We would like to thank the Children&#8217;s Hospital at Montefiore, Department of Pediatrics, for providing support for these studies. We would like to thank the National Alopecia Areata Foundation for providing financial support for conducting studies with C3H/HeJ engrafted mice.

伦理学声明:所有涉及动物的研究必须由该机构IACUC批准（为此如下数据，协议批准了蒙蒂菲奥里IACUC，协议＃11-6-240和＃13-7-100）。动物提供了浅麻醉指示摄影过程中保持他们仍然是唯一的目的，有需要这种协议没有痛苦的过程。 1.采集照片的<ol><li>看设定对焦和现场使用的纸张与印刷文字凝胶成像仪。验证跨区域拍照光源均匀性。为了帮助确保均匀性，使用凝胶成像仪具有一个内置的光源，用于反射摄影。不要使用透照光源，因为这将创建一个不适合进一步的灰度分析动物的剪影。 注:这将将鼠标稍微失焦，其中跨过感兴趣区域（ROI）的区域提供了光学平均，并会有助于减少量子错误的利息非常小（ 即 &lt;10像素）的区域。感兴趣较大区域将不会影响此次要聚焦调整，也不会受到影响，在动物的大小差异。 </li><li>麻醉用氯胺酮（100毫克/毫升）/赛拉嗪（20毫克/毫升）的动物（2:1），因为这提供了快速麻醉效果和快速恢复和最适合拍摄多个动物。 注:麻醉证实当动物仍足以允许摄影。由于动物通常在10-15分钟这个浅麻醉恢复，兽医已经不推荐使用眼药膏的。 </li><li>放置麻醉动物在垂直取向凝胶成像仪（如接近平行越好）。 <ol><li>对于背照片，将动物的四肢伸展俯卧位。 </li><li>对于腹面照片，将动物在仰卧位，照顾这些动物不是横向旋转。 </li&gt; </ol></li><li>将在拍下区域灰度标准。 </li><li>关闭检修门。重要提示:环境光可以引入变化曝光。 </li><li>设置光圈的曝光哪个地方采集的线性范围内的感兴趣的区域（读出的F-停止）。 注意:大多数的系统将显示在图像饱和。 </li><li>拍摄照片。 </li><li>变化的F-停止向其中通过增加或由1减小光圈，这样，无论是标准的和感兴趣的区域保持在采集（参考的线性范围放置的线性范围内的感兴趣的区域的另一曝光设置F-停止）。 </li><li>拍摄照片。 </li><li>一旦拍摄完成后，将动物在加温台和监控，直到他们能保持胸骨斜卧。回到动物的笼子。返回组动物一次所有的动物完全恢复的动物饲养。 </li></ol> 2.定量光吸收的&lt;/ P&gt; <ol><li>使用提供的对动物的图像感兴趣区域标记软件用于凝胶成像仪。 <ol><li>对于整个动物背视图中，使用矩形或椭圆形的图像从上肢到下肢延伸，横向延伸尽可能使得盒的任何部分延伸超过该动物的背部， 如图1A所示 。 注意:一个也标记感兴趣的使用徒手画图工具的区域。 </li><li>对于整个动物腹面观中，使用2矩形:1覆盖骨盆区域，一个覆盖胸部区域， 如图1B所示 。 </li><li>对于感兴趣的， 即，给药位置，标记为适当的较小的区域。 </li></ol></li><li>对灰度吸收基准利率标记区域（S）。 </li><li>从每个感兴趣的标记区域的记录吸收。 </li></ol> 3.分析吸收水平obtained表示感兴趣的区域，可能需要进行归一化的背景标准照片比较。暴露于吸收的关系是双对数（ 即，该关系是log（曝光之间线性）和log（吸收））。使用这种关系，感兴趣的区域的吸收可以是归一化到一个标准的背景值，这将允许吸收直接不同的照片，其中包括那些在不同时间点（一个研究协议范围内，即，连续测量）拍摄之间进行比较。用于执行这些校正的过程详述在任选的步骤3.1和3.2。 <ol><li>日志（曝光）使用来自灰度吸收标准得到的数值（可选）绘制的曲线与日志（吸收）。 </li><li> （可选）调整的变化每张照片的标准如下: <ol><li>选择F-停止阅读和F-STOP（如步骤1.6确定），以供参考（如在下定决心TEP 1.8）。 </li><li>计算标准平均吸收所有的照片在阅读F-停止。这平均值为参考标准值（RSV）。 </li><li>计算在每个照片中的阅读和参考光圈设置之间的吸收差值为标准（Δ-S）和为每个定义的ROI（Δ-ROI-1，Δ-ROI-2 ... ..delta-ROI-X）的</li><li>计算校正吸收每个ROI如下:在阅读F-停止更正吸收率（ROI-X）=吸收率（ROI-X）+（RSV - 吸收的标准，在阅读F-停止）*（Δ-ROI-X / Δ-S） </li></ol></li><li>编译的实验数据，并用标准的统计方法对连续变量进行数据分析（ 即，方差分析，T检验）。 </li></ol>

<ol>
	<li>McAndrews, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+Hair+Loss+Association.">American Hair Loss Association.</a> , (2011).</li><li>Safavi, K. H., Muller, S. A., Suman, V. J., Moshell, A. N., Melton, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+alopecia+areata+in+Olmsted+County,+Minnesota,+1975+through+1989.">Incidence of alopecia areata in Olmsted County, Minnesota, 1975 through 1989.</a> Mayo Clin Proc. 70 (7), 628-633 (1995).</li><li>Hussein, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy-induced+alopecia:+new+developments.">Chemotherapy-induced alopecia: new developments.</a> South Med J. 86 (5), 489-496 (1993).</li><li>Trueb, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy-induced+hair+loss.">Chemotherapy-induced hair loss.</a> Skin Therapy Lett. 15 (7), 5-7 (2010).</li><li>Sato, N., Leopold, P. L., Crystal, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+adenovirus-mediated+expression+of+Sonic+hedgehog+gene+on+hair+regrowth+in+mice+with+chemotherapy-induced+alopecia.">Effect of adenovirus-mediated expression of Sonic hedgehog gene on hair regrowth in mice with chemotherapy-induced alopecia.</a> J Natl Cancer Inst. 93 (24), 1858-1864 (2001).</li><li>McGarvey, E. L., Baum, L. D., Pinkerton, R. C., Rogers, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychological+sequelae+and+alopecia+among+women+with+cancer.">Psychological sequelae and alopecia among women with cancer.</a> Cancer Pract. 9 (6), 283-289 (2001).</li><li>Baxley, K. O., Erdman, L. K., Henry, E. B., Roof, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alopecia:+effect+on+cancer+patients'+body+image.">Alopecia: effect on cancer patients' body image.</a> Cancer Nurs. 7 (6), 499-503 (1984).</li><li>Stough, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychological+effect,+pathophysiology,+and+management+of+androgenetic+alopecia+in+men.">Psychological effect, pathophysiology, and management of androgenetic alopecia in men.</a> Mayo Clin Proc. 80 (10), 1316-1322 (2005).</li><li>Ogunmakin, K. O., Rashid, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alopecia:+the+case+for+medical+necessity.">Alopecia: the case for medical necessity.</a> Skinmed. 9 (2), 79-84 (2011).</li><li>Olsen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+randomized+clinical+trial+of+5%+topical+minoxidil+versus+2%+topical+minoxidil+and+placebo+in+the+treatment+of+androgenetic+alopecia+in+men.">A randomized clinical trial of 5% topical minoxidil versus 2% topical minoxidil and placebo in the treatment of androgenetic alopecia in men.</a> J Am Acad Dermatol. 47 (3), 377-385 (2002).</li><li>Rossi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1+mg+daily+administration+on+male+androgenetic+alopecia+in+different+age+groups:+10-year+follow-up.">1 mg daily administration on male androgenetic alopecia in different age groups: 10-year follow-up.</a> Dermatol Ther. 24 (4), 455-461 (2011).</li><li>Gibbons, R. D., Fiedler-Weiss, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer-aided+quantification+of+scalp+hair.">Computer-aided quantification of scalp hair.</a> Dermatol Clin. 4 (4), 627-640 (1986).</li><li>Fleming, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+a+structural+analysis+of+dermatoscopic+imagery.">Techniques for a structural analysis of dermatoscopic imagery.</a> Comput Med Imaging Graph. 22 (5), 375-389 (1998).</li><li>Chamberlain, A. J., Dawber, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+evaluating+hair+growth.">Methods of evaluating hair growth.</a> Australas J Dermatol. 44 (1), 10-18 (2003).</li><li>. <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 Kodak/Tiffen Q-13 Gray Scale*. , .</li><li>Freyschmidt-Paul, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+alopecia+areata+in+C3H/HeJ+mice+with+the+topical+immunosuppressant+FK506+(Tacrolimus).">Treatment of alopecia areata in C3H/HeJ mice with the topical immunosuppressant FK506 (Tacrolimus).</a> Eur J Dermatol. 11 (5), 405-409 (2001).</li><li>Katikaneni, R., Ponnapakkam, T., Matsushita, O., Sakon, J., Gensure, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+and+prevention+of+chemotherapy-induced+alopecia+with+PTH-CBD,+a+collagen-targeted+parathyroid+hormone+analog,+in+a+non-depilated+mouse+model.">Treatment and prevention of chemotherapy-induced alopecia with PTH-CBD, a collagen-targeted parathyroid hormone analog, in a non-depilated mouse model.</a> Anticancer Drugs. 25 (1), 30-38 (2014).</li><li>Peters, E. M., Foitzik, K., Paus, R., Ray, S., Holick, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+strategy+for+modulating+chemotherapy-induced+alopecia,+using+PTH/PTHrP+receptor+agonist+and+antagonist.">A new strategy for modulating chemotherapy-induced alopecia, using PTH/PTHrP receptor agonist and antagonist.</a> J Invest Dermatol. 117 (2), 173-178 (2001).</li><li>Katikaneni, R., Ponnapakkam, T., Seymour, A., Sakon, J., Gensure, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parathyroid+hormone+linked+to+a+collagen+binding+domain+promotes+hair+growth+in+a+mouse+model+of+chemotherapy+induced+alopecia+in+a+dose-dependent+manner.">Parathyroid hormone linked to a collagen binding domain promotes hair growth in a mouse model of chemotherapy induced alopecia in a dose-dependent manner.</a> Anti-cancer drugs. 25 (7), 819-825 (2014).</li></ol>

Authors have nothing to disclose.

在这份报告中的详细描述，提供了一种新技术，用于量化脱发在啮齿类动物。此技术利用的凝胶成像仪进行图像采集和分析，设备这是在大多数实验室容易获得。测量已被证明是健壮到微小变化在技术（ 图4 - 5），并且是公相关，在治疗模型视觉毛发损失（ 图6）的程度。这些技术已被成功地用于在实验性疗法的评价为脱发17。 ?...

脱发（脱发）可以是心理和情感上令人痛心的事件有多个原因。男性型脱发是脱发的最常见的原因，35岁1影响大约三分之二男性。脱发的一个类似的模式可以在女性多囊卵巢综合征被观察到。在这两种病症中，脱发是雄激素介导的。脱发也可发生自身免疫病，斑秃，从而影响人口2的1.7％。脱发可发生的一些医学治疗，如化疗3的副作用。化疗患者的高比例（65-85％）经历某种程度的脱发4,5。脱发的心理影响已经得到很好的研究，在化疗的设置。化疗引起的脱发会导致焦虑，抑郁，消极的身体形象，降低自尊和幸福6,7减少的感觉。高PE女性癌症患者rcentage（47-58％）认为脱发是化疗最痛苦的方面，以及高达8％的跌幅治疗恐惧脱发4,6。也有在雄激素性脱发的证据来支持治疗，以减少脱发8,9-心理甚至医学后果。同样，斑秃已报道有严重的心理后果2和脱发的斑片状性质可以导致更不愉快的美容效果比脱发的最其他原因。 虽然药物轻度抗雄激素效应（ 即，安体舒通）曾使用了有限的成功为治疗脱发，首先有效的药物治疗脱发的米诺地尔是10。这种降压具有引起毛发生长的观察副作用，现在用作局部治疗许多形式的脱发。然而，反应往往是不完整的，有些科目只显示缓慢荷兰国际集团的脱发，而不是实际的再生10。非那雄胺是一种竞争性拮抗剂II型5α还原酶其阻断睾酮转化为二氢睾酮，致使在局部全身雄激素阻断牺牲改善雄激素性脱发。应答率与长期（10年以上）的治疗是在50％左右11。总体而言，尽管相当多的研究在这方面，目前还没有适当的治疗脱发。 几十年来，科学家和临床医生已经检查测量的头皮头发生长的临床试验的方法。用的药物治疗脱发的发展，出现了用于测量头发的生长，并且具体地，对治疗的反应的可靠的，经济的和微创手段更加需要。图像分析技术对头发密度的患者的脱发症的准确定量，得到在过去的一致和有效的结果使用各种techniqu的ES，包括分析数字化图像12，每根毛发的图像分析和皮损13，和显微镜扫描量化毛传中定义的头皮区域14。 不幸的是，虽然上述的方法提供了效力的改进的评价对头发生长促进干预在临床试验中，这些方法还没有被应用到啮齿动物的研究中的临床前调查。我们的目标是开发一个一致的和可靠的方法来量化脱发小鼠，这将允许研究者能够更准确地评估和比较新的治疗方法对各种形式的脱发。我们已经开发使用的设备在大多数实验室，这将使头发密度的小鼠棕色或黑色的头发快速，可靠的量化一应俱全的方法。这种方法已在化疗诱导的脱发，斑秃，并从waxin脱发的小鼠模型被测试克。详细的协议提出了进行这些测量，包括C57BL / 6的验证数据和C3H /小鼠品系HeJ小鼠。作为这种技术依赖于从在毛干色素检测光的吸收，它不能被用于检测的毛发生长小白鼠或白化小鼠。

<table border="0" cellpadding="0" cellspacing="0" width="623">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:121px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">KODAK Gel Logic 100 Imaging System&#160;&#160;</td>
			<td style="width:121px;">Eastman Kodak Company, Rochester, NY, USA</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Gel imager for obtaining and analyzing photographs, must have built in light source for reflective photography.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">The Kodak/Tiffen Q-13 Gray Scale</td>
			<td style="width:121px;">Imatest, http://www.imatest.com/docs/q13/</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Greyscale standard</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">C57BL/6J Mice</td>
			<td style="width:121px;">&#160;Jackson Laboratories, Bar Harbor, Maine</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Mice for representative study</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">C3H/HeJ engrafted mouse</td>
			<td style="width:121px;">&#160;Jackson Laboratories, Bar Harbor, Maine</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Mice for representative study</td>
		</tr>
	</tbody>
</table>

a new technique for quantitative analysis of hair loss in mice using grayscale analysis

脱发是它可以发生在许多不同的条件，包括男性型脱发，多囊性卵巢综合征和斑秃脱发的常见形式。脱发，也可能发生由于化疗的癌症患者的副作用。在这项研究中，我们的目标是开发一个一致的和可靠的方法来量化脱发小鼠，这将允许研究者准确地评估和比较新的治疗方法，这些不同形式的脱发。该方法利用一种标准的凝胶成像仪获取和处理的小鼠的图像，测量光吸收，这发生在粗糙成比例的黑色（或灰色）的头发上的鼠标的量。已量化以这种方式的数据然后可以使用标准的统计技术（ 即，方差分析，T检验）分析。这种方法是在化疗诱导的脱发，斑秃和脱发的小鼠模型从打蜡测试。在这份报告中，详细的协议是PRESented用于执行这些测量，其中包括来自C57BL / 6的验证数据和C3H /小鼠HeJ小鼠品系。这种新技术提供了许多优点，包括应用的相对简单性，依赖于设备这是在大多数研究实验室容易获得的，并且施加一个客观，定量评估这比主观评价更加健壮。改善头发生长的量化小鼠会改善脱发模型研究和促进有前途的新治疗方法的评价在临床前研究。

用的C3H / HeJ小鼠植入小鼠，脱发的小鼠模型斑秃16这种技术进行了验证。这些动物发展出进步逐渐随着时间的推移全球性脱发。然而，脱发发生在补丁，并且可以变化从一个鼠标到下，引入相当大的变化，并且会阻碍的定性评估。该模型提供了一个机会，在对小鼠的有效性进行评估的不同位置，以测试光密度的测量值之间的相关性。 如果使用的是第一个问题是措施不?...

Watch this Scientific Journal Video about A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis at JoVE.com

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

新技术脱发的定量分析小鼠使用灰度分析

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

a-new-technique-for-quantitative-analysis-hair-loss-mice-using

Research

JoVE Journal

Biology

8.9K 次观看.  Children's Hospital at Montefiore. Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

视频: A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

随机使用时移视频显微镜细胞迁移的定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

定量健身分析工作流程

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

采用先进的3D荧光显微镜定量分析自噬

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

多蛋白复合物的比较分析新方法基于<sup&gt; 15</sup&gt;Ñ代谢标记和定量质谱

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

新技术脱发的定量分析小鼠使用灰度分析

摘要

探索更多视频

此视频中的章节