热痛测试使用Thermostimulator TSA的II 1。简介<ol><li>这个程序使用该协议II，市售热感官分析仪，可提供与热电极（梅多克先进的医疗系统，达勒姆，数控）的热刺激皮肤。中央单位产生和传输信号的热电极。这个信号通常在一台笔记本电脑连接到中央处理单元上存储的专有软件控制。此外，隶属中央单位，是一个手持式的控制，允许一个研究课题，停止加热的热电极。 </li><li>交付七个连续和相同的热刺激，刺激间的时间间隔30秒。热电极基底温度设定在一个舒适的35℃其中热电极加热速率设定为1℃/ s，而在它冷却下来的速度是在10 ° C / s的设置热电极，最高温度设定在52 ° C。 </li></ol> 2。准备和训练科目<ol><li>为了准备科目为热痛测试座在躺椅舒适，提供足够的头部，胳膊和腿支持。椅子也应该允许方便地访问研究的部位。 </li><li>测试设施应在舒适的室温，并提供一个相当的和中立的环境中，心无旁鹜。 </li><li>在实验开始之前，所有的参与者应在热痛测试的所有方面训练有素。培训才能进行实验前的任何时间。 </li><li>应仅限于那些收集数据所需的的研究课题和调查人员之间的相互作用。戏说和小谈气馁，因为他们可能会危及数据的一致性。研究对象被告知的相互作用会故意有点单调和实验过程中的中性。 </li><li>保持明确的指示，简单，一致的。强调，疼痛是主观的，有没有正确或错误的答案。这是最重要的是一致的。定义为热痛阈值点之间的“大约是痛苦的”和“刚刚成为痛苦的。” </li><li>这是关键的一个主题确切知道什么期望在测试会话。任何“惊喜”，可能会导致数据不太一致，因为混杂因素，如焦虑，可能会产生。例如，如果7个热疼痛刺激是用来确定热痛阈值，这个问题应该知道这一点。 </li><li>在任何时候，这个问题可以阻止任何热推手持式控制按钮的热电极。在控制会降低主体的焦虑，使数据更可靠。 </li><li>培训会议提供了一个机会来判断一个研究课题的表现。它可能是最好的列车在两到四个测试周期科目，使他们个人的疼痛阈值不超过0.8 ° C，并且最好由不超过0.6 °之间的最后两个测试周期彗星。变化未能执行一贯的主题通常是从研究中排除。 </li></ol> 3。确定疼痛阈值<ol><li>要开始疼痛阈值的测试周期，标志着主体的皮肤上的热电极安置。在这个实验中，将相同发炎的患处皮肤微透析技术用于测试的疼痛，以了解疼痛测试发布的生化物​​质。 </li><li>然后，启动每个周期的热电极放置到明显的皮肤面积。匹配尽可能密切标记的热电极的位置。允许皮肤和热电极之间的温度平衡至35℃ - 温度，安全不会造成任何痛苦。这将需要大约5秒钟，将显示在计算机屏幕上阅读的35℃。 </li><li>在开始之前，让手持控制主体。 </li><li>使用常数和温和的压力，以保持与主体的皮肤充分接触，在测试过程中的热电极的整个表面。 </li><li>现在启动预编程的测试算法。热电极将开始加热1 ° C / S只要受到的痛苦经验，他们会按手持设备。 </li><li>热电极温度引起疼痛的手持设备上的按钮按下后，会自动进行注册，将开始下降率在10 ° C /秒下降到基准温度35 ° C. </li><li>温度探测器将自动保持30秒，在它开始之前再次上升1在35 ° C ° C / S，直到问题再次推手持设备的按钮。就在热电极的温度大约是再增加，提醒，这是即将发生的主体。这将保持集中的主题。 </li><li>这个序列将反复进行，直到已注册的7个温度记录。平均计算这些坦佩rature读数，这是热痛阈值。 </li></ol>与间断压力传感器的机械痛测试 1。简介<ol><li>我们使用定制校准穿插压力传感器，因为它们提供了比传统的使用和市售的冯弗雷头发更一致的结果。探头均采用相同的圆柱形不锈钢钢丝（直径为240微米），安装在不同重量的铜杆（1，2，4，8，16，32，49，64和81克），在更广泛的移动的自由手铝管举行。 </li><li>相同的一般实验条件下，热痛测试概述，适用于机械痛测试。该科目应做好充分的准备和训练。尤其重要的是保证学科探头生硬，不会刺穿皮肤。这将有助于受试者放松，并提供一致的数据。 </li></ol> 2。确定的机械痛阈<ol><li>要确定机械痛阈值，首先用钢丝尖端的最轻的探针垂直于皮肤在实验发炎的病变中心。 </li><li>手持铝管，然后降低对皮肤不接触。这样一来，整个重量的铝管内的铜杆取决于钢丝头。 </li><li>研究的课题，然后指示探头是否会导致疼痛说“是”或“无”。 </li><li>如果题目的答案“没有”，然后在相同的探针或明年较重的探头将被用于未来。该算法被称为递增刺激序列。随机确定下一个探测是否是较重的或相同的重量。这将保持一个受蒙蔽的算法。 </li><li>适用，直至“是的。”这个系列中的最后两个探头的平均体重为第一偏转点记录的题目答案在递增序列的探针。 </li><li>后的题目答案“是的，”开始使用降刺激序列，并使用一个打火机的探头或与以前相同的探针。随机确定下一个探测是否从轻或者同等重量。这将保持一个受蒙蔽的算法。 </li><li>套用一个递减的序列中进行探测，直到问题回答“没有”。平均体重在这个序列中的最后两个探头构成第二偏转点。 </li><li>当题目的答案“没有”，切换回递增刺激序列。之间的递增和递减的序列，直到七个挠度点记录备用。这七个百分点的平均机械痛阈值。 </li></ol>

<ol>
	<li>Angst, M. S., Clark, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comment+on+Koltzenburg+et+al.:+Differential+sensitivity+of+three+experimental+pain+models+in+detecting+the+analgesic+effects+of+transdermal+fentanyl+and+buprenorphine.">Comment on Koltzenburg et al.: Differential sensitivity of three experimental pain models in detecting the analgesic effects of transdermal fentanyl and buprenorphine.</a> Pain. 126, 165-174 (2006).</li><li>Angst, M. S., Clark, J. D., Carvalho, B., Tingle, M., Schmelz, M., Yeomans, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+profile+in+human+skin+in+response+to+experimental+inflammation,+noxious+stimulation,+and+administration+of+a+COX-inhibitor:+A+microdialysis+study.">Cytokine profile in human skin in response to experimental inflammation, noxious stimulation, and administration of a COX-inhibitor: A microdialysis study.</a> Pain. , (2008).</li><li>Angst, M. S., Koppert, W., Pahl, I., Clark, J. D., Schmelz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+infusion+of+the+mu-opioid+agonist+remifentanil+in+humans+causes+hyperalgesia+during+withdrawal.">Short-term infusion of the mu-opioid agonist remifentanil in humans causes hyperalgesia during withdrawal.</a> Pain. 106, 49-57 (2003).</li><li>Lotsch, J., Angst, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mu-opioid+agonist+remifentanil+attenuates+hyperalgesia+evoked+by+blunt+and+punctuated+stimuli+with+different+potency:+a+pharmacological+evaluation+of+the+freeze+lesion+in+humans.">The mu-opioid agonist remifentanil attenuates hyperalgesia evoked by blunt and punctuated stimuli with different potency: a pharmacological evaluation of the freeze lesion in humans.</a> Pain. 102, 151-161 (2003).</li></ol>

本视频演示了为评估人类志愿者发炎的皮​​肤的热量和机械痛阈值算法。对疼痛的感知是主观的。收集充足的疼痛数据，需要一个合适的测试设备（如宁静和安慰），与在建的数据质量控制措施（重测信度），严格遵守，以一个标准化的研究协议，并避免混杂因素的研究参与者的全面培训（如紧张，焦虑）。可靠的主观数据对疼痛的敏感性，与镇痛药的管理和局部释放伤害性介质，如机械数据的收集，如干预配对到疼痛的机制提供了有价值的见解和手段，以防止或减轻。

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>TSA-II Neuro Sensory Analyzer</td>
<td>Tool</td>
<td>Medoc Advanced Medical Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Puntuated Pressure Probes</td>
<td>Tool</td>
<td>Custom-made</td>
<td>&nbsp;</td>
<td>Alternatively, commercially available von Frey Hairs can be used.</td>
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determining heat and mechanical pain threshold in inflamed skin of human subjects

在以前的文章中我们已经证明皮肤微透析技术在人体的伤害性和炎症的组织特异性生物化学集合的可视化实验杂志。在这篇文章中，我们将展示痛苦的测试往往是在串联使用微透析程序的范例。结合微透析的痛苦测试，提供之间的行为和生化数据，允许负责确定关键的生化物质的产生和传播疼痛的关键环节。 两种型号唤起人类研究参与者发炎的皮​​肤疼痛。第一种模式唤起与热刺激援助疼痛。此处所述的热诱发疼痛主要是介导的小，非髓周边伤害性神经纤维（C -纤维）。第二种模式唤起通过穿插的压力刺激疼痛。间断的压力引起的疼痛主要是介导的小，外围伤害性髓神经纤维（A -三角洲纤维）。这两款机型是机制不同的和独立研究的两个主要的外围神经纤维参与疼痛信号的人群伤害性处理。 热痛诱发的TSA二，市售的感觉热分析仪（梅多克先进的医疗系统，达勒姆，数控）的援助。刺激的配置和交付是与特定的软件援助进行处理。 Thermodes不同大小和形状，但在原则上由一个金属板，可以在各种速率和不同的时间段进行加热或冷却。算法评估热诱发疼痛是多方面的。在此处所示的实验中，研究参与者被要求说明在什么时候，他们开始经历痛苦而热电极与皮肤接触是在预定率在温度不引起疼痛开始加热。在其中一个主题开始经历痛苦的​​热电极温度构成热痛阈值。 机械痛诱发穿插探头。这种探头是市售几个厂家（冯弗雷毛）。然而，冯弗雷毛发的准确性受到批评，许多研究者使用自定义标点压力探头。八个自定义作出穿插不同的权重的探测器，在这里显示的实验是在连续的顺序应用，过程称为上下的算法，以确定知觉偏转点，即从没有疼痛的感觉感觉疼痛或反之亦然变化。平均体重造成了知觉偏转构成的机械痛阈值。

Watch this Scientific Journal Video about Determining heat and mechanical pain threshold in inflamed skin of human subjects at JoVE.com

Determining heat and mechanical pain threshold in inflamed skin of human subjects

评估人类研究对象的实验发炎的皮肤的热和机械痛阈值算法。这两个痛苦的测试范例独立研究两大周围神经纤维传输疼痛人口的伤害性处理，即非髓C纤维和小髓一个增量纤维。

确定人类受试者皮肤发炎的热和机械痛阈

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific ...

determining-heat-mechanical-pain-threshold-inflamed-skin-human

Research

JoVE Journal

Biology

20.7K 次观看.  Stanford University School of Medicine. 评估人类研究对象的实验发炎的皮肤的热和机械痛阈值算法。这两个痛苦的测试范例独立研究两大周围神经纤维传输疼痛人口的伤害性处理，即非髓C纤维和小髓一个增量纤维。

视频: Determining heat and mechanical pain threshold in inflamed skin of human subjects

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

使用微机电系统（MEMS）开发诊断工具

科研

生物学

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

小鼠皮肤移植

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

大鼠心脏中植入的工程组织

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis. In some women the severe episodes of cyclic pain change and the resultant pain becomes continuous and this condition becomes known as Chronic Pelvic Pain. This state can be present even after the appropriate medical or surgical therapy has been instituted. It can be associated with pain and tenderness in the muscles of the abdomen wall and intra-pelvic muscles leading to severe dyspareunia. Additional symptoms of irritable bowel and interstitial cystitis are common. A common sign of the development of this state is the emergence of cutaneous allodynia which emerges from the so-called viscero-somatic reflex. A simple bedside test for the presence of cutaneous allodynia is presented that does not require excessive time or special equipment. This test builds on previous work associated with changes in sensation related to gall bladder function and the viscera-somatic reflex(1;2).
The test is undertaken with the subject s permission after an explanation of how the test will be performed. Allodynia refers to a condition in which a stimulus that is not normally painful is interpreted by the subject as painful. In this instance the light touch associated with a cotton-tipped applicator would not be expected to be painful. A positive test is however noted by the woman as suddenly painful or suddenly sharp. The patterns of this sensation are usually in a discrete pattern of a dermatome of the nerves that innervate the pelvis.
The underlying pathology is now interpreted as evidence of neuroplasticity as a consequence of severe and repeating pain with changes in the functions of the dorsal horns of the spinal cord that results in altered function of visceral tissues and resultant somatic symptoms(3). 
The importance of recognizing the condition lies in an awareness that this process may present coincidentally with the initiating condition or after it has been treated. It also permits the clinician to evaluate the situation from the perspective that alternative explanations for the pain may be present that may not require additional surgery.

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

A demonstration of the bedside test for cutaneous allodynia and its clinical implications.

示范皮肤触感在慢性盆腔疼痛协会

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis.  In some women the severe episodes of cyclic pain change and the ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 &#181;m&#160;x 200 &#181;m&#160;x 200 &#181;m using LED illumination, to 5 mm&#160;x 5 mm&#160;x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.

Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object.

的弱散射主题内嵌数字全息显微镜（DIHM）

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

小鼠胚胎皮肤和Live成像黑素细胞迁移的体外培养

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

间隙连接通讯：天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

A Buoyancy-based Method of Determining Fat Levels in Drosophila

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat in Drosophila, but most are expensive and/or laborious and have clear limitations. Here, we present a method to quickly and cheaply determine organismal fat levels in L3 Drosophila larvae. The technique relies on the differences in density between fat and lean tissues and allows for rapid detection of fat and lean phenotypes. We have verified the accuracy of this method by comparison to body fat percentage as determined by neutral lipid extraction and gas chromatography coupled with mass spectrometry (GCMS). We furthermore outline detailed protocols for the collection and synchronization of larvae as well as relevant experimental recipes. The technique presented below overcomes the major shortcomings in the most widely used lipid quantitation methods and provides a powerful way to quickly and sensitively screen L3 larvae for fat regulation phenotypes while maintaining the integrity of the larvae. This assay has wide applications for the study of metabolism and fat regulation using Drosophila.

A Buoyancy-based Method of Determining Fat Levels in Drosophila

Here we present a method to measure organismal fat levels in the third instar (L3) larval stage of Drosophila melanogaster. This method exploits the comparatively low density of fat tissue to differentiate between larvae with altered fat stores. Buoyancy-based analysis is a valuable tool for rapid, reproducible, and economical screening.

确定脂肪水平在基于浮力法<em&gt;果蝇</em

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat ...