قبل يوم واحد من التجربة : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تسمح BME (خفض عامل النمو ، Cultrex ، Trevigen) لذوبان الجليد على الجليد أو على 4 درجات مئوية ؛ إذابة الجليد مرة واحدة يبقي على منع اعادة ترسيخ. </li></ol> يوم من التجربة : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الفئران (6-7 أسابيع من العمر) وسيتم تخدير ونزفت بها. </li><li style=";text-align:right;direction:rtl"> إزالة الأبهر الصدري في طبق بتري معقمة مليئة PBS الباردة. </li><li style=";text-align:right;direction:rtl"> أداء التنظيف الميكانيكية من الأنسجة المحيطة الدهون. علما ، وتجنب تجفيف الأبهر في أي وقت. </li><li style=";text-align:right;direction:rtl"> استخدام شفرة جراحية ، شريحة الأورطي بالتساوي إلى حلقات 1 مم. يجب أن تكون موحدة وخفض نظيفة. </li><li style=";text-align:right;direction:rtl"> نقل حلقات لبرنامج تلفزيوني الباردة العذبة والحفاظ على الجليد في جميع الأوقات. </li><li style=";text-align:right;direction:rtl"> نصائح قبل استخدام ماصة المبردة ، إضافة إلى كل بئر في صفيحة ال 48 أيضا انخفاضا مقربة من BME 150 مل الباردة. السماح للهبوط BME لترسيخ عند 37 درجة مئوية لمدة 20-30 دقيقة. </li><li style=";text-align:right;direction:rtl"> حلقة الأبهر مكان واحد في مركز أعلى كل قبة. احتضان لمدة 10 دقيقة عند 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> على رأس كل حلقة تضيف BME 150 مل. احتضان لمدة 20-30 دقيقة عند 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> إعداد المتوسط ​​: البطانية مصل الإنسان المتوسطة الحرة (GIBCO ، كارلسباد ، كاليفورنيا) تستكمل مع FCS 2 ٪ ، والوحدات 50 / البنسلين مل و 50 ميكروغرام / مل الستربتوميسين (Cellgro). </li><li style=";text-align:right;direction:rtl"> إضافة إلى كل من 500 مل تحتوي المتوسطة إما : <ol class="lalpha" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عينات تجريبية. </li><li style=";text-align:right;direction:rtl"> الإيجابية التحكم : غشائي تكملة نمو الخلية (رسم القلب ، وتركيز النهائي 200 ميكروغرام / لتر ، العلوم البيولوجية دينار بحريني). </li><li style=";text-align:right;direction:rtl"> السيطرة السلبية : متوسطة وحدها. </li></ol></li><li style=";text-align:right;direction:rtl"> لوحة احتضان عند 37 درجة مئوية لمدة 12 يوما ، وخواتم صورة بين 6 و 12 يوما بموجب معيار المرحلة المجسام التباين. </li><li style=";text-align:right;direction:rtl"> اختياري : جمع supernatants. </li></ol> ملاحظات : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إبقاء ظروف معقمة في كل الأوقات. </li><li style=";text-align:right;direction:rtl"> تجنب إدخال فقاعات في BME. </li></ol>

<ol>
	<li>Goodwin, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+assays+of+angiogenesis+for+assessment+of+angiogenic+and+anti-angiogenic+agents.">In vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents.</a> Microvascular Research. 74, 172-183 (2007).</li><li>Masson, V., Devy, L., Grignet-Debrus, C., Bernt, S., Bajou, K., Blacher, S., Roland, G., Chang, Y., Fong, T., Carmeliet, P., Foidart, J. M., Noel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+aortic+ring+assay:+a+new+approach+of+the+molecular+genetics+of+angiogenesis.">Mouse aortic ring assay: a new approach of the molecular genetics of angiogenesis.</a> Biol. Proced. Online. 4, 24-31 (2002).</li></ol>

الأبهر مقايسة الدائري هو أداة مفيدة على طريقة لتقييم عائية وكذلك العوامل المضادة للعائية. السفن التي تنبثق من حلقات الأبهر تجنيد خلايا العضلات الملساء وpericytes اقترانه الأنبوب الخلية البطانية ، وهذا يعني أنها تشبه تشريحيا إلى neovessels في الجسم الحي. علاوة على ذلك ، ?...

aortic ring assay

الأوعية الدموية ، يرتبط تنتشر الأوعية الدموية من الأوعية الدموية الموجودة مسبقا مع كل من العمليات الطبيعية والمرضية. المقايسات الأوعية الدموية المختلفة تنطوي على دراسة الخلايا البطانية في ظروف الثقافة الفردية (1). في مقايسة حلقة الأبهر هو نموذج الأوعية الدموية التي تقوم على الثقافة الجهاز. في هذا الاختبار والأوعية عائية تنمو من شريحة من الشريان الأبهر (معدلة من (2)). باختصار ، هو استئصاله الماوس الأبهر الصدري ، تتم إزالة طبقة الدهون والبرانية ، ونحن على استعداد الحلقات ما يقرب من 1 ملم في الطول. هي جزء لا يتجزأ ثم حلقات الفردية في قبة صغيرة من مادة صلبة استخراج مصفوفة الطابق السفلي (BME) ، ويلقي داخل الآبار الفردية لوحة 48 - جيدا. يمكن أن تكون عوامل وعائية مثبطات تكوين الأوعية الدموية وأضاف مباشرة إلى الحلقات ، ويمكن استخدام مختلط يشارك في حلقات ثقافة الأبهر وأنواع الخلايا الأخرى لدراسة الآثار نظير الصماوي عائية. ومن الملاحظ تنتشر عن طريق التفتيش تحت stereomicroscope على مدى فترة من 6-12 أيام. ينصح بشدة بسبب الاختلاف الكبير الناجم عن مخالفات في قطاعات الأبهري ، والتجريب في plicates - 6. ويتم رصد جميع أنحاء Neovessel ثمرة التجربة وتصويرها باستخدام المجهر المرحلة ، ويتم جمع supernatants لقياس العوامل ذات الصلة وعائية المضادة عائية ، علامات موت الخلايا والنتريت.

Watch this Scientific Journal Video about Aortic Ring Assay at JoVE.com

Aortic Ring Assay

الأوعية الدموية ، يرتبط تنتشر الأوعية الدموية من الأوعية الدموية الموجودة من قبل ، مع كل من العمليات الطبيعية والمرضية. نحن هنا تدل على وجود عصابة مقايسة الأبهر الذي يسمح potentiators عائية ومثبطات المضافة مباشرة إلى حلقات الأبهر في الثقافة. تنتشر ويمكن تحديدها عن طريق التفتيش ثمرة neovessel الحلقات الأبهر على مدى فترة من 6-12 أيام.

الأبهر خاتم الفحص

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis ...

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31.0K Views.  Ben-Gurion University. الأوعية الدموية ، يرتبط تنتشر الأوعية الدموية من الأوعية الدموية الموجودة من قبل ، مع كل من العمليات الطبيعية والمرضية. نحن هنا تدل على وجود عصابة مقايسة الأبهر الذي يسمح potentiators عائية ومثبطات المضافة مباشرة إلى حلقات الأبهر في الثقافة. تنتشر ويمكن تحديدها عن طر...

Video: Aortic Ring Assay

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase 

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of abdominal aortic aneurysm (AAA) ever since it was first described in rats by Anidjar and colleagues.1 The rationale for its development was based on the disrupted nature of elastin observed in AAAs. This rat model has been modified to produce AAAs in the infrarenal aortic region of mice.2 The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice. Moreover, it is a viable platform to test potential therapeutic agents for AAA. In this video, we demonstrate the elastase infusion AAA procedure used in our laboratory. 
Mice are anesthetized using 2.5% isoflurane, and a laparotomy is performed under sterile conditions. The abdominal aortais isolated with the assistance of an operating stereomicroscope (Leica). After placing temporary ligatures around the proximal and distal aorta, an aortotomy is created at the bifurcation with the tip of a 30-gauge needle. A heat-tapered segment of PE-10 polyethylene tubing is introduced through the aortotomy and secured. The aortic lumen is subsequently perfused for 5-15 minutes at 100 mm Hg with saline containing type I porcine pancreatic elastase (4.5 U/mL; Sigma Chemical Co.). After removing the perfusion catheter, the aortotomy is repaired without constriction of the lumen.

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase

This video shows how to induce abdominal aortic aneurysms (AAA) in mice via transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta. The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice.

خلق الفئران التجريبية تمدد الأوعية الدموية الأبهري البطني مع إيلاستاز

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of ...

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

المستعرضة في انقباض الأورطي الفئران

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

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 ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

السذاجة الكبار الخلايا الجذعية عزل من المرحلة الابتدائية الثقافات الخلايا الليفية الإنسان

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

فائقة الدقة التصوير من Cytokinetic Z الدائري في البكتيريا تعيش طريق سريع منظم 3D الإضاءة المجهري (F3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

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

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

العزلة والثقافة الابتدائية من خلايا الفأر الأورطي غشائي

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

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

قدرة التمايز للخلايا الدهنية ارتشاح الابهري البشرية السلف

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

عزل وثقافة الخلايا البطانية الأبهرية الأولية من الخنازير المصغرة

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

This article describes the isolation of mouse aortic valve cells by a two-step collagenase procedure. Isolated mouse valve cells are important for performing different assays, such as this in vitro calcification assay, and for investigating the molecular pathways leading to aortic valve mineralization.

عزل خلايا الصمام الخلالي الماوس لدراسة تكلس الصمام الأبهري في المختبر

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) ...

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

This paper describes an explant culture-based method for the isolation and culturing of primary, patient-specific human aortic smooth muscle cells and dermal fibroblasts. Furthermore, a novel method is presented for measuring cell contraction and subsequent analysis, which can be used to study patient-specific differences in these cells.

عزل خلايا العضلات الملساء الأبهرية الأولية الخاصة بالمريض وقياسات الانقباض شبه الكمية في الوقت الفعلي في المختبر

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in ...