La extracción de sangre de las ratas a través de la vena safena y por punción cardiaca Nota: Todos los procedimientos deben ser aprobados por el cuidado de los animales de su institución y el empleo. 1. Extracción de sangre de la vena safena <ol><li> Este procedimiento se realiza sin anestesia y se necesitan dos personas, una que se encarga de la rata, y el que lleva a cabo el sorteo. </li><li> Hacer un cono de una toalla o mantener la rata con guantes de manipulación de roedores, dejando una pata trasera al descubierto. </li><li> Afeitarse la parte posterior de la pierna con una cortadora eléctrica hasta que la vena safena es visible. Afeitarse un área lo suficientemente grande, por lo que no tiene pelo entre en contacto con el sitio de punción. Use una pequeña cantidad de crema para las manos no aromático para mantener el pelo afeitado no lejos del sitio de la punción. </li><li> Hacer un punto de compresión en la base de la pierna para hacer el bulto de la vena safena (similar al uso de un torniquete para extraer sangre de un humano o un animal de gran tamaño). Punción de la vena con una aguja 20G y saca la sangre a medida que sale con un microvette. De bombeo con la pierna le ayudará a sacar más sangre. Cuando haya recogido suficiente cantidad de sangre, coloque compresas limpias en el sitio de punción para detener el sangrado. </li></ol> 2. Extracción de sangre por punción cardiaca <ol><li> Este procedimiento requiere anestesia y es terminal. Los animales deben ser sacrificados inmediatamente al final de una punción cardíaca. </li><li> Prepare una jeringa de 5 ml con una aguja 23G1. </li><li> Profundamente anesthesize la rata y el cheque para la anestesia por la falta de movimiento espontáneo, la tasa de respiración lenta, y la falta de respuesta a los estímulos (como presionar un dedo del pie). Para garantizar la anestesia prolongada, coloque una jeringa que contiene una toalla de papel empapado en anestésico volátil en la nariz de las ratas durante el procedimiento. </li><li> Coloque el ratón sobre la espalda, de espaldas a usted. </li><li> Si usted es diestro, coloque el dedo índice izquierdo a nivel de las costillas inferiores, sin aplicar ninguna presión. El corazón se encuentra aproximadamente 1 cm por encima de este punto, ligeramente hacia la derecha. </li><li> La celebración de la jeringa en un ángulo de 45 grados, inserte la aguja entre dos costillas y ver una gota de sangre para entrar en la aguja. Esto es una indicación de que está en el corazón. Sin mover la jeringa, tire del émbolo para llenar la jeringa. Una vez que la jeringa se llena, con cuidado de desconexión de la aguja y se vacía en un tubo. La jeringa puede ser re-unido a la aguja para la elaboración de más sangre. Debe ser posible llegar a 5-10 ml de sangre de una rata 120-180 g. </li><li> Inmediatamente la eutanasia a la rata. </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>

La extracción de sangre de la vena safena es una manera conveniente de obtener pequeñas cantidades de sangre sin necesidad de anestesia. Si el muestreo repetido es necesario, consulte las normas para asegurarse de que no sacan mucha sangre de una rata. La extracción de sangre por punción cardiaca es una manera conveniente de obtener grandes cantidades de sangre, pero esto es un procedimiento terminal. El animal debe ser sacrificado al final de la extracción de sangre. Estas técnicas (como todas las técnicas de los ani...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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drawing blood from rats through the saphenous vein and by cardiac puncture

La extracción de sangre de los roedores es necesario para un gran número de los estudios in vitro e in vivo. Los sitios de extracción de sangre son numerosas en los roedores: retro-orbital sinusal, la vena yugular, vena maxilar, la vena safena, el corazón. Cada técnica tiene sus ventajas y desventajas, y algunos no han sido aprobados más en algunos países (por ejemplo, retro-orbital se basa en Holanda). Una discusión de las diferentes técnicas para la extracción de sangre están disponibles 3.1. Aquí se presentan dos técnicas para la extracción de sangre de las ratas, cada uno con sus aplicaciones específicas. Extraer la sangre de la vena safena, siempre y cuando se hace correctamente, provoca angustia mínima en los animales y no requiere anestesia. Esta técnica permite repetir sorteos de pequeñas cantidades de sangre, como necesarios para los estudios farmacocinéticos 4,5, la determinación de la química del plasma, o recuento sanguíneo 6. La perforación cardíaca permite la obtención de grandes cantidades de sangre de un solo animal (hasta 10 ml de sangre puede ser extraída de una rata de 150 g). Esta técnica es muy útil por lo tanto, como un procedimiento terminal cuando la extracción de sangre de la safena interna no sería una muestra suficientemente grande. Usamos punción cardiaca cuando necesitamos una cantidad suficiente de suero de una cepa específica de las ratas a crecer T líneas de linfocitos in vitro 4-9.

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

La extracción de sangre son necesarias en un gran número de estudios, por ejemplo para estudiar el perfil farmacocinética de un compuesto. En este sentido, se muestra cómo extraer la sangre de las ratas mediante dos técnicas: extracción de sangre de la vena safena o por punción cardiaca.

La extracción de sangre de las ratas a través de la vena safena y por punción cardiaca

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 vistas.  University of California, Irvine (UCI). La extracción de sangre son necesarias en un gran número de estudios, por ejemplo para estudiar el perfil farmacocinética de un compuesto. En este sentido, se muestra cómo extraer la sangre de las ratas mediante dos técnicas: extracción de sangre de la vena safena o por punción cardiaca.

Video: 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.

Extracción de sangre del cangrejo herradura de América, 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 ...

Investigación

Biología

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.

Aislamiento y cultivo de células endoteliales pulmonares de ratones recién nacidos

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.

Seguimiento de las actividades de quinasa y fosfatasa través del ciclo celular por Proporcional FRET

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.

Microscopia intravital de la microcirculación en el músculo de ratón Cremaster para el análisis de la migración de la célula madre periféricas

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.

Eficiente iPS Generación Celular de sangre usando episomas y HDAC inhibidores

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.

Toma de muestras de sangre de la vena lateral de la cola de la rata

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.

Generación y Cultura de Sangre Consecuencia endoteliales Las células de la sangre periférica humana

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.

Análisis de la transferencia dependiente de la unión Gap de miARN con 3D-FRAP Microscopía

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.

Un método sencillo y eficaz para la manipulación In Vivo de Gene cardiaco específico por inyección intramiocárdica en ratones

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.

Aislamiento, caracterización y diferenciación de células cardiacas desde el corazón de ratón adulto

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 ...

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Subtitles

La extracción de sangre de las ratas a través de la vena safena y por punción cardiaca