Name: isolation of myofibroblasts from mouse and human esophagus
Uploaded: 2015-01-18T14:00:00
Description: Watch this Scientific Journal Video about Isolation of Myofibroblasts from Mouse and Human Esophagus at JoVE.com

Authors have nothing to disclose.

프로토콜은 동물 실험, 이론적 근거와 연구의 목적을 수행하기 위해, 남부 캘리포니아 기관 동물 관리 및 사용위원회의 대학에 의해 제출하여 승인을했다. 취소 확인 된 인간의 식도 표본에서 차 문화의 설립을위한 프로토콜은 남부 캘리포니아 심사위원회 (Institutional Review Board)의 대학에 의해 승인되었다. 1. 마우스 또는 인간의 식도를 얻습니다 <ol><li...

<ol>
	<li>Stappenbeck, T. S., Miyoshi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+stromal+stem+cells+in+tissue+regeneration+and+wound+repair.">The role of stromal stem cells in tissue regeneration and wound repair.</a> Science. 324 (5935), 1666-1669 (2009).</li><li>Powell, D. W., Pinchuk, I. V., Saada, J. I., Chen, X., Mifflin, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+cells+of+the+intestinal+lamina+propria.">Mesenchymal cells of the intestinal lamina propria.</a> Annu Rev Physiol. 73, 213-237 (2011).</li><li>Rieder, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-Helper+2+Cytokines,+Transforming+Growth+Factor+beta1,+and+Eosinophil+Products+Induce+Fibrogenesis+and+Alter+Muscle+Motility+in+Patients+With+Eosinophilic+Esophagitis.">T-Helper 2 Cytokines, Transforming Growth Factor beta1, and Eosinophil Products Induce Fibrogenesis and Alter Muscle Motility in Patients With Eosinophilic Esophagitis.</a> Gastroenterology. 146 (5), 1266-1277 (2014).</li><li>Powell, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+cells+important+in+health+and+disease.">Paracrine cells important in health and disease.</a> The American Journal of Physiology. 277 (1 Pt. 1), C1-9 (1999).</li><li>Powell, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+subepithelial+myofibroblasts.">Intestinal subepithelial myofibroblasts.</a> The American Journal of Physiology. 277 (2 Pt. 1), C183-201 (1999).</li><li>Shaker, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cells+participate+in+the+murine+esophageal+mucosal+injury+response.">Stromal cells participate in the murine esophageal mucosal injury response.</a> American Journal of Physiology Gastrointestinal and Liver Physiology. 304 (7), 662-672 (2013).</li><li>Shaker, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epimorphin+deletion+protects+mice+from+inflammation-induced+colon+carcinogenesis+and+alters+stem+cell+niche+myofibroblast+secretion.">Epimorphin deletion protects mice from inflammation-induced colon carcinogenesis and alters stem cell niche myofibroblast secretion.</a> The Journal of Clinical Investigation. 120 (6), 2081-2093 (2010).</li><li>Kalabis, J., et al. <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+characterization+of+mouse+and+human+esophageal+epithelial+cells+in+3D+organotypic+culture.">Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture.</a> Nature Protocols. 7 (2), 235-246 (2012).</li><li>Khalil, H., Nie, W., Edwards, R. A., Yoo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+primary+myofibroblasts+from+mouse+and+human+colon+tissue.">Isolation of primary myofibroblasts from mouse and human colon tissue.</a> Journal of Visualized Experiments. (80), (2013).</li><li>Rieder, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastroesophageal+reflux+disease-associated+esophagitis+induces+endogenous+cytokine+production+leading+to+motor+abnormalities.">Gastroesophageal reflux disease-associated esophagitis induces endogenous cytokine production leading to motor abnormalities.</a> Gastroenterology. 132 (1), 154-165 (2007).</li></ol>

조직의 크기에 적합한 닦지 소화 회 뮤린 또는 인체 조직을 사용할 때 차 배양 생성 기법은 일반적으로 유사하다. 뮤린 조직의 경험은 상술 한 방법을 사용하는 것이 일관되게 차 배양의 성공 확립 될 것을 제안한다. 프로토콜 내에서 중요한 단계는 수술 장비의 살균 및 조직 문화의 표준 멸균 기술을 다음과 같습니다. 마우스의 나이는 중요한 단계입니다. 8~12일 된 신생아는 지속적으로 성공 차 ?...

상피 기질 상호 작용 재생 점막, 보수, 섬유증 및 발암 1,2- 포함한 위장관 다양한 기능의 조절에 관여한다. 이러한 상호 작용은 가장 작은 창자 및 결장에서 연구되었다 마찬가지로 식도 점막 질환 3에서 역할을 수행 할 수있다. 장과 대장 기질 세포라고 근육 섬유 모세포의 하위 집단은 조직 손상, 염증을 중재에 참여하고 4,5를 복구하는 입증되었다. 원위 위장관에서 이러한 스핀들 모양의 세포는 상피와 고유 층 사이의 계면에서 기저막에 인접하고, α-SMA 및 멘틴으로 정의 된 양,-팬 아세포 부정하고, 약한 양성 또는 최근 desmin 5 음. 식도 기질은 엄격하게 세포 또는 분자 수준에서 특징되지 않았습니다. 쥐의 식도에있는 우리의 작업은 취소했다monstrated의 α-SMA 편평 상피 6 때때로 밑의 식도 기질과 멘틴 세포. 상피 세포 - 기질 상호 작용 등의 위식도 중재 부상 (6)과 호산 구성 식도염 3과 식도 점막 질환에 연루되어있다. 섬유 성 협착은 위장 섬유증의 발병에 연루되어 식도 부상과 간질 세포의 알려진 합병증이다. 이러한 세포의 분리 미친 신호 전달 경로를 조사하는 데 필요한 연구를 수행하는 데 도움이됩니다. 이 제출는 이러한 상호 작용을 매개하는 신호 전달 경로에 대한 지식을 기존의 격차가 해결 될 수 있도록 α-SMA 긍정적 인, 멘틴 긍정적 인 근육 섬유 모세포의 차 문화를 구축하는 데 필요한 기술을 제공합니다. 기재된 기술이 성공적 일차 결장 뮤린 근섬유 7 furthe를 확립 저자 사용되고R 6은 뮤린 및 인간 근섬유 같은 식도 기질 세포의 확립을위한 장치. 여기에서 우리는 설정하고 마우스 또는 미래의 기능 연구에 사용하기 전에 인간의 식도에서 설립이 문화를 특징 짓는 데 필요한 조건을 설명합니다. 배양액을 성장시키고, 그 최대 15 계대 이용 될 수있다. 아래에 설명 된 방법을 통해 분리 및 차 문화의 설립은 근섬유 표현형과 간질 세포를 생성; , vimentin에 양성하고, 약한 양성 또는 최근 desmin에 음성 및 아세포 네거티브 SMA를 α. 이 표현형은 주로 멘틴 양성 식도 섬유 아세포의 표현형, 음의 α-SMA 3 근층 점막 (6)의 α-SMA 양성, 멘틴 부정적인 표현형 구별된다.

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			<td>Sigma Aldrich</td>
			<td>H6648</td>
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			<td>GIBCO, Invitrogen</td>
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			<td>Sigma-Aldrich&#160;</td>
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			<td>GIBCO, Invitrogen&#160;</td>
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			<td>Sigma-Aldrich</td>
			<td>S1876</td>
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			<td>Sigma)</td>
			<td>F42442</td>
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			<td>Roche</td>
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			<td>Corning</td>
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			<td>Sigma-Aldrich</td>
			<td>E9644</td>
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			<td>Corning</td>
			<td>&#160;25-052-Cl</td>
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			<td>Sigma Aldrich</td>
			<td>G9023</td>
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			<td>abcam</td>
			<td>ab7817</td>
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			<td>111-225-144</td>
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			<td>ebiosciences</td>
			<td>48-0319-410</td>
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			<td>ebiosciences&#160;</td>
			<td>17-0909-41&#160;</td>
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			<td>559763</td>
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			<td>BD Pharmigen</td>
			<td>2136662</td>
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			<td>Motic AE31 inverted microscope</td>
			<td>Motic AE31 inverted microscope</td>
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			<td>Nuaire Biosafety Cabinet and Incubators</td>
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			<td>Thermo Scientific</td>
			<td>177437</td>
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			<td height="20" style="height:20px;">Eppendorf Centrifuge 5810R</td>
			<td>Eppendorf Centrifuge 5810R</td>
			<td>Eppendorf Centrifuge 5810R</td>
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			<td height="20" style="height:20px;">Olympus Vacuum-Driven Filter System&#160;</td>
			<td>Genesee Scientific&#160;</td>
			<td>25-227</td>
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			<td height="20" style="height:20px;">Nikon Eclipse TE300 Fluorescent Microscope&#160;</td>
			<td>Nikon Eclipse TE300 Fluorescent Microscope (Tokyo, Japan)&#160;</td>
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			<td>Corning</td>
			<td>3516</td>
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			<td height="20" style="height:20px;">T25 Flasks&#160;</td>
			<td>TRP&#160;</td>
			<td>90026</td>
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			<td height="20" style="height:20px;">T75 Flasks&#160;</td>
			<td>Corning</td>
			<td>43064</td>
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			<td>Dissection Scissors</td>
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			<td>Dissection Forceps</td>
			<td>Dissection Forceps</td>
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			<td height="20" style="height:20px;">Single Tipped Q-Tips&#160;</td>
			<td>Kendall</td>
			<td>540500</td>
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		<tr height="20">
			<td height="20" style="height:20px;">T75 Flasks</td>
			<td>Corning</td>
			<td>43064</td>
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			<td height="20" style="height:20px;">Metamorph software (Molecular Devices)</td>
			<td>Molecular Devices</td>
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			<td height="20" style="height:20px;">FACSCAlibur&#160;</td>
			<td>BD Bioscience&#160;</td>
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			<td height="20" style="height:20px;">FACSVerse</td>
			<td>BD Bioscience</td>
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isolation of myofibroblasts from mouse and human esophagus

설치류와 인간의 식도 근육 섬유 모세포는 소화 효소를 통해 생성된다. 신생아 (8~12일 세) 쥐의 식도, 수확 다진, 세척하고, 25 분 동안 콜라게나 제와 디스 파제와 소화 효소 실시한다. 인간 식도 절제 표본 근층 고유 층과 외막의 벗겨, 나머지의 점막 다진되고, 최대 6 시간 동안 콜라게나 제와 디스 파제 및 소화 효소를 실시. 배양 된 세포는 α-SMA 및 멘틴 및 명시 최근 desmin 약하게 또는 전혀 표현한다. 배양 조건은 상피 세포, 조혈, 또는 내피 세포의 성장에 도움이되지 않습니다. 문화 순도는 또한 잠재적 오염 조혈 및 내피 세포의 세포 표면 마커의 발현을 유세포 평가에 의해 확인된다. 설명 된 기술은 간단하며 비 hematopoieitc 비 내피 간질 세포의 일관된 발생을 초래한다. 이 기술의 한계의 사용에 내재즉, 분자 생물학 연구의 주요 문화, 피할 수없는 변화는 다른 쥐 또는 인간 전반에 걸쳐 문화 사이에 발생했습니다. 그러나 차 배양 세포주에 비해 생체 상태의 대표성 반사된다. 이러한 방법은 연구자들에게 집단 간의 비교를 가능하게 분리 할 수​​있는 능력과 다른 임상 및 실험 조건에서 배양 기질 세포를 제공한다. 특성화 식도 기질 세포는 식도 상피의 질환 - 간질 상호 작용을 조사하는 연구에 사용될 수있다.

기계 및 소화 효소를 사용하여 격리 식도 기질 세포는 처음 도금 6 웰 플레이트에서 배양된다. 도금 시간 내에 거꾸로 현미경으로 세포의 시험은 판 바닥 (그림 1A)에 느슨하게 부착 혼합 세포 현탁액을 보여줍니다. 다음 24 시간에 걸쳐, 접시 바닥에 견고하게 부착 방추형 세포가 혼합 된 세포 현탁액 돋 셀 웰 오일 내에서의 전체 영역을 커버 및 형태 유지, 성공적으로 계대 수 .These <str...

Watch this Scientific Journal Video about Isolation of Myofibroblasts from Mouse and Human Esophagus at JoVE.com

Isolation of Myofibroblasts from Mouse and Human Esophagus

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

마우스 및 인간의 식도에서 근섬유 아세포의 분리

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

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Research

JoVE Journal

Biology

Isolation of Genomic DNA from Mouse Tails

마우스 뒷면에서 게놈 DNA의 분리

연구

생물학

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

사람의 피부에서 성인 상피 줄기 세포의 분리 및 문화

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

인간 Lipoaspirates에서 지방 유래 줄기 세포를 수동으로 분리

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

마우스와 인간의 대장 조직에서 주요 근육 섬유 모세포의 분리

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

마우스 저항 동맥의 미세 혈관 내피 튜브의 분리

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

성인 마우스 앞니의 치아 상피 줄기 세포의 분리 및 문화

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

인간 혈청에서 작은 비 암호화 RNA를의 분리

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

높은 처리량 마이크로 호흡 측정을위한 마우스 골격근의 최소 수량에서 미토콘드리아의 분리

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

신생아 및 성인 마우스 피부의 1 차 마우스 각질 세포의 분리 및 배양

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

성인 심장 섬유 아세포 및 근섬유 아세포의 격리 및 특성화

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...