سحب الدم من الفئران عن طريق الوريد الصافن والبزل القلب ملاحظة : يجب الموافقة على جميع الإجراءات من قبل رعاية الحيوانات المعهد الخاص واستخدام لجنة. 1. سحب الدم من الوريد الصافن <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يتم تنفيذ هذا الإجراء دون تخدير ويتطلب شخصين ، واحد الذي يعالج الفئران ، وأحد الذين ينفذ التعادل. </li><li style=";text-align:right;direction:rtl"> جعل مخروط من منشفة أو الاستمرار على الفئران مع قفازات القوارض المناولة ، وترك أحد أطرافه الخلفية المكشوفة. </li><li style=";text-align:right;direction:rtl"> حلق الخلفي من الساق مع الانتهازي الكهربائية حتى الوريد الصافن مرئيا. يحلق على مساحة واسعة بما فيه الكفاية ، بحيث لا شعر سوف تأتي في اتصال مع موقع الثقب. استخدام كمية صغيرة من المستحضر جهة غير عطرة للحفاظ على الشعر غير حلق بعيدا عن موقع الثقب. </li><li style=";text-align:right;direction:rtl"> جعل نقطة ضغط على قاعدة الساق لجعل الوريد الصافن من انتفاخ (مشابه لاستخدام عاصبة عند سحب الدم من الإنسان أو الحيوان الكبرى). ثقب الوريد باستخدام إبرة 20G وحلج القطن في الدم لأنه يخرج باستخدام microvette. وسوف تضخ مع الساق تساعد في جذب مزيد من الدماء. عندما كنت قد جمعت ما يكفي من الدم ، ضغط عقد النظيفة على موقع البزل لوقف النزيف. </li></ol> 2. سحب الدم من خلال ثقب في القلب <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> هذا الإجراء يتطلب التخدير والطرفية. يجب أن يتم التخلص فورا الحيوانات في نهاية ثقب في القلب. </li><li style=";text-align:right;direction:rtl"> إعداد محقنة 5 مل مع إبرة 23G1. </li><li style=";text-align:right;direction:rtl"> anesthesize عميقا للفئران ، والاختيار للتخدير بسبب قلة الحركة العفوية ، وبطء معدل التنفس ، وعدم الاستجابة للمحفزات (مثل معسر في احد اصابع القدم). لضمان التخدير لفترة طويلة ، ومكان للبرميل حقنة تحتوي على منشفة ورقية غارقة في مخدر المضطربة على أنف الفئران خلال هذا الإجراء. </li><li style=";text-align:right;direction:rtl"> وضع فأر على ظهرها ، تواجه بعيدا عنك. </li><li style=";text-align:right;direction:rtl"> إذا كنت اليد اليمنى ، ضع السبابة اليسرى في مستوى أدنى من أضلاعه ، ودون تطبيق أي ضغط. وسوف يكون موجودا في قلب ~ 1 سم فوق هذه النقطة ، قليلا إلى اليمين. </li><li style=";text-align:right;direction:rtl"> عقد المحقنة الخاص بزاوية 45 درجة ، تضاف إبرة بين ضلعين ومشاهدة لقطرة من الدم إلى حيز الإبرة. هذا هو إشارة إلى أن كنت في القلب. حقنة دون تحريك الخاص بك ، سحب على المكبس لملء الحقنة. بمجرد حقنة مليئة ، بعناية فصلها من الإبرة وتفريغها في أنبوب. ويمكن بعد ذلك المحقنة يعاد تعلق على إبرة لسحب المزيد من الدم. ينبغي أن يكون من الممكن استخلاص 50-10 مل من الدم من الفئران ز 120-180. </li><li style=";text-align:right;direction:rtl"> الموت ببطء على الفور الفئران. </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>

سحب الدم من الوريد الصافن هو وسيلة مريحة للحصول على كميات صغيرة من الدم من دون تخدير. إذا كان مطلوبا أخذ العينات المتكررة ، والتحقق من لوائح لضمان عدم سحب الدم الكثير من الفئران. الرسم في الدم عن طريق ثقب في القلب هو وسيلة مريحة للحصول على كميات كبيرة من ...

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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

سحب الدم من القوارض من الضروري بالنسبة لعدد كبير من كلا في التجارب المختبرية والدراسات المجراة. مواقع عديدة توجه الدم في القوارض : قلب الرجعية المدارية الجيوب الأنفية ، وحبل الوريد ، عرق الفك العلوي ، الوريد الصافن. كل أسلوب له مزاياه وعيوبه ، وعدم الموافقة على بعض أي أكثر في بعض البلدان (مثل الرجعية المدارية تلفت في هولندا). مناقشة أساليب مختلفة للرسم والدم 1-3 المتاحة. هنا ، نقدم اثنين من التقنيات لسحب الدم من الفئران ، مع كل التطبيقات الخاصة. سحب الدم من الوريد الصافن ، شريطة ان يتم ذلك بشكل صحيح ، يدفع الحد الأدنى من الضائقة في الحيوانات و لا يتطلب التخدير. هذا الأسلوب يسمح المتكررة تلفت كميات صغيرة من الدم ، مثل الحاجة للدراسات الدوائية 4،5 ، وتحديد البلازما والكيمياء ، أو تهمة الدم 6. ثقب في القلب تسمح (ويمكن استخلاص ما يصل الى 10 مل من الدم من الفئران 150 غ) جمع كميات كبيرة من الدم من حيوان واحد. هذه التقنية مفيدة للغاية ولذلك كإجراء المحطة عند سحب الدم من الصافن لن تقدم عينة كبيرة بما فيه الكفاية. نستخدم ثقب في القلب عندما نحتاج كميات كافية من المصل من سلالة معينة من الفئران أن تنمو خطوط اللمفاويات T 4-9 في المختبر.

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

توجه الدم ضرورية في عدد كبير من الدراسات ، على سبيل المثال لدراسة ملف الدوائية للمجمع. هنا ، ونحن لشرح كيفية سحب الدم من الفئران باستخدام اثنين من تقنيات : سحب الدم من الوريد الصافن أو من خلال ثقب في القلب.

سحب الدم من الفئران عن طريق الوريد الصافن والبزل القلب

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

57.2K Views.  University of California, Irvine (UCI). توجه الدم ضرورية في عدد كبير من الدراسات ، على سبيل المثال لدراسة ملف الدوائية للمجمع. هنا ، ونحن لشرح كيفية سحب الدم من الفئران باستخدام اثنين من تقنيات : سحب الدم من الوريد الصافن أو من خلال ثقب في القلب.

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

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

تخصيب خلايا NK من دم الانسان مع مجموعة من تقنيات RosetteSep StemCell

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

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.

كوكتيل من الدم والسرطان حدوة الحصان الأميركي ، الليمول 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 ...

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.

العزلة والثقافة في الخلايا البطانية الرئوي من الفئران حديثي الولادة

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.

رصد كيناز والفوسفاتيز الأنشطة من خلال دورة الخلية التي Ratiometric الحنق

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.

حيوي داخلي المجهري للدوران الأوعية الدقيقة في العضلات المشمرة ماوس لتحليل الهجرة الجذعية الطرفية المحمولة

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.

كفاءة الجذع الجيل خلية من الدم عن طريق Episomes وHDAC المانعون

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.

أخذ عينات الدم من الوريد الذيل الأفقي من الفئران

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

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.

تحليل الفجوة تعتمد على تقاطع نقل ميرنا مع المجهر 3D فراب

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.

طريقة بسيطة وفعالة في فيفو التلاعب بالجينات الخاصة بالقلب بالحقن إينتراميوكارديال في الفئران

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.

عزل وتوصيف والتفريق في القلب من الخلايا الجذعية من قلب الماوس الكبار

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 ...