TMS支持的哈伦信托奖学金（GWU）在研究过程中。经费实地收集和育种研究片形吸虫和 E。 delphinii提供了由国家科学基金会资助DEB 0642438 JTL。

1。材料制备<ol><li>准备用硼硅玻璃毛细管和微管拉拔器的针头（1-2微米的头和锥度3-4毫米）。仪器设置：斜坡：561速度：20;热：560;延迟：1;拉：100压力：500。 </li><li>准备在收集解决方案：Grace的中等（GM）辅以10％的胎牛血清（FBS）和抗凝血剂缓冲区的20％（98毫米的NaOH，186 mM的氯化钠，1.7 mM的EDTA和41 mM的柠檬酸酸，pH值4.5的60％ ）。在无菌条件下，将上述溶液制备新鲜，并在任何时候都在冰上保存。 </li><li>准备的孵化缓冲溶液：90％的GM补充有10％的胎牛血清（FBS）。该解决方案还准备了新鲜的冰在任何时候都保持。 </li><li>准备一个总共至少15微升×昆虫的收集和孵化溶液的两个数。 </li></ol> 2。注射采集解决方案的毛毛虫<ol><li>麻醉倒数第二个插件低温在冰上20分钟前血细胞提取焦油幼虫。 </li><li>适合一个干净毛细管进入管件，它连接到一个20毫升的玻璃注射器（ 图2）的针（16号）的钝端。让管中收集解决方案，打开该实验室的长椅上，慢慢地填充毛细管的缓冲区。毛细管通常填充有约10-15微升收集溶液，毛虫（约1μl溶液每10毫克毛虫总身体质量的大小取决于=身体总质量的0.01％）。休息了一球粘土，是摆在解剖显微镜下针。 </li><li>毛毛虫放在培养皿中。应被定位在其一侧，使气门是清晰可见的（ 图3）的昆虫。 </li><li>在解剖显微镜下观察的同时，保持到位的毛虫与对钳子，放置的针接近一个气孔，其中在第一节角质层一般较柔和。轻轻按下毛细管针对人体的昆虫。一旦针穿透角质层，注入溶液的总体积到毛毛虫（ 图3）。重要的是要避免气泡注入到毛虫。毛毛虫的身体会在注射后膨胀（ 例如 ，增加量的20％），但不应破裂或渗出。 </li><li>返回毛虫冰，定位昆虫从伤口（ 图3）中漏出的背面，以防止缓冲器。 </li><li>重复步骤2.1-2.4血淋巴中提取用于为每个毛毛虫。在这个阶段，让所有的昆虫，冰上冷却30分钟，然后再继续。 </li></ol> 3。血细胞的提取<ol><li>在解剖显微镜下，将毛毛虫的培养皿中。 </li><li>浅（长2-3毫米）切口在后区的毛毛虫使用无菌scalpe的<html>升（ 图3）。请注意，不要破坏肠道或其他内部器官的目的是到穿刺的角质层仔细，收集缓冲/血淋巴的组合包含个人昆虫的血细胞。 </li><li>挤压的毛毛虫，灵活的塑料镊子轻轻地使用，同时收集10微升移液器吸头的血淋巴组合（约10-20微升），再小心，不要伤到内部组织（ 图3）。血淋巴收集到的液滴从毛毛虫的身体挤压。扔掉的毛毛虫的遗体。 </li><li>为单独的标记硅化的0.5 ml微量离心管收集血淋巴每一个人毛虫，。 </li><li>孵化溶液等体积的（约10-20微升）添加到每个血淋巴样品收集从个别毛​​虫。将冰血淋巴样品在任何时候都。 </li><li>重复步骤3.1-3.5实验中所用的每个毛毛虫。 </li></ol>文件“&gt; 4。血细胞的定量<ol><li>混合血淋巴样品和孵化解决方案之前血细胞计数。这种治疗方法可以适当的血细胞分离，并防止结块。 </li><li>使用干净的移液枪枪头转移10μL血淋巴样品的孵育液混合到一个血球计数仪。 </li><li>使用的化合物显微镜，计数和至少15的大正方形（面积为1平方：0.04毫米2，深度：0.1毫米），中央网格的血细胞计数仪（ 图3）的记录中的血细胞数量。这些值添加到获得的15个正方形中的血细胞的数量的总和。 </li><li>血细胞计数仪和盖玻片彻底清洗瓶用蒸馏水和去离子水冲洗干净并擦干Kimwipes（金佰利），每个样品之​​间。 </li><li>每个血淋巴样品重复步骤4.1-4.4。 </li><li>计算血细胞数的平均值，每毫升的血淋巴volume为每个毛毛虫，如下所示： </li></ol>血细胞计数仪网格的每个正方形的总体积为：（0.04毫米2面积）×（0.1 mm深度）= 0.004毫米3×15平方= 0.06毫米3 =总体积的所有15个平方。 （血细胞总数的计数/计数所有15个正方形）的总体积的15平方的总和×2稀释因子=血细胞数/ mm 3的 ×1,000 =血细胞/毫升的血淋巴中的数目。

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没有利益冲突的声明。

从医学上重要的昆虫，鳞翅目模式昆虫血细胞中提取先前已报道的方法9，14。血细胞提取方法适于根据昆虫种类，发育阶段的昆虫和其形态特征。例如，可以容易地进行从烟草天蛾幼虫的血淋巴中分离，通过剪断腹部15的端部的弯曲角附近。因为刺蛾幼虫（刺蛾科）缺乏​​这方面腹部的结构，我们已经开发了一种独特的协议为小型现场采集的毛毛虫血淋巴中提取。此方法?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>微管拉马</td><td>萨特仪器</td><td> P-1000 </td><td></td></tr><tr><td>高硼硅玻璃管</td><td>萨特仪器</td><td> B100-50-10 </td><td> OD：1.0，ID：0.50毫米</td></tr><tr><td> Grace的中</td><td>西格玛</td><td> G8142 </td><td></td></tr><tr><td>胎牛血清</td><td>赛默飞世尔科技</td><td> SH3007102 </td><td>热灭活</td></tr><tr><td>杜蒙＃5镊子</td><td>精细科学的工具</td><td> 11252-40 </td><td></td></tr><tr><td> Neubauer血球</td><td>豪塞尔科学</td><td> 3200 </td><td></td></tr><tr><td>塑料tubiNG </td><td>三技术</td><td> TT-3-32OD </td><td> OD：3/32“，ID：1/32 </td></tr><tr><td>玻璃医用注射器</td><td>财神越</td><td> D-97877 </td><td> 50毫升的量</td></tr><tr><td>平端针</td><td>小零件</td><td> NE-162PL-25 </td><td> 16号×1“的长度</td></tr></tbody></table>

a simple protocol for extracting hemocytes from wild caterpillars

昆虫血细胞（相当于哺乳动物白血细胞）的昆虫的整个生命周期的1几种生理过程中发挥重要的作用。在幼虫阶段的昆虫，属于鳞翅目（飞蛾和蝴蝶），双翅目（真苍蝇）的订单，血细胞形成的淋巴腺（一个专门的造血器官）或胚胎干细胞，可以通过到成年阶段。参与胚胎血细胞在细胞迁移过程中的发展和趋化调控在炎症过程中。他们还参与细胞凋亡和胚胎发育2是必不可少的。血细胞介导的细胞手臂的昆虫先天免疫反应，包括多种功能，如细 ​​胞扩散，细胞聚集，形成结节，细胞吞噬功能和外国侵略者的封装3。他们还负责编排特定的昆虫体液防御感染过程中，如公关最大制本抗菌肽和其他效应分子4，5。栉孔扇贝血细胞的形态和功能主要是研究遗传或生理昆虫模型，包括果蝇， 果蝇 6，7，蚊子埃及伊蚊和冈比亚按蚊 8日，9日和烟草天蛾，烟草天蛾 10，11。然而，目前的资料很少存在的多样性，分类，形态和功能的血细胞非模式昆虫物种，特别是那些从野生12。 在这里，我们描述了一个简单而有效的协议，提取野生毛毛虫血细胞。我们使用：，倒数第二龄Lithacodes片形吸虫 （黄肩蛞蝓蛾）（ 图1）和Euclea delphinii（多刺的橡木蛞蝓）毛毛虫（鳞翅目：刺蛾科），并表明，足量的血淋巴（昆虫血）可以分离和血细胞的数目计数从个别幼虫。此方法可用于有效地研究在这些物种以及从外地收获在其他相关的鳞翅目毛虫的血细胞类型，或它可以容易地结合旨在探讨血细胞的功能与微生物或寄生生物13感染后的免疫学测定方法。

本文描述的协议允许的最小体积的10-20微升从个别毛虫昆虫血淋巴的集合。血细胞收集使用此方法是无细胞结块，黑化缺陷，组织碎片或其他污染物。因此血细胞可以很容易地观察和显微镜下计数，和几种昆虫可以观察到，在短短的Hosur。我们观察到，在我们的样本中的血细胞的大部分包括浆细胞3（血球扩频不对称）（ 图4）。目前，我们正在表征不同的血细胞类型中发现这?...

Watch this Scientific Journal Video about A Simple Protocol for Extracting Hemocytes from Wild Caterpillars at JoVE.com

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

昆虫的血细胞进行了许多重要的功能，包括免疫和非免疫，昆虫生长发育的各个阶段的。我们目前的知识来自血细胞类型和功能对抗虫基因模型的研究。在这里，我们提出了野生毛毛虫血细胞提取，量化和可视化的方法。

一个简单的协议提取血细胞从野生毛毛虫

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

a-simple-protocol-for-extracting-hemocytes-from-wild-caterpillars

Research

JoVE Journal

Biology

16.1K 次观看.  The George Washington University. 昆虫的血细胞进行了许多重要的功能，包括免疫和非免疫，昆虫生长发育的各个阶段的。我们目前的知识来自血细胞类型和功能对抗虫基因模型的研究。在这里，我们提出了野生毛毛虫血细胞提取，量化和可视化的方法。

视频: A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and species-rich microbial population that assists in the degradation of lignocellulose predominantly into acetate, the key nutrient fueling termite metabolism (Odelson &amp; Breznak, 1983).  Within these microbial populations are bacteria, methanogenic archaea and, in some ("lower") termites, eukaryotic protozoa. Thus, termites are excellent research subjects for studying the interactions among microbial species and the numerous biochemical functions they perform to the benefit of their host.  The species composition of microbial populations in termite guts as well as key genes involved in various biochemical processes has been explored using molecular techniques (Kudo et al., 1998; Schmit-Wagner et al., 2003; Salmassi &amp; Leadbetter, 2003). These techniques depend on the extraction and purification of high-quality nucleic acids from the termite gut environment.  The extraction technique described in this video is a modified compilation of protocols developed for extraction and purification of nucleic acids from environmental samples (Mor  et al., 1994; Berthelet et al., 1996; Purdy et al., 1996; Salmassi &amp; Leadbetter, 2003; Ottesen et al. 2006) and it produces DNA from termite hindgut material suitable for use as template for polymerase chain reaction (PCR).

Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes

This video illustrates the technique for extracting DNA from the species of microbes resident in the termite hindgut. The preparation of a wet mount slide, which is useful for visualizing the gut microbial community is also illustrated, and a tour through the species-rich gut environment is given.

从白蚁肠道微生物（Zootermopsis Angusticollis）中提取的DNA和可视化肠道微生物

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and ...

科研

生物学

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

蚊子养育的议定书“（冈比亚按蚊）

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

焦磷酸测序：一个准确的基因分型的简单方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

培养大鼠颈上神经节（SCG）的交感神经元的议定书

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

一个基于PCR的基因分型方法来区分野生型和观赏品种白茅</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

一个简单的生物测定血管内皮生长因子的评价

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤：一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

A Method for Extracting Pigments from Squid Doryteuthis pealeii

A protocol for the extraction of pigments from the nanostructured granules in squid Doryteuthis pealeii chromatophores is presented.

一种从鱿鱼提取色素的方法<em&gt; Doryteuthis pealeii</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

胰腺胰岛与小鼠分离的简单高效协议

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

一种从人间充质干细胞中分离小细胞外囊泡的简单台式过滤方法

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...