병원 아마토 루시타노, 카스텔루 브랑쿠 포르투갈. 병원 수사 마틴스, Guarda 포르투갈.

<ol><li> 동맥은 3~7cm의 UC 작품에서 얻을 수 있습니다. </li><li> 동맥을 둘러싸는 와튼의 젤리는 신중하게 가위로 절단하여 제거되었습니다. </li><li> 0.6 - 1mm의 외경의 무딘 엔드 바늘은 해부 동맥의 한쪽 끝을에서 7mm에 대해 삽입했다. </li><li> 혈관 내부에 남아있는 혈액을 제거하려면 동맥은 수직으로 개최 약 20-40 ML, 생리 생리 식염수 (PSS)와 perfused, 바늘에 연결된 20 ML 주사기를 사용하고 있습니다. 필요한 경우,이 세척 단계는 여러 번 반복되었다. </li><li> 동일한 절차는 태아 소 혈청 (FBS)의 10 %를 보충 RPMI 1640을 사용하여 반복했다. </li><li> 혈관 중 하나는 말단는 폐쇄되었습니다. 10 ML 주사기는 바늘에 연결되고 collagenase 유형 I (행크의 버퍼 소금 솔루션 (HBSS) 800 단위 / ML) 3 ML과 perfused했다. 그럼 다른 extremityof은 혈관도 폐쇄되었습니다. </li><li> 인산은 37 15 분 ° C 동요와 대한 생리 식염수 (PBS)를 버퍼로 배는 incubated되었습니다. </li><li> 선박의 끝을는 무료 SMCs를 수집하기 위해 상처 나서. 동맥은 5 FBS의 %와 50 ML 튜브에 항생과 보충 DMEM - F12의 약 20-40 ML과 perfuse되었습니다. </li><li> 튜브는 상온 (250g, 8 분)에서 centrifuged되었고, 세포 펠렛은 FBS의 5 %를 보충 DMEM - F12 5 ML에 resuspended되었습니다. </li><li> 세포 현탁액은 T - 플라스크 콜라겐 - 코팅에 전송하고 37 incubated되었습니다 ° C, 5 % CO 2 humidified 분위기에 따라. </li><li> 5 16 H, 비 점착 성의 세포와 세포 drebis이 제거되었습니다 후 37 문화 매체의 5 ML이 추가되었으며 세포 incubated되었습니다 ° C, 5 % CO 2 humidified 분위기에 따라. </li><li> SMCs가 합류에 도착할 때까지 세포 배양 매체는 2 3 일마다 변경되었습니다. </li><li> 세포가 합류를 획득 후 그들은 두 개 또는 세 25cm 2 T - flasks에 씨앗을 품고 있었다. </li></ol> 대청소 <ol><li> 샵스 컨테이너에서 바늘 폐기. </li><li> 큰 생물 학적 주사기 용기에 배출. </li><li> 작은 생물 학적 가방에 모든 조직을 넣어. -20에서 가방과 동결을 닫고 ° 소각까지 섭씨. </li><li> 적어도 10 분 virucide의 모든 악기를 만끽해보세요. </li><li> 냉장고에 사용되지 않은 미디어를 반환합니다. </li><li> 생물 학적 폐기물의 장갑 폐기. </li></ol>

<ol>
	<li>Van Rijen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctions+in+human+umbilical+cord+endothelial+cells+contain+multiple+connexins.">Gap junctions in human umbilical cord endothelial cells contain multiple connexins.</a> The American journal of physiology. 272 (1 PT 1), C117-C117 (1997).</li><li>Mart&#237;n de Llano, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedure+to+consistently+obtain+endothelial+and+smooth+muscle+cell+cultures+from+umbilical+cord+vessels.">Procedure to consistently obtain endothelial and smooth muscle cell cultures from umbilical cord vessels.</a> Translational Research. 149 (1), 1-9 (2007).</li><li>Jaffe, E. A., Nachman, R. L., Becker, C. G., Minick, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+human+endothelial+cells+derived+from+umbilical+veins.+Identification+by+morphologic+and+immunologic+criteria.">Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria.</a> J Clin Invest. 52 (11), 2745-2756 (1973).</li><li>Cairr&#227;o, E., Santos-Silva, A., Alvarez, E., Correia, I., Verde, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+human+umbilical+artery+smooth+muscle+cells+expressing+functional+calcium+channels.">Isolation and culture of human umbilical artery smooth muscle cells expressing functional calcium channels.</a> In Vitro Cellular &#38; Developmental Biology-Animal. 45 (3), 175-184 (2009).</li></ol>

SMC는 분리, 압축시키는 것 약품과 함께 미래를 assays를위한 모델로 사용 구조적, L - 타입 칼슘 채널을 특징 혈관 기능의 세포 및 분자 측면을 연구하고 조직 공학을 사용하실 수 있습니다. SMCs 및 내장 컴퓨터가 인간에 사용되는 세미 합성 혈관의 생산에 사용될 수있는 새로운 biomaterials의 발전을위한 기본입니다.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM-F12</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>095K8311</td>
<td>&nbsp;</td>
<tr>
<td>Fetal bovine serum (FBS)</td>
<td>&nbsp;</td>
<td>Biochrom-AG</td>
<td>0117T</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>3103806</td>
<td>&nbsp;</td>
<tr>
<td>Collagen</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>067K2302</td>
<td>&nbsp;</td>
<tr>
<td>Antibiotic</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 &mu;L/ 1 mL of medium</td>
</tr>
<tr>
<td>Scissors</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Sterilized</td>
</tr>
<tr>
<td>Needle</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Diameter of 0.6-1mm</td>
</tr>
<tr>
<td>Syringe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 - 20 mL</td>
</tr>
<tr>
<td>Physiological Saline Solution (PSS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>20-40 mL per artery</td>
</tr>
<tr>
<td>Collagenase type I</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>047K1052</td>
<td>3 mL per artery</td>
</tr>
<tr>
<td>Hank's Buffered Salt Solution(HBSS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Diluted collagenase </td>
</tr>
<tr>
<td>Phosphate buffered saline solution (PBS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Warmed to 37&deg;C</td>
</tr>		</tbody>
		</table>
		</div>

isolation of human umbilical arterial smooth muscle cells (huasmc)

인간의 탯줄 (UC)는 쉽게 바로 출산 후 얻을 수있다 생물 학적 샘플입니다. 이것은 생물 학적 샘플, 대부분의 시간이다 삭제하고 컬렉션은 신생아지나 어머니의 건강에 어떠한 추가 위험을 의미하지는 않습니다. 또한 아무런 윤리적 문제가 제기되지 않습니다. UC 한 정맥 내피 세포와 모두있는 (내장 컴퓨터) 2 동맥에 의해 구성되어있다<sup&gt; 1</sup&gt;와 평활근 세포 (SMCs)<sup&gt; 2</sup&gt; 혈관의 주요 세포 구성 요소의 두, 격리 수 있습니다. 이 프로젝트에서는 SMCs는 이에 따라 UC의 동맥의 효소 치료 이전에 자페에 의해 설명하는 실험 절차 후에 얻은되었습니다<em&gt; 외</em<sup&gt; 3</sup&gt;. 세포 분리 후 그들은 DMEM - F12은 태아 소 혈청의 5 %를 보완하고 여러 구절에 대한 교양했던 T - 플래시에 보관했다. 세포는 서로 다른 세대에서 형태학의 다른 phenotypic 특성을 유지. 본 연구의 목적은 압축시키는 것 약물과 함께 미래를 assays를위한 모델로 사용하기 위해서는 부드러운 근육 세포를 분리하는 것이었다, 분리 및 구조적 혈관 기능의 세포 및 분자 측면을 연구, L - 타입 칼슘 채널을 특징<sup&gt; 4</sup&gt; 및 조직 공학을 사용할 수 있습니다.

Watch this Scientific Journal Video about Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC at JoVE.com

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

탯줄 코드는 여러 가지 방법으로 부드러운 근육 세포를 분리하는 데 사용됩니다. 이 작품에서 우리는 고립 평활근 세포에 효소 처리를 사용합니다.

인간의 탯줄 아티 평활근 세포 절연 (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

isolation-of-human-umbilical-arterial-smooth-muscle-cells-huasmc

Research

JoVE Journal

Biology

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

13.9K 조회수.  Universidade da Beira Interior. 탯줄 코드는 여러 가지 방법으로 부드러운 근육 세포를 분리하는 데 사용됩니다. 이 작품에서 우리는 고립 평활근 세포에 효소 처리를 사용합니다.

비디오: Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

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, 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 often do not resemble vessels in vivo. Here we demonstrate an optimized 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. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

인간 제대 베인 내피 세포의 절연 (HUVEC)

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

연구

생물학

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

초점 칼슘 2 +</sup평활근에서> 과도 감지

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

인간 탯줄의 분리 및 동물 혈청 무료 확장 Mesenchymal Stromal 세포 (MSCS)와 내피 콜로니 형성 전구 세포 (ECFCs)을 유래

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

패치 클램프 전기 생리학 및 압력 Myography를 사용하여 간선 평활근 Kv7 칼륨 채널 기능을 탐색

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

근육 위성 세포의 분리, 문화, 및 이식

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

신생아 쥐의 폐 동맥 평활근 세포의 분리

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

세포 내 칼슘의 두 광자 이미징<sup&gt; 2 +</sup&gt; 고립 된 쥐 대동맥의 취급 및 내피 세포에서 산화 질소 생산과 부드러운 근육 세포

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

배양 된 평활근 세포의 근질 그물 자리 내 칼슘 변동 FRET 측정을 기반으로 공 촛점 이미지

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

쥐 관상 동맥 혈관 평활근 세포의 분리

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, fresh, and isolated DSM cells presents an important technical challenge whose achievement provides optimal cells for subsequent functional and molecular studies. The method developed and elaborated herein, successfully used by our group for over a decade, describes dissection of human urinary bladder specimens obtained from open bladder surgeries followed by an enzymatic two-step treatment of DSM pieces and mechanical trituration to obtain freshly isolated DSM cells. The initial step involves dissection to separate the DSM layer (also known as muscularis propria) from mucosa (urothelium, lamina propria, and muscularis mucosa) and the adjacent connective, vascular, and adipose tissues present. The DSM is then cut into pieces (2-3 mm x 4-6 mm) in nominal Ca2+-containing dissection/digestion solution (DS). DSM pieces are next transferred to and sequentially treated separately with DS containing papain and collagenase at ~37 &#176;C for 30-45 min per step. Following washes with DS containing enzyme-free bovine serum and trituration with a fire-polished pipette, the pieces release single DSM cells. Freshly isolated DSM cells are ideally suited for patch-clamp electrophysiological and pharmacological characterizations of ion channels. Specifically, we show that the TRPM4 channel blocker 9-phenanthrol reduces voltage-step evoked cation currents recorded with the amphotericin-B perforated patch-clamp approach. DSM cells can also be studied by other techniques such as single cell RT-PCR, microarray analysis, immunocytochemistry, in situ proximity ligation assay, and Ca2+ imaging. The main advantage of utilizing single DSM cells is that the observations made relate directly to single cell characteristics revealed. Studies of freshly isolated human DSM cells have provided important insights characterizing the properties of various ion channels including cation-permeable in the urinary bladder and will continue as a gold standard in elucidating DSM cellular properties and regulatory mechanisms.

We describe a method for preparation of the single freshly isolated detrusor smooth muscle cells from human urinary bladder specimens employing a two-step enzymatic procedure. The obtained viable DSM cells can be studied by various single cell techniques including the described amphotericin-B patch-clamp electrophysiology to reveal physiological and pharmacological properties.

9-페난트롤에 민감한 양이온 전류의 특성화를 위해 갓 분리된 인간 분리기 평활근 세포의 준비 및 활용

Detrusor smooth muscle (DSM) cells present within the urinary bladder wall ultimately facilitate urine storage and voiding. Preparation of the viable, ...