMM系统的结肠推进运动结肠炎诱导neuroplastic变化的影响，可以追究。该系统在监测，评估通过简单的测量时间，粪便颗粒传播给定的距离推进运动的结肠运动的传统方法有几个优点。以往的研究，在结肠的炎症引起的神经可塑性都表示有颗粒推进（ 菩提树等，2003 年 。; 林登等，2005）的速度在减少，但最近的调查，使用的GIMM系统已经透露了更多（强等 ，2010），复杂的变化。例如，颗粒推进率是下降的远端结肠发炎溃烂地区，但在邻近地区，这不是由早前的“秒表”的方法确认的现象加速蠕动。其他研究也显示，在肠道炎症的活跃期，可以恢复与抑制COX - 2（ 菩提树等 ，2004），但改变蠕动持续超越解决炎症的COX - 2不敏感（Krauter等，2007） 。此外，在这些后来的研究中，由GIMM系统生成的时空地图显示，在炎症后的动物，有助于推进率下降观察结肠的蠕动，逐步格局。 豚鼠远端结肠推进运动的实验结论，代表一个高吞吐量的测量测试化合物和病理条件下，对结肠运动的影响，此外，GIMM系统提供了一个简单而直观的方法来评估在体外的结肠推进运动。它可以连续测量颗粒的推进，以及一代的时空地图研究自发的活动模式。与传统方法相比，它也可以产生更复杂的现象，可以定量和重新分析多种方式，数字文件保存到系统和方便。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Krebs&#x2019; Solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane</td>
<td>Anesthesia</td>
<td>Webster Veterinary</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Epoxy-coated fecal pellet </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>native guinea pig pellet dried and epoxy (black nail polish) coated</td>
</tr>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micro Scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Stainless Steel Pins</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Beakers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>500 mL</td>
</tr>
<tr>
<td>Gas Tank</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>95% O2/5% CO2</td>
</tr>
<tr>
<td>Timer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gastrointestinal Motility Monitor</td>
<td>&nbsp;</td>
<td>Catamount Research and Development</td>
<td>&nbsp;</td>
<td><a href="http://www.catamountresearch.com/products/gimm.htm">http://www.catamountresearch.com/products/gimm.htm</a></td>
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gastrointestinal motility monitor (gimm)

肠胃蠕动监视器（GIMM;野猫的研究与发展;圣奥尔本斯，VT）是在体外系统，监视在豚鼠远端结肠的隔离段推进运动。整个系统由一台电脑，摄像机，照明器官浴槽，循环泵的蠕动和恒温水浴，和自定义GIMM软件来记录和分析数据。与传统监测结肠的蠕动方法相比，GIMM系统可连续，定量评价蠕动。豚鼠远端结肠是沐浴在温暖，含氧Krebs液，并在口头端插入粪粒沿结肠段约2毫米/秒的速度推进。被捕获的粪便颗粒沿段出发的电影，GIMM软件可以被用来跟踪的粪便颗粒的进展。推进运动的速率可为整个段或特定区域的任何利益。除了丸引起的蠕动模式的分析，时空地图可以从捕获的视频段的建造评估自发运动活动模式。该系统的应用，包括药理评价受体激动剂和拮抗剂推进运动的影响，以及评估进行的病理生理条件，如炎症或压力，导致变化的。豚鼠远端结肠推进运动实验，使用的GIMM系统，非常简单，简单易学，它提供了一个可靠和可重复性的方法评估推进运动。

Watch this Scientific Journal Video about Gastrointestinal Motility Monitor GIMM at JoVE.com

Gastrointestinal Motility Monitor GIMM

（GIMM）肠胃蠕动监视器在豚鼠远端结肠的结肠运动的评价是一个简单和简单易学的方法，定量评价消化道推进运动。

肠胃蠕动监视器（GIMM）

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility ...

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Unit 1

Gastrointestinal Motility Monitor (GIMM)

Drugs Affecting Gastrointestinal Motility

SCIENTISTS IN ACTION

30.6K 次观看.  The University of Vermont. （GIMM）肠胃蠕动监视器在豚鼠远端结肠的结肠运动的评价是一个简单和简单易学的方法，定量评价消化道推进运动。

视频: Gastrointestinal Motility Monitor GIMM

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

使用琼脂糖凝胶为基础的微流体装置的评估神经干细胞的运动

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

科研

生物学

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

在啮齿类动物呼吸道上皮纤毛运动功能的高分辨率成像的体外方法

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

准备，成像和定量的细菌表面运动性测定

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

幼虫活动的测量<em&gt;果蝇</em&gt;活动监视器

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues. When compared to previous techniques the video recording and STmap approach provides a more comprehensive understanding of gastrointestinal motility.

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Recently available video recording and spatiotemporal mapping (STmap) techniques make it possible to visualize and quantify both propagating and mixing patterns of intestinal motility. The goal of this protocol is to explain the generation and analysis of STmaps using the GastroIntestinal Motility Monitoring (GIMM) system.

运动性的时空制图<em&gt;离体</em肠子&gt;准备工作

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

高分辨率法监测IRF3的磷酸化依赖性激活

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

利用pHluorin标记的受体监视亚细胞定位和贩运

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

使用前 Vivo 共聚焦显微镜可视化淋巴节点结构和细胞定位

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Measuring Crop Motility and Food Passaging in Drosophila

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. Disordered GI motility and gastric emptying cause multiple diseases and symptoms. As a powerful genetic model organism, Drosophila can be used in GI motility research. The Drosophila crop is an organ that contracts and moves food into the midgut for further digestion, functionally similar to a mammalian stomach. Presented is a protocol to study Drosophila crop motility using simple measurement tools. A method for counting crop contractions to evaluate crop motility and a method for detecting the distribution of food dyed blue between the crop and gut using a spectrophotometer to investigate the effect of the crop on food passaging is described. The method was used to detect the difference in crop motility between control and nprl2 mutant flies. This protocol is both cost-efficient and highly sensitive to crop motility.

Measuring Crop Motility and Food Passaging in Drosophila

The goal of this protocol is to measure crop contraction and quantify food distribution in the Drosophila gut.

测量果蝇的作物运动性和食物通度

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. ...

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

使用实时细胞代谢通量分析仪监测成骨细胞生物能量学

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...