وقدمت الدعم المالي لمعظم الدراسات التي أجراها مجلس البحوث الطبية السويدية (منحة 5942).

<ol>
	<li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+models+of+angiogenesis.">In vivo models of angiogenesis.</a> J. Cell. Mol. Med. 10, 588-612 (2006).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+testing+with+angiogenesis+models.">Drug testing with angiogenesis models.</a> Expert Opin. Drug. Discov. 3, 533-549 (2008).</li><li>Norrby, K., Jakobsson, A., Sorbo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast-cell-mediated+angiogenesis:+a+novel+experimental+model+using+the+rat+mesentery.">Mast-cell-mediated angiogenesis: a novel experimental model using the rat mesentery.</a> Virchows Arch. B Cell. Pathol. Incl. Mol. Pathol. 52, 195-206 (1986).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cells+and+angiogenesis.">Mast cells and angiogenesis.</a> APMIS. 110, 355-371 (2002).</li><li>Albertsson, P., Lennernas, B., Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+meteronomic+chemotherapy:+modulation+of+angiogenesis+mediated+by+VEGF-A.">On meteronomic chemotherapy: modulation of angiogenesis mediated by VEGF-A.</a> Acta Oncol. 45, 144-155 (2006).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+apo-lactoferrin+enhances+angiogenesis+mediated+by+vascular+endothelial+growth+factor+A+in+vivo.">Human apo-lactoferrin enhances angiogenesis mediated by vascular endothelial growth factor A in vivo.</a> J. Vasc. Res. 41, 293-304 (2004).</li><li>Norrby, K., Mattsby-Baltzer, I., Innocenti, M., &amp;amp, T. u. n. e. b. e. r. g., S, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orally+administered+bovine+lactoferrin+systemically+inhibits+VEGF(165)-mediated+angiogenesis+in+the+rat.">Orally administered bovine lactoferrin systemically inhibits VEGF(165)-mediated angiogenesis in the rat.</a> Int. J. Cancer. 91, 236-240 (2001).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-molecular-weight+heparins+and+angiogenesis.">Low-molecular-weight heparins and angiogenesis.</a> APMIS. 114, 79-102 (2006).</li><li>Norrby, K., Nordenhem, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dalteparin,+a+low-molecular-weight+heparin,+promotes+angiogenesis+mediated+by+heparin-binding+VEGF-A+in+vivo.">Dalteparin, a low-molecular-weight heparin, promotes angiogenesis mediated by heparin-binding VEGF-A in vivo.</a> APMIS. 118, 949-957 (2010).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+and+de+novo+mammalian+angiogenesis.">Basic fibroblast growth factor and de novo mammalian angiogenesis.</a> Microvasc. Res.. 48, 96-113 (1994).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor+and+de+novo+mammalian+angiogenesis.">Vascular endothelial growth factor and de novo mammalian angiogenesis.</a> Microvasc. Res. 51, 153-163 (1996).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+density+in+terms+of+number+and+length+of+microvessel+segments+per+unit+tissue+volume+in+mammalian+angiogenesis.">Microvascular density in terms of number and length of microvessel segments per unit tissue volume in mammalian angiogenesis.</a> Microvasc. Res. 55, 43-53 (1998).</li></ol>

الإعلان عن أي تضارب في المصالح.

وقد قدم في عام 1986 فحص (3) ولقد تم تطويرها تباعا وصقلها في مختبرنا 7،10-12. كما نوقشت في ورقات الاستعراض (دعوة) 1 ، 2 ، مقايسة يقارن جيدا مع الثدييات الأخرى في مقايسة الأوعية الدموية في الجسم الحي الصدد ولا سيما إلى ميزات هامة مثل النسيج اختبار الكبار أصلا أوعية دموية ويفتقر إلى الأوعية الدموية الفسيولوجية ؛ الحد الأدنى من الصدمة ، إن وجد ، هو لحقت النسيج ؛ تقاس المتغيرات الكمية حقا ، والذي يسمح التحليل الإحصائي السليم بشأن الاستجابة للجرعة والدراسات الجزيئية النشاط ؛ تنتشر الأوعية الدموية يحدث ، وهو غلبة في الأنسجة الطبيعية والأورام ، ويتم تجميع البيانات بسهولة السمية في الفئران التي تنمو بقوة من الناحية الفسيولوجية في مرحلة البلوغ. قد تتضمن ميزات غير ملائم حقيقة أن مقايسة نسبيا تستغرق وقتا طويلا ، وخصوصا عندما تقدير عدد وطول قطاعات microvessel الفردية وبراعم microvessel ، وأنه لا يسمح real-time/serial الملاحظات ، وأنه لا يصلح بالمرة لالفئران منذ العديد من النوافذ المساريقي في الفئران عدم microvessels الأصل المحتملة من الشعيرات الدموية الجديدة التي يمكن أن تنشأ. هي ، في الوقت الحقيقي الملاحظات ومع ذلك ، تمكين بواسطة المجهر intravital حيث مفلطحة النوافذ سليمة المساريقي exteriorized لكن على مدى مرحلة المجهر بينما الحيوان هو تخدير ، كما نوقش في 1. وقد تم تحقيق تقدم كبير في مجال البحوث الأوعية الدموية في العقود الماضية باستخدام نماذج مثل micropocket القرنية ، والغشاء فرخ مشيمائي ، والمقايسات SC Matrigel المكونات ، والتي هي أدوات مفيدة لكنها محدودة في نهاية المطاف. التطبيقات السريرية في المستقبل من العلاجات المضادة للعائية وعائية الموالية - يستلزم إنشاء والتحقق من صحة أكثر حساسية وأهمية من الناحية البيولوجية ، ونماذج ما قبل السريرية الكمية حقا ، مثل تلك التي وصفها في التقرير الحالي.

مخازن والكواشف 0.5 حل M - تريس حمض الهيدروكلوريك (الرقم الهيدروجيني 7.8) المخزون يذوب تريس (hydroxymethyl) aminomethan (60،57 غ) في ماء مقطر ~ 900 مل ويضاف ~ 45 مل من حمض الهيدروكلوريك 25 ٪ (تركزت) لتحقيق درجة الحموضة 7.8 تليها لتمييع 1 ليتر ماء مقطر مع. تريس العازلة المالحة (TBS ، الرقم الهيدروجيني 7.8) يضاف الى 200 مل من محلول كلوريد الصوديوم الاسهم 18 ز المذابة في ماء مقطر ~ 1800 مل مع تعديل الرقم الهيدروجيني إلى 7.8 باستخدام محلول المخزون ، تليها التخفيف إلى 2 لتر ماء مقطر مع. TBS (الرقم الهيدروجيني 7.4) يضاف الى 200 مل من محلول كلوريد الصوديوم الاسهم 18 ز الذائبة في المذابة في ماء مقطر ~ 1800 مل مع تعديل الرقم الهيدروجيني إلى 7.4 باستخدام محلول المخزون ، تليها التخفيف إلى 2 لتر ماء مقطر مع. التربسين الحل (0.05 ٪ التربسين ، 0.1 ٪ CaCl 2) يحتوي على 0.1 و 0.2 التربسين ز ز 2 CaCl في TBS 200 مل (7.8 درجة الحموضة). حل السكروز 2.5 ٪ يحتوي على 5 غرام في السكروز TBS 200 مل (7.8 درجة الحموضة). محلول المخزون من أزيد الصوديوم 1 ٪ في TBS (الرقم الهيدروجيني 7.4) يحتوي على 1 غرام نا آزيد في 100 مل مع TBS (الرقم الهيدروجيني 7.4). تمييع العازلة : 0.1 ٪ BSA ، 0.01 ٪ نا آزيد (مستعملة لإضعاف الأجسام المضادة في عدة Vectastain ABC). يحتوي على 0.1 غ ألبومين المصل البقري (BSA) و 1 مل من 1 ٪ نا آزيد ، مخففة الى 100 مل مع TBS (الرقم الهيدروجيني 7.4). ABC مجموعة Vectastain PK 4002 ، ABC البيروكسيداز الماوس مفتش مصل طبيعي الحصان ؛ حجب المصل ؛ حل الصفراء : 3 مل التخفيف drops/10 العازلة. Biotinylated الأجسام المضادة ، ومكافحة الفأر مفتش ؛ حل الزرقاء : 1 تخفيف مل drop/10 العازلة. ABC - الكاشف : حل البرتقالي (2) ، بالإضافة إلى قطرات البني الحل 2 مل drops/10 TBS (الرقم الهيدروجيني 7.4). مزيج هذه الحلول قبل 20 دقيقة من الاستخدام! الماوس (وحيدة النسيلة) لمكافحة الفئران البطانة (بيوسورس الدولية ، كاماريللو ، كاليفورنيا ، الولايات المتحدة الأمريكية) <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> كتالوج # </td><td> ARU0181 </td></tr><tr><td> استنساخ # </td><td> OX MRC - 43 </td></tr></tbody></table> يتم توجيه هذه الأجسام المضادة ضد خلايا بطانة الأوعية الدموية في جميع الفئران وذلك باستثناء تلك الموجودة في الدماغ. Vectastain البيروكسيداز أفيدين البيوتين مجمع (ABC) طقم طقم يحتوي جسم الابتدائي ، الثانوي biotinylated الأضداد ، أفيدين - biotinylated مجمع انزيم (ABC) ، والركيزة الانزيم. انظر <a href="http://www.vectorlabs.com/">www.vectorlabs.com</a> أو <a href="http://www.immunkemi.se/">www.immunkemi.se</a> عن البروتوكولات. ألبومين المصل البقري (BSA) (سيغما الدريخ ، وأنواع # A - 4503). DAB = 3،3 &#39;- Diaminobenzidine Isopac (سيغما الدريخ ، وأنواع # D 9015). التربسين من البنكرياس البقري (سيغما الدريخ ، وأنواع # T 4665). Vectastain البيروكسيداز ABC - عدة ، الفأر مفتش (Immunkemi F &amp; D AB ، Jarfalla ، السويد)

rat mesentery angiogenesis assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Watch this Scientific Journal Video about Rat Mesentery Angiogenesis Assay at JoVE.com

Rat Mesentery Angiogenesis Assay

لم تكن طبيعية نسيج أوعية دموية الكبار الثدييات التي تفتقر الأوعية الدموية الفسيولوجية والتي تتعرض لتدخل جراحي يستخدم لدراسة : (ط) بدء وتطوير الأوعية الدموية داخل الصفاق التالية من وكلاء الإدارة الاختبار ، و (ب) تعديل الأوعية الدموية التالية للادارة الشاملة اختيار وكلاء الاختبار.

الفئران الفحص مساريق الأوعية الدموية

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Research

JoVE Journal

Biology

13.8K Views.  University of Gothenburg. لم تكن طبيعية نسيج أوعية دموية الكبار الثدييات التي تفتقر الأوعية الدموية الفسيولوجية والتي تتعرض لتدخل جراحي يستخدم لدراسة : (ط) بدء وتطوير الأوعية الدموية داخل الصفاق التالية من وكلاء الإدارة الاختبار ، و (ب) تعديل الأوعية الدموية التالية للادارة الشاملة اختي...

Video: Rat Mesentery Angiogenesis Assay

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

كريستوفر هيوز : حدث في نموذج المختبر لدراسة الأوعية الدموية (مقابلة)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

الفحص الأمثل الفايبرين الخرزة جل لدراسة الأوعية الدموية

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. 
Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20&deg;C.

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

إعداد عامل نمو الخلايا T من Splenocytes الجرذ

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

رسم الخرائط البصرية القلوب الجرذ Langendorff - perfused

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

الابتدائية ثقافة الكبار Myocytes قلب فأر

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Intracellular Refolding Assay

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding1 and have a crucial role in promoting cell survival upon stress insults like heat shock2, nutrient starvation and exposure to chemicals/poisons3. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells4 as well as neurodegeneration5. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy7 and neurodegenerative disorders9. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones10,11. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene 11 (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45&deg;C for 30 minutes. Upon recovery at 37&deg;C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).

In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity.

الخلايا Refolding الفحص

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

جرذ الاهتمام لأمور ظاهرية مساريق: نموذج للتحقيق في حيوية الخلوي شارك في الأوعية الدموية

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

البطانية أنبوب تشكيل خلية الفحص ل<em&gt; في المختبر</em&gt; دراسة الأوعية الدموية

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

تروية دقيقة التقنية المعنية بالتحقيق في تنظيم Microvessel النفاذية في الفئران مساريق

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

في فيفو القلوية المذنب الفحص وأنزيم المعدلة القلوية المذنب الفحص لقياس فواصل حبلا الحمض النووي والحمض النووي الضرر التأكسدي في كبد الفئران

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...