感谢陆军医科大学第二附属医院神经外科的现场支持。资助:本研究由中国自然科学基金（82104696）资助。

本研究的实验符合动物伦理学的"3R"原则，并已获得陆军医科大学实验动物福利和民族委员会（AMUWEC20234543）的批准。
1.啮齿动物身体固定装置的制备
<ol><li>基于中国实用新型专利9设计一种小动物支架。</li>
	<li>通过3D打印技术生产设备。</li>
	<li>准备一根 2 m 长的绳子，分成 6 根绳子。将这 6 根绳索穿过固定器上的孔。利用弹簧加载扣固定绳索末端，便于调整环尺寸。 注意:固定器可以由任何配备基于设计图纸的 3D 打印技术的公司制造。该器件的设计图如 图1所示。本研究中使用的固定器是由 3D 打印机通过一种称为熔融沉积建模 （FDM） 的打印方法生产的，也称为熔融长丝制造 （FFF）10。这种增材制造方法通过选择性地将熔化形式的材料沉积在预定路径中，逐层打印设计的零件。该工艺使用长丝形式的热塑性聚合物来创造最终的物理结构。本研究中使用的固定装置可以同时固定三只动物，需要六根绳索，每只动物需要一对绳索:一根用于颈部，一根用于躯干。</li>
</ol>2.啮齿动物的制备
<ol><li>购买3只Sprague-Dawley大鼠（10-40g，7-45日龄）和3只C57 BL / C小鼠（5-40g）。</li>
	<li>在昼夜循环12小时，环境温度为21-23°C，湿度为45%-60%的房间内饲养动物。定期对饲养环境进行消毒通风，让动物自由进食、饮水。</li>
</ol>3. 将啮齿动物固定在固定器上（俯卧位）
<ol><li>将啮齿动物的头部放在固定器前面的U形孔中，使啮齿动物处于俯卧位。将啮齿动物的身体穿过两个绳环。</li>
	<li>将绳索系在啮齿动物的脖子和腹部，确保它不会逃脱，但不要太紧，以免引起呼吸窘迫和死亡。<ol><li>关键步骤是将颈部固定到位。在啮齿动物的脖子和绳子之间留出 2-5 毫米的间隙。确保绳子不会太紧而无法勒死动物或太松而无法逃脱。</li>
	</ol></li>
	<li>在设备中以相同的方式修复其他啮齿动物。</li>
	<li>重复上述固定步骤（3.1-3.2）3天，以尽量减少固定过程引起的潜在应激反应。连续 3 天重复固定程序，每次持续 5 分钟。 注意:为确保啮齿动物在固定过程中不会过度紧张，我们连续 3 天重复该过程。每次治疗每天持续 5 分钟，为小鼠或大鼠提供充足的时间来适应该程序并最大限度地减少任何潜在的不适。</li>
	<li>从第 4 天开始 Strat EA。</li>
	<li>为防止疾病传播，请将无菌棉签或棉球浸入 75% 的酒精中，并在每次使用前后彻底擦拭支架以清洁和消毒。处理啮齿动物时，请使用手套和其他防护设备，以避免潜在的健康风险。</li>
</ol>4. 在小鼠或年轻大鼠中执行 EA （MS6）
<ol><li>选择 0.25 mm x 13 mm 一次性无菌针灸针。</li>
	<li>按解剖位置找到穴位，并确认穴位处的插入深度。 注意: MS6 位于头部的外侧。头部的这个区域穿过神聪（Ex-hn1）和玄里（GB6）之间的连接，也称为前顶叶（Du21）。MS6对应于头皮上大脑皮层中央前回的表达。Ex-hn1 位于头部前后中线中点后 0.5 cm 处。GB6位于耳朵前部的中点和眼睛的末端角。滑车上上神经、眶上神经、枕神经和耳颞神经分布在其中。MS6主要用于治疗中风和脑病，如运动障碍。</li>
	<li>在3只啮齿动物中，从前申冲（Exhn1）到玄礼（GB6）沿皮下，分别在3/5深度处将双侧无菌针头拧入MS6。</li>
	<li>将连接电缆的一侧连接到 EA 仪器。</li>
	<li>将包含正负EA夹的连接电缆的另一侧连接到左右MS6上的针灸针柄。</li>
	<li>打开 EA 仪器。<ol><li>旋转按钮（功能/选择开关）设置频率和功能模式。</li>
		<li>将 频率 设置为 10 Hz，然后选择 连续波 模式。</li>
		<li>旋转相应的按钮（调整强度）以设置强度。每个调整强度按钮控制其下方连接的电线的啮齿动物的电流强度。将电流强度调整到啮齿动物可接受的范围，通常为 1-2 mA，持续 5 分钟。 注意: 在手术过程中监测啮齿动物的呼吸和行为非常重要，以确保它们不会感到任何痛苦或不适。如果大鼠似乎在挣扎或疼痛，则应立即停止该程序。</li>
	</ol></li>
</ol>5. 将啮齿动物固定在固定器上（仰卧位）
<ol><li>将啮齿动物的头部放在固定器前面的U形孔中，使啮齿动物处于俯卧位。将啮齿动物的身体穿过两个绳环。</li>
	<li>将绳子系在啮齿动物的脖子和腹部，确保它不会逃脱，但不要太紧以至于导致呼吸窘迫和死亡。 注意:固定过程中要注意的要点与步骤3.2相同。</li>
	<li>以同样的方式修复其他啮齿动物。</li>
	<li>与步骤3.4一样，重复上述固定步骤3天，以减少啮齿动物的应激反应。</li>
	<li>从第 4 天开始 Strat EA。 注意: 按照步骤 3.6 中所述进行消毒。</li>
</ol>6. 执行 EA （ST25）
<ol><li>选择 0.25 mm x 13 mm 一次性无菌针灸针。</li>
	<li>按解剖位置找到穴位，并确认穴位处的插入深度。 注意: ST25 位于脐部水平。在啮齿动物中定位这个点需要首先测量啮齿动物爪子的长度。ST25 位于肚脐旁边爪子长度的 2/3。例如，如果测量的啮齿动物爪子为 0.6 cm，则 ST25 位于脐部左右 0.4 cm。</li>
	<li>在3只啮齿动物上，分别以4-8mm的深度缓慢扭曲并穿透无菌针头进入ST25双侧。</li>
	<li>将连接电缆的一侧连接到 EA 仪器。</li>
	<li>将包含正负EA夹的连接电缆的另一侧连接到左右ST25的针灸针柄。</li>
	<li>打开 EA 仪器。<ol><li>旋转按钮（功能/选择开关）设置频率和功能模式。</li>
		<li>将 频率 设置为 10 Hz，然后选择 连续波模式。</li>
		<li>旋转相应的按钮（调整强度）以设置强度。每个调整强度按钮控制其下方连接的电线的老鼠的电流强度。将电流强度调整到啮齿动物可接受的范围，通常为 1-2 mA，持续 5 分钟。 注:观察结果与步骤4.6相同。</li>
	</ol></li>
</ol>

使用 3D 打印支架对小动物进行体电针

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提交人声明不存在相互竞争的利益冲突。

电针 （EA） 是一种针灸形式，涉及在针灸针上使用电刺激11。该技术涉及使用特定强度和频率的微脉冲电流来刺激穴位并达到增强的治疗效果3.然而，EA的愈合机制是有限的，需要大量的基础研究来证明1,2,3。啮齿动物模型通常用于基本的 EA 研究 8,12,13<...

<a href="https://app.jove.com/t/6632">This corrects</a> the article <a href="https://dx.doi.org/10.3791/65648">10.3791/65648</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/65648">Bulk Electroacupuncture Operation for Mice or Young Rats</a>. Figure 3 was updated.

Erratum: Bulk Electroacupuncture Operation for Mice or Young Rats

电针疗法是一种独特的方法，将针灸针插入头皮并连接到电机以刺激特定穴位 1。与手动刺激不同，EA 通过稳定特定频率和波形来更好地控制刺激，以实现最佳治疗效果2。据调查，中国81.2%的医疗机构使用EA或手针灸治疗脑瘫、神经痛、面瘫等疾病。尽管很受欢迎，但EA的具体疗效机制仍然未知，这阻碍了其在神经系统疾病康复治疗中的推广和应用1,2,3。需要进一步的研究来充分了解EA的有效性机制，并促进其在神经系统疾病康复治疗中的应用。
随着针灸在世界范围内的影响力不断扩大，许多研究已经研究了对啮齿动物模型（例如大鼠或小鼠模型）进行 EA 的机制 1,2,3。在EA的啮齿动物实验中经常会遇到几个问题。首先是如何在没有麻醉的情况下固定啮齿动物，因为对清醒受试者进行的针灸更能反映临床实践。此外，一些实验要求动物保持清醒以观察治疗效果2,3。另一个挑战是准确定位小鼠或大鼠的穴位，这些穴位与人类的穴位相对应。目前，许多学者正在研究实验啮齿动物穴位的精确定位4,5。在该协议中，选择了MS6和ST25，它们已通过人体解剖定位的转换在啮齿动物中明确定义。MS6 通常用于治疗某些脑部疾病，例如帕金森病6。ST25 通常用于治疗胃肠道问题，例如腹泻7.选择这两个穴位主要是为了展示如何在仰卧位和俯卧位上有效地固定啮齿动物。此外，这些穴位已被广泛研究，并为研究目的提供了重要的见解 6,7。
以前固定一只大鼠进行实验的方法不仅耗时，而且很难由一个人处理 8.此外，由于动物的不合作，在实践中成功率相对较低。因此，迫切需要创建一个易于建立的具有稳定特性的动物模型，以提高实验效率。在这篇文章中，介绍了一种用于小动物的3D打印支架，可以很容易地固定多只啮齿动物，导致运动受限。本文的目的是对一批年轻大鼠或小鼠进行EA治疗，重点研究小鼠批量限制、识别和刺激MS6和ST25穴位的策略。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D printing batch mice fixator</td>
			<td>MESH INVENT</td>
			<td>Custom made</td>
			<td>The fixator produced by 3D printer. The printing method is called fused deposition modeling (FDM), also known as fused filament fabrication (FFF).&#160;&#160;</td>
		</tr>
		<tr>
			<td>Electroacupuncture instrument</td>
			<td>Hwato, Suzhou Medical Appliance Factory</td>
			<td>SDZ-III</td>
			<td>www.Hwato-med.com</td>
		</tr>
		<tr>
			<td>Disposable sterile acupuncture needle</td>
			<td>Suzhou Medical Appliance Factory</td>
			<td>N/A</td>
			<td>0.25 mm x 13 mm</td>
		</tr>
	</tbody>
</table>

bulk electroacupuncture operation for mice or young rats

电针（EA）被广泛用于治疗各种健康状况。然而，EA治疗的潜在机制尚不清楚，阻碍了其推广。机理研究需要小鼠或大鼠模型来解决这个问题。然而，这些动物不服从实验过程，这很耗时。为了解决这些问题，我们设计了3D打印的小动物身体散装固定器，以提高EA动物实验的效率。该视频详细介绍了如何使用固定器对小鼠或年轻大鼠进行批量 EA。对于穴位的选择，选取顶点颞部（MS6头部）和天枢点（ST25腹部）的前斜线，验证俯卧位和仰卧位固定装置的效果。使用3D打印的小动物支架可以同时固定和治疗多个啮齿动物，从而减少实验所需的时间和资源。这种技术可以通过3D打印不同尺寸的动物模型应用于其他动物模型，并有可能用于各种固定条件。该设备有利于促进EA的实验科学研究。

我们设计的动物身体固定器的平面图如图1所示。此外，还提交了该固定器的 3D 图形模型，以提供设计的全面视图（补充文件 1）。这是一种允许 3 只啮齿动物固定并同时执行 EA 的设备。大鼠和小鼠在清醒状态下被限制在固定器内，俯卧位和仰卧位都可以牢固地固定，而不会对啮齿动物造成伤害（图2和图3</strong...

Watch this Scientific Journal Video about Bulk Electroacupuncture Operation for Mice or Young Rats at JoVE.com

作者聚焦:使用 3D 打印支架同时进行多次电针手术

在这里，我们提出了一种使用三维（3D）打印支架在小鼠或年轻大鼠中进行高效和快速电针（EA）的方案。该技术允许在多只动物身上同时操作，节省时间并提高实验效率。

小鼠或幼鼠的批量电针操作

Electroacupuncture (EA) is widely used to treat various health conditions. However, the underlying mechanism of EA treatment remains unclear, hindering ...

在本视频中，我们介绍了一种在多只清醒的小鼠或大鼠身上使用电针的有效方法。在膀胱研究中，动物在排尿，但它们不合作，针灸只是消耗时间。另一个问题是关于麻醉，这在针灸的临床上不适用。为了解决这些问题，我们展示了使用3D打印支架对多只清醒的小鼠或大鼠进行电针灸的有效方法。我们的方案展示了一种更有效、更安全的方式来同时持有多只动物，这与其他意味着固定的方法不同，这种方法不仅节省了时间，而且提高了实验成功率。此外，该设备设计为易于操作，可由一个人操作。ProTech和设备有利于促进电针的实验科学研究。

bulk-electroacupuncture-operation-for-mice-or-young-rats

Research

JoVE Journal

Biology

Bulk Electroacupuncture Operation for Mice or Young Rats

557 次观看.  Army Medical University. 在本视频中，我们介绍了一种在多只清醒的小鼠或大鼠身上使用电针的有效方法。在膀胱研究中，动物在排尿，但它们不合作，针灸只是消耗时间。另一个问题是关于麻醉，这在针灸的临床上不适用。为了解决这些问题，我们展示了使用3D打印支架对多只清醒的小鼠或大鼠进行电针灸的有效方法。我们的方案展示了一种更有效、更安全的方式来同时持有多只动物，这与?...

视频: 作者聚焦:使用 3D 打印支架同时进行多次电针手术

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

微探针：操作和使用的局部表面处理

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

科研

生物学

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

为贴壁细胞培养的氧气插入的制备及操作

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

手动约束和常见的化合物在小鼠和大鼠的给药途径

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

剖检诊断，并选择在大鼠和小鼠的组织和样品收集

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Ovariectomy and 17&beta;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17&beta;-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17&beta;-estradiol level/administration method profoundly influences the experimental results1-3. We have in a series of studies characterized the different modes of 17&beta;-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17&beta;-estradiol4-6. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration7, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17&beta;-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.

Ovariectomy and 17&#946;-estradiol Replacement in Rats and Mice: A Visual Demonstration

Ovariectomy and subsequent replacement of 17&beta;-estradiol by means of silastic capsules and peroral Nutella are demonstrated in rats and mice.

在大鼠和小鼠的卵巢切除和17β-雌二醇更换:一个可视化示范

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

数字核酸定量实验分析的简单堆积读数

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

The goal of this video is to demonstrate how to perform&#160;automated DNA extraction from formalin-fixed paraffin-embedded (FFPE) reference standard cell lines and digital droplet PCR&#160;(ddPCR) analysis to detect rare mutations in a clinical setting. Detecting mutations in FFPE samples demonstrates the clinical utility of ddPCR in FFPE samples.

采用数字液滴PCR技术检测BRAF V600E突变的福尔马林固定石蜡包埋的参考标准细胞系

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

乳酸脱氢酶螯合测定--一种简便、可靠的测定哺乳动物细胞中自噬性螯合活性的方法

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...