准备试剂<ol><li>含BSA（0.5％），和2毫米EDTA PBS缓冲区。由于气泡可以阻止磁珠分离柱，缓冲区，并储存于2-8 ° C，使用前需要脱气。 </li><li>我们用RPMI 1640培养基中含有5％的小鼠血清。培养液中不应该包含BSA或FCS，这些化合物会改变细胞刺激的特异性。 </li><li>小鼠IL - 17分泌含量 - 细胞富集和从美天旎生物技术检测试剂盒。该套件包含以下几部分组成的IL - 17的捕捉试剂，IL - 17检测抗体（生物素），抗生物素- PE，和反PE微球。 </li></ol>刺激的脾细胞<ol><li>此协议是执行中存在的消极和积极的控制，如未刺激的脾细胞和T细胞的染液，。该协议是使用无菌技术进行。 </li><li>准备一个单细胞悬液，小鼠的脾细胞中分离使用gentleMACS™Dissociator。通过细胞计数，细胞浓度应预先确定。颗粒的细胞在室温下10分钟，在200 × g。 </li><li>离心后，吸出上清液用吸管关闭沉淀。不要调迁管，以避免损失颗粒。 </li><li>现在，重悬的细胞在培养液中，然后添加到良好。添加足够的介质浓度为每毫升1000万细胞和细胞每平方厘米500万。 </li><li>为了刺激免疫反应在悬浮细胞，我们添加离子霉素（1微克/毫升）和PMA（10毫微克/毫升）样品和混合解决方案，通过向上和向下轻轻吹打。然后井相应的标记。 </li><li>现在，我们将孵育3小时，我们的细胞，在37℃，没有搅拌，开始的刺激期。继续从刺激开始到IL - 17的分析，3小时，所以相应的计划。 </li><li>要停止分泌，细胞被放置在冰和我们收集的刺激细胞轻轻吹打冷缓冲区向上和向下。细胞，然后转移从井管，洗第二次。 </li><li>为了确保所有细胞都收集，它是一个好主意，在显微镜下检查你的菜。如果细胞仍然保持连接，您可以收集冲洗用冷水缓冲菜剩下的细胞。采用预分离过滤器，可去除细胞悬液中的任何细胞团块。 </li></ol>标签捕获试剂细胞<ol><li>这是最重要的要注意，这个实验工程优化，如果低于2％的IL - 17分泌细胞都存在。 </li><li>如果预计的IL - 17分泌细胞浓度大于2％，相应地调整卷。 <img src="/files/ftp_upload/old/1037_2008_12_8_6/figure8.png" alt="图8" width="500" height="162" /></li><li>此过程中的陷阱是一个潜在的交叉污染，渔获物试剂，它可以在标签发生时的双特异抗体非分泌性T细胞和陷阱从邻近的淋巴细胞分泌IL - 17的结合，从而产生误报。 </li><li>为了规避这个问题，关键是凉爽下来之前，标签和冷缓冲区的工作细胞缓慢的IL - 17的扩散和避免这种情况的交叉污染。此外，以维持细胞的冷，他们必须保持在一个定义的浓度。 </li><li>要开始标签的过程中，我们使用一百万个细胞，在15毫升的Closable管。如果要使用更高的手机号码，只需规模相应的所有卷。一旦已获得的最佳细胞浓度，加入10毫升冷缓冲洗细胞。 </li><li>离心10分钟在冷冻离心机（2-8℃）在300 × G细胞。 </li><li>离心后，完全用吸管吸出上清液。不要倒出上清液，因为这会导致细胞的损失和不准确的卷。重复的洗涤步骤，包括加入冷的缓冲液，离心和愿望10毫升。 </li><li>现在，我们有一个所需纯度的颗粒，在80μL冷培养基重悬细胞。给它们加上标签，现在我们将添加20μL小鼠IL - 17捕获试剂。 </li><li>在冰上5分钟的孵育细胞。 </li><li>在冰上5分钟的潜伏期后，取出管和稀释在10毫升37℃的温暖中期的细胞。然后安全MACSmix管肩管和管在37 ° 45分钟下连续运动的彗星。提高温度至37 ° C将重新启动细胞因子的分泌。 </li></ol>与IL - 17的检测抗体（生物素）和抗生物素PE标记的细胞<ol><li>经过45分钟的分泌期在37 ° C，立即在冰上放置管。这将阻止细胞因子的分泌。从这里就可以保持细胞在冰上的关键。与寒冷的缓冲区工作将避免交叉污染渔获物试剂。 </li><li>填充管冷缓冲区和离心机在300xg在2-8 ° C为10分钟。完全吸出上清液。重复清洗步骤有更多的时间。 </li><li>细胞沉淀悬浮于80μL冷缓冲管置于冰上。 </li><li>为了避免非特异性结合的抗体，我们建议除了加入10μlFCR阻断剂和5分钟的冰孵化。然后，我们添加20μL小鼠IL - 17检测抗体（生物素），结合生物素和10分钟在冰上孵育。 </li><li>再次，洗在300xg加入10毫升冷缓冲区和离心机在2-8 ° 10分钟之前我们已经做了的细胞。 </li><li>现在，吸出上清液，并在80冷缓冲液重悬细胞沉淀。 </li><li>加入我们的二级抗体，抗生物素- PE 20μL。 </li><li>细胞和二次抗体溶液混合，并再次孵育10分钟就冰管。 </li><li>一旦这第二个孵化完成后，用10毫升冷缓冲区和离心机的细胞。 </li><li>现在可以沉淀悬浮于500μL冷缓冲区。 </li><li>如果要作进一步的下游分析分离细胞，它们可以被磁标记与反PE的微珠和失散的手动或自动使用Mac列和MACS分离。 </li><li>现在，我们已经完成了脾的标签，并准备好进行分析。 </li></ol>流式细胞仪分析<ol><li>对于我们的分析，我们会比较刺激和未刺激的脾细胞，用流式细胞仪。 </li><li>之前，流式细胞仪分析，细胞染色为0.5微克/毫升与碘化丙啶门出死细胞。 MACSQuant™分析器装载每个样品20细胞，现在我们要看看样品中的相对抗体反应的散点图。稀有细胞分析的两个重要的考虑因素 - 如IL - 17阳性细胞 - 流式细胞仪检测： <ul><li>淋巴细胞上设置一个门，前向散射与侧散点图</li><li>出死细胞（碘化丙啶染色）和B细胞（这可能会导致非特异性背景染色）浇注，进一步提高检测的灵敏度</li></ul></li><li>我们用CD4 - APC抗体检测T细胞和CD45R/B220-PerCP抗体检测的B细胞。我们看到了样品的前部和侧散射性质和应用对淋巴细胞人口的门。 </li><li>第二个门被应用到染色的死细胞和彩色B细胞（Y轴）。这些细胞是T细胞的分析排除。 </li><li>在这个例子中，基本上是我们的阴性对照，我们可以看到，有很少IL - 17分泌的CD4阳性T细胞在未刺激样本 - 约0.003％（图4）。在刺激T细胞的人口来看，我们可以看到大量的CD4阳性T细胞分泌IL - 17 - 约0.367％（图5A）前分离磁选后MACS科技（图5B）和60.76％。 </li></ol>

所有的协议和数据表www.miltenyibiotec.com。 警告 试剂含有叠氮化钠。在酸性条件下的叠氮化钠产量hydrazoic酸，这是毒性极强。叠氮化合物，应与运行前丢弃的水稀释。这些预防措施建议，以避免在管道爆炸国情的发展可能的存款。

Th17细胞在适应性免疫和炎症反应中发挥不可或缺的作用，并在推动自身免疫性炎症的重要组成部分。 此视频文件，如何使用美天旎的鼠标IL - 17的分泌含量 - 细胞富集和检测试剂盒（PE）。执行此过程时，重要的是要在您的起始细胞制备的IL - 17分泌细胞的频率估计。在适当的细胞浓度的标签和细胞因子的分泌期防止其他细胞的交叉污染，在您的暂停，并确保可靠的结果。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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		</thead>
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<tr>
<td>Mouse IL-17 Secretion Assay Cell Enrichment and Detection Kit (PE) </td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-094-213</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_98_1005_Mouse_IL_17_Secretion_Assay_Cell_Enrichment_and_Detection_Kit.aspx">http://www.miltenyibiotec.com/en/PG_98_1005_Mouse_IL_17_Secretion_Assay_Cell_Enrichment_and_Detection_Kit.aspx</a>
</td>
</tr>
<tr>
<td>RPMI 1640</td>
<td>Medium</td>
<td>Miltenyi Biotec</td>
<td>130-091-440</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_243_293_RPMI_1640.aspx">http://www.miltenyibiotec.com/en/PG_243_293_RPMI_1640.aspx</a></td>
</tr>
<tr>
<td>CD4-APC</td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-091-611</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_599_327_CD4.aspx">http://www.miltenyibiotec.com/en/PG_599_327_CD4.aspx</a></td>
</tr>
<tr>
<td>CD8a-FITC</td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-091-605</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_599_328_CD8a.aspx">http://www.miltenyibiotec.com/en/PG_599_328_CD8a.aspx</a></td>
</tr>
<tr>
<td>Propidium Iodide Solution</td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-093-233</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx">http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx</a></td>
</tr>
<tr>
<td>FcR Blocking reagent, mouse </td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-092-575</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_601_435_FcR_Blocking_Reagent_mouse_.aspx">http://www.miltenyibiotec.com/en/PG_601_435_FcR_Blocking_Reagent_mouse_.aspx</a></td>
</tr>
<tr>
<td>Dead Cell Removal Kit </td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-090-101</td>
<td>&nbsp;</td>
<tr>
<td>MACSmix Tube Rotator</td>
<td>Instrument</td>
<td>Miltenyi Biotec</td>
<td>130-090-753</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx">http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx</a></td>
</tr>
<tr>
<td>gentleMACS&#x2122; Dissociator</td>
<td>Instrument</td>
<td>Miltenyi Biotec</td>
<td>130-093-235</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx">http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx</a></td>
</tr>
<tr>
<td>autoMACS Pro Starting Kit</td>
<td>Instrument</td>
<td>Miltenyi Biotec </td>
<td>130-092-545</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_651_679_autoMACS_Pro_Starting_Kit.aspx">http://www.miltenyibiotec.com/en/PG_651_679_autoMACS_Pro_Starting_Kit.aspx</a></td>
</tr>
<tr>
<td>MACS Columns</td>
<td>Tool</td>
<td>Miltenyi Biotec</td>
<td>&nbsp;</td>
<td><a href="http://www.miltenyibiotec.com/en/NN_115_Columns_at_a_glance.aspx">http://www.miltenyibiotec.com/en/NN_115_Columns_at_a_glance.aspx</a></td>
</tr>
<tr>
<td>MACS Separator</td>
<td>Tool</td>
<td>Miltenyi Biotec</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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detection and isolation of viable mouse il-17-secreting t cells

互助委员会的细胞因子的分泌检测技术，允许在可行的细胞因子分泌细胞的单细胞水平和敏感隔离分泌的细胞因子的检测。为了标签IL - 17分泌细胞，小鼠脾单细胞悬液，准备和刺激，在37 ° C至PMA /离子霉素诱导细胞因子的分泌。要停止分泌细胞，然后放置在冰暴露的IL - 17的捕获试剂，结合细胞表面的白细胞和IL - 17的CD45的分泌和细胞表面附近捕获，因为它是一个双特异抗体。分泌，然后重新开始提高温度至37 ° C和IL - 17是由Catch试剂被困。分泌，然后又停了下来，放置在冰细胞。要检测出被困IL - 17，第二个IL - 17特异性抗体结合，以生物素和抗生物素- PE抗体细胞培养。细胞现在可以直接通过流式细胞仪分析或准备与反PE标记的微球随后标签隔离和浓缩。

Watch this Scientific Journal Video about Detection and Isolation of Viable Mouse IL-17-Secreting T Cells at JoVE.com

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

此过程描述了小鼠Th17细胞的白细胞，积极分泌刺激后，IL - 17的检测和隔离。

可行的小鼠IL - 17分泌性T细胞的检测和隔离

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable ...

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detection-and-isolation-of-viable-mouse-il-17-secreting-t-cells

Research

JoVE Journal

Biology

11.1K 次观看.  Miltenyi Biotec,GmbH.  ChineseVersion JapaneseVersion 

视频: Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

分离的CD4 +从小鼠淋巴节点使用美天旎MACS纯化的T细胞

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

科研

生物学

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

瓣膜内皮细胞的分离

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

鼠标涎腺干细胞的分离

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

小鼠气管基底细胞和粘膜下腺体的导管细胞分离

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

从食虫植物组织分离存活的多细胞腺的，猪笼草</i

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

鼠标冠状动脉内皮细胞的分离

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.