从老鼠血液通过大隐静脉和心脏穿刺“ 注 ：所有的程序必须由研究所的动物护理和使用委员会批准。 1。从大隐静脉抽血<ol><li>执行此过程无需麻醉，需要两个人，一个人处理的大鼠，并执行平局。 </li><li>走出一条毛巾，一个圆锥体或鼠与灭鼠处理手套，留下一个后肢暴露。 </li><li>剃须电动微调的腿后面，直到大隐静脉可见。剃须一个足够大的面积，所以会在穿刺部位的接触，无毛。使用少量的非芳香洗手液，以保持非剃光头发远离穿刺部位。 </li><li>压缩点的腿基地，使大隐静脉凸出（类似于使用止血带，从一个人或一个大型动物血液时）。使用20G针头静脉穿刺和血舀出来，因为它使用一个microvette。抽水的腿，将有助于吸引更多的血液。当你已经收集了足够的血液，保持一个干净的压缩穿刺部位止血。 </li></ol> 2。心脏穿刺抽血<ol><li>这个过程需要麻醉和终端。必须立即动物安乐死在心脏穿刺结束。 </li><li>准备与23G1针5毫升注射器。 </li><li>深anesthesize大鼠和麻醉检查缺乏自发的运动，呼吸速度缓慢，缺乏刺激（如捏脚趾）。为了确保长时间的麻醉，放置一个针筒，含有挥发性麻醉手术过程中浸泡在老鼠的鼻子的纸巾。 </li><li>将大鼠在它的后面，面临着来自你走。 </li><li>如果你是右撇子，你的左手食指放在水平最低的肋骨，不应用任何压力。将位于心脏〜1厘米，上面这点稍微向右。 </li><li>保持在一个45度角的注射器，插入两根肋骨和手表之间的一滴血液来进针针。这是一个迹象表明，你在心里。而不移动您的注射器，拉填充注射器的柱塞。一旦注射器是全面的，仔细断开它从针头和空管。注射器就可以重新连接到针吸取更多的血液。它应该有可能借鉴从120-180克大鼠5-10毫升的血液。 </li><li>立即安乐死的老鼠。 </li></ol>

<ol>
	<li>Van Herck, H., Baumans, V., Brandt, C. J. W. M., Boere, H. A. G., Hesp, A. P. M., Van Lith, H. A., Schurink, M., Beynen, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sampling+from+the+retro-orbital+plexus,+the+saphenous+vein+and+the+tail+vein+in+rats:+comparative+effects+on+selected+behavioural+and+blood+variables.">Blood sampling from the retro-orbital plexus, the saphenous vein and the tail vein in rats: comparative effects on selected behavioural and blood variables.</a> Laboratory Animals. 35, 131-139 (2001).</li><li>Luzzi, M., Skoumbourdis, E., Baumans, V., Conte, A., Sherwin, C., Kerwin, A., Lang, T., Morton, D., Barley, J., Moreau, E., Weilenmann, R. F., Reinhardt, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collecting+blood+from+rodents:+a+discussion+by+the+laboratory+animal+refinement+and+enrichment+forum.">Collecting blood from rodents: a discussion by the laboratory animal refinement and enrichment forum.</a> Animal Technology and Welfare. 4, 99-102 (2005).</li><li>Angelow, O., Schroer, R. A., Heft, S., James, V. C., Noble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+two+methods+of+bleeding+rats:+the+venous+plexus+of+the+eye+versus+the+vena+sublingualis.">A comparison of two methods of bleeding rats: the venous plexus of the eye versus the vena sublingualis.</a> Jounal of Applied Toxicology. 4, 258-260 (2006).</li><li>Beeton, C., Wulff, H., Barbaria, J., Clot-Faybesse, O., Pennington, M., Bernard, D., Cahalan, M. D., Chandy, K. G., Beraud, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+blockade+of+T+lymphocyte+K++channels+ameliorates+experimental+autoimmune+encephalomyelitis,+a+model+for+multiple+sclerosis.">Selective blockade of T lymphocyte K+ channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis.</a> Proc. Natl. Acad. , 13942-13947 (2001).</li><li>Beeton, C., Pennington, M. W., Wulff, H., Singh, S., Nugent, D., Crossley, G., Khaytin, I., Chen, C. Y., Calabresi, P. A., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+effector+memory+T+cells+with+a+selective+peptide+inhibitor+of+Kv1.3+channels+for+therapy+of+autoimmune+diseases.">Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases.</a> Mol. Pharmacol. , 1369-1381 (2005).</li><li>Beeton, C., Wulff, H., Standifer, N. E., Azam, P., Mullen, K. M., Pennington, M. W., Kolski-Andreaco, A., Wei, E., Grino, A., Counts, D. R., Wang, P. H., LeeHealey, C. J., Andrews, B. S., Sankaranarayanan, A., Homerick, D., Roeck, W. W., Tehranzadeh, J., Stanhope, K. L., Zimin, P., Havel, P. J., Griffey, S., Knaus, H. G., Nepom, G. T., Gutman, G. A., Calabresi, P. A., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kv1.3+channels+are+a+therapeutic+target+for+T+cell+mediated+autoimmune+diseases.">Kv1.3 channels are a therapeutic target for T cell mediated autoimmune diseases.</a> Proc. Natl. Acad. Sci. USA. , 17414-17419 (2006).</li><li>Beeton, C., Barbaria, J., Devaux, J., Benoliel, A. -. M., Gola, M., Sabatier, J. -. M., Bernard, D., Crest, M., Beraud, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+blocking+of+voltage-gated+K++channels+treats+experimental+autoimmune+encephalomyelitis+and+inhibits+T-cell+activation.">Selective blocking of voltage-gated K+ channels treats experimental autoimmune encephalomyelitis and inhibits T-cell activation.</a> J. Immunol. , 936-944 (2001).</li><li>Devaux, J., Forni, C., Beeton, C., Barbaria, J., Beraud, E., Gola, M., Crest, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+basic+protein-reactive+T+cells+induce+conduction+failure+in+vivo+but+not+in+vitro.">Myelin basic protein-reactive T cells induce conduction failure in vivo but not in vitro.</a> Neuroreport. , 317-320 (2003).</li><li>Beeton, C., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+monitoring+of+adoptive+delayed+type+hypersensitivity+in+rats.">Induction and monitoring of adoptive delayed type hypersensitivity in rats.</a> Journal of Visualized Experiments. 8, (2007).</li></ol>

吸取大隐静脉的血液，是一种方便的方法得到少量的血，不需麻醉。如果需要重复抽样，检查法规，以确保您不画从一个老鼠太多血。 绘图血液由心脏穿刺是一种方便的方式来获得大量的血液，但是这是一个终端程序。被安乐死的动物必须在抽血结束。 这些技术（如活的动物所有的技术），应首先做一名兽医或兽医技术员，谁就能以评估动物是妥善处理，并不会受到不必要的痛苦和/或遇险的存在。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Needle 20G 1/2</td>
<td>Tool</td>
<td>Becton Dickinson</td>
<td>305176</td>
<td>For puncturing the saphenous vein</td>
</tr>
<tr>
<td>Microvette 300</td>
<td>Tool</td>
<td>Sarstedt</td>
<td>20.1308.100</td>
<td>To collect blood from the saphenous vein</td>
</tr>
<tr>
<td>Electric trimmer</td>
<td>Tool</td>
<td>Braintree</td>
<td>CLP-32130</td>
<td>&nbsp;</td>
<tr>
<td>Needle 23 G 1</td>
<td>Tool</td>
<td>Becton Dickinson</td>
<td>305145</td>
<td>For cardiac puncture</td>
</tr>
<tr>
<td>5 ml plastic syringe, slip tip</td>
<td>Tool</td>
<td>Fisher</td>
<td>14-826-12</td>
<td>For cardiac puncture</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
					</div>

drawing blood from rats through the saphenous vein and by cardiac puncture

绘制从啮齿类动物的血液，是必要的，无论在体外和体内研究的大量。网站绘制的血液是在啮齿类动物中数不胜数：眼窝窦，颈内静脉，上颌静脉，大隐静脉，心脏。每种技术都有其优点和缺点，和一些在一些国家没有批准任何更多的（例如，在荷兰的眼窝平）。讨论抽血不同的技术可用1-3。在这里，我们目前从老鼠的血液，两种技术各有其特定的应用程序。 从大隐静脉抽血，只要做得好，诱导动物最小的窘迫和不需要麻醉。这种技术可以反复吸引了少量的血，如需要4,5药代动力学研究，确定等离子体化学，或血球计数6。 心脏穿刺可收集大量的血液从单一的动物（最多10毫升血液，可以从一个150克的老鼠绘制）。因此，这种技术是非常有用的，作为一个终端程序，吸取血隐时将无法提供足够大的样本。我们用心脏穿刺，当我们需要从具体的增长在体外培养 4-9的T淋巴细胞的线条大鼠应变足够数量的血清。

Watch this Scientific Journal Video about Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture at JoVE.com

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

抽血是在大量研究的必要，例如，研究化合物的药代动力学轮廓。在这里，我们展示了如何绘制血液从大鼠使用两种技术：从大隐静脉或心脏穿刺抽血。

从老鼠血液通过大隐静脉和心脏穿刺“

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: ...

drawing-blood-from-rats-through-saphenous-vein-cardiac

Research

JoVE Journal

Biology

57.3K 次观看.  University of California, Irvine (UCI). 抽血是在大量研究的必要，例如，研究化合物的药代动力学轮廓。在这里，我们展示了如何绘制血液从大鼠使用两种技术：从大隐静脉或心脏穿刺抽血。

视频: Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

从美国的马蹄蟹，鲎波吕斐摩斯采血车

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

科研

生物学

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

从血液高效的iPS细胞生成使用附加体和HDAC抑制剂

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

从大鼠的侧尾静脉血液取样

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

人外周血代文化血液的产物内皮细胞

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

一种简便、有效的心肌注射小鼠心脏特异基因操作方法--体内

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...