사만다 Khoury와 파멜라 Ajuyah는 호주 석사 과정을 지원합니다. 우리는 또한 사만다 Khoury 자신의 추가 지원에 대한 번역 상 암 연구 네트워크, 로위 암 연구 센터, 뉴 사우스 웨일즈 대학과 북부 번역 상 암 연구 단위를 인정하고 싶습니다.

참고 : 건강한 환자 또는 암을 가진 환자에서 인간의 혈청 샘플, 로얄 프린스 알프레드 병원 시드니 (프로토콜 번호 X10-0016 및 HREC/10/RPAH/24) 기술 대학에서 승인 된 인간의 윤리 프로토콜에서 동의 얻어졌다 시드니. 혈청 샘플은 다양한 시드니 병원에서 수술 전 환자로부터 수집 및 -80 ° C.에 저장에 넣었다 혈청에서 1. 작은 RNA 격리 전체 RNA는 트라이 RT 시약의 LS 프로토콜의 변형 된 버전을 사용하여 인간 혈청으로부터 제조 하였다. <ol><li> 얼음에 냉동 혈청 샘플을 해동 한 다음 레이블 microcentrifuge 관에 갓 해동 혈청 400 μl를 전송합니다. </li><li> 자유롭게 된 RNase H 2 O 100 μL로 혈청을 희석하고 1 ㎎ / ㎖의 농도에서 단백질 분해 K을 추가한다. </li><li> 프로 테 K.에 의해 단백질의 소화를 할 수 있도록 20 분 동안 37 ° C에서 알을 품다 </li><li> 티오, 완전한 용해를 보장 트라이 시약 RT LS의 1.5 배의 양 및 4 - 브로 모 아니 솔의 100 μl를 추가합니다. </li><li> 간단히, 균질 반전 5 초 동안 반복 피펫 팅을 수행하고 표시된 2 ㎖ 무거운 위상 잠금 튜브로 전송할 수 있습니다. </li><li> 4 ℃에서 20 분 동안 12,000 × g에서 균질 스핀 </li><li> 정중 신선한 DNA LOBIND 튜브로 얻어진 수용액의 적어도 1 ㎖를 가만히 따르다. 유기 계면과는 위상 잠금 튜브의 흰색 겔 아래 포획한다. </li><li> 수용액에 글리코겐의 5.0 μL (5 ㎎ / ㎖) 및 100 % 이소프로판올 500 μl를 추가 반전하여 혼합하고, -20 ℃에서 O / N을 품어 </li><li> 다음 O / N 배양, 4 ° C에서 원심 분리기에서 12,000 × g에서 20 분 동안 원심 분리기 샘플. </li><li> 맑은 상층 액을 버리고 4 ° C에서 원심 분리기에서 16,000 × g에서 2 분간 &quot;플래시&quot;스핀을 수행합니다. </li><li> 조심스럽게 분명 졸을 제거피펫을 사용하여 펠렛을 주변의 ution. </li><li> 10 분 동안 10,000 × g에서 70 %의 에탄올과 원심 분리기 1 ㎖로 펠렛을 씻으십시오. 세척 솔루션을 가만히 따르다 및 세척 단계를 반복합니다. </li></ol> 솔루션에 2. RNA 재 부상 <ol><li> 펠렛이 완전히 용해 보장 된 RNase H 2 O 자유로운 10 μL에 펠렛을 재현 탁. RNA가 완전히 용해되어 있는지 확인하기 위해 샘플을 5 분 동안 55 ° C로 가열 할 수있다. 이 시간 동안 반복 피펫으로 시료를 혼합. 높은 총 RNA 수율의 경우, 동일 환자에서 두 가지 RNA 준비를 풀. </li><li> UV-VIS 분광 광도계를 사용하여 재현 탁 RNA 정량과 2100 Bioanalyzer를 사용하여 RNA의 품질을 평가합니다. 다운 스트림 응용 프로그램에 사용하기 위해 -80 ° C에서 풀링 된 RNA 샘플을 저장합니다. </li></ol>

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공개 아무것도 없다.

마이크로 RNA의 인구는 혈청에서 발견 된 총 RNA의 약 0.4 ~ 0.5 %를 구성한다. 또한 인간 혈청에서 발견 높은 단백질 함량이있다. RNA를 개선하고 단백질과 페놀 양을 줄이기 위해 우리가 여러 단계의 추가와 함께 기존의 Chomczynski 방식 9을 수정 한 오염. 우리 연구실에서 수행 할 때 전체 RNA는 표준 트라이 시약 RT-LS (분자 연구 센터)를 사용하여 혈청에서 분리 하였다 그러?...

최근, 인간의 질병의 조기 진단을위한 새로운 바이오 마커 발견 성장 밀기가 있었다. 많은 관심 잠재적 마커와 같은 마이크로 RNA 2 (의 miRNA 또는 미르)의 작은 RNA를 사용에 초점을 맞추고있다. 이러한 작은 RNA를가 혈청 및 연구 등의 체액에서 발견되는 그들이 분해에 탄력과 다양한 환경 조건 (3)의 범위에서 안정적 보여 주었다. 이러한 기능, 혈청 또는 순환의 miRNA가 이상적인 바이오 마커 4, 5 감안할. 현재 생체 액에서 작은 RNA의 분리에 대한 두 가지 접근 방법이있다. 두 번째 방법은 페놀 및 아니 디늄 티오 시아 네이트 시약 7과의 오랜 프로토콜을 사용하면서 첫 번째 방법은, 작은 RNA를 6 바인딩 및 용출 컬럼 기반 기술을 사용한다. 우리는 인간의 혈청에서 작은 RNA를 격리하는 간단한 효과, 열이없는 프로토콜을 개발했습니다. 분리 된 RNA는 즉시 주입니다DNA 올리고 뉴클레오티드의 배열과 RNA의 염기 서열을 포함하여 다운 스트림 애플리케이션에서 ately 사용할. 혈청에서 RNA를 분리하는 페놀 계 방법을 사용할 때 우리는 몇 가지 문제에 직면했기 때문에이 프로토콜이 개발되었다. 전통적인 Chomczynski 방법은 자주 대부분의 상용 공급 업체에서 제공 시약의 범위와 대부분의 실험실에서 사용된다. 그러나 그들의 광범위한 사용을 고려, 엄격한 가이드 라인은 지속적으로 특정 혈액이나 혈청, 체액에서 고품질의 RNA를 생산하기 위해 개발되지 않았습니다. 혈청에서 RNA를 분리와 관련된 일반적인 문제는 낮은 RNA 수율 및 격리, 특히 페놀 동안 사용 된 시약의 오염이 (가) 있습니다. 우리의 접근 방식은 정량 PCR (qPCR에)와 RNA 서열 분석 등의 다운 스트림 분석을위한 고품질의 RNA를 제공하기 위해 이러한 페놀 오염 물질을 제거한다. 우리는 더 miRNA의 배열에서이 RNA를 테스트했습니다.

<table border="0" cellpadding="0" cellspacing="0" style="width:852px;" width="851">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Name of the Material/Equipment&#160;</td>
			<td style="width:223px;">Company&#160;</td>
			<td style="width:123px;">Catalog Number&#160;</td>
			<td style="width:248px;">Comments/ Description (optional)&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Tri-Reagent RT LS</td>
			<td style="width:223px;">Molecular Research Centre, USA</td>
			<td style="width:123px;">TR 118</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:259px;">RNase free H20</td>
			<td style="width:223px;">GIBCO Invitrogen</td>
			<td style="width:123px;">10977-023</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Proteinase K&#160;</td>
			<td style="width:223px;">Finnzymes, Finland</td>
			<td style="width:123px;">EO0491</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Heavy Phaselock Tube</td>
			<td style="width:223px;">5PRIME</td>
			<td>2302830</td>
			<td style="width:248px;">2ml capacity</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">DNA Lobind tube</td>
			<td style="width:223px;">Eppendorf</td>
			<td style="width:123px;">0030 108.078</td>
			<td style="width:248px;">1.5ml capacity</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Glycogen&#160;</td>
			<td style="width:223px;">Invitrogen, USA</td>
			<td style="width:123px;">10814-010</td>
			<td style="width:248px;">5mg/ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">RNA grade Isopropanol&#160;</td>
			<td style="width:223px;">Sigma Aldrich, USA</td>
			<td style="width:123px;">I9516</td>
			<td style="width:248px;">100%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Refrigerated Centrifuge</td>
			<td style="width:223px;">John Morris</td>
			<td style="width:123px;">-</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Nanodrop UV-Vis spectrophotometer</td>
			<td style="width:223px;">Thermo Fisher Scientific, USA</td>
			<td style="width:123px;">-</td>
			<td style="width:248px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">RNA grade Ethanol</td>
			<td style="width:223px;">Sigma Aldrich, USA</td>
			<td style="width:123px;">E7023</td>
			<td style="width:248px;">70%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:259px;">Agilent 2100 Bioanalyser</td>
			<td style="width:223px;">Agilent, USA</td>
			<td style="width:123px;">-</td>
			<td style="width:248px;">-</td>
		</tr>
	</tbody>
</table>

isolation of small noncoding rnas from human serum

RNA와 그 표현의 분석은 많은 실험실에서 일반적인 기능입니다. 중요한 것은 포유 동물 세포에서 발견되는 마이크로 RNA와 같은 작은 RNA를의 등장입니다. 이 작은 RNA를은 성장, 발달 및 죽음과 많은 관심과 같은 중요한 경로를 제어하는​​ 강력한 유전자 규제 체액에서의 표현에 지시 된입니다. 이것은 암 혈청 바이오 마커로서의 가능성 응용 프로그램으로 인간의 질병에서의 조절 장애에 의한 것입니다. 그러나, 혈청에서의 miRNA 발현의 분석은 문제가 될 수있다. 대부분의 경우 혈청의 양을 제한하고, 혈청 작은 RNA를은 0.4 ~ 0.5 % 1을 구성하는 총 RNA의 낮은 금액을 포함합니다. 따라서 혈청에서 품질의 RNA의 충분한 양의 분리는 연구자들에게 중요한 도전 오늘입니다. 이 기술 문서에서는, 우리는 DNA 배열 또는 qPCR의 분석도 충분한 RNA를 얻기 위해 인간의 혈청 400 μl를 사용하는 방법을 보여줍니다.이 방법의 dvantages은 단순하고 고품질의 RNA를 생성 할 수있는 능력이다. 그것은 작은 RNA를 정제에 대한 전문적인 열을 필요로하지 않습니다 및 일반 실험실에있는 일반 시약 및 하드웨어를 사용합니다. 우리의 방법은 동시에 고품질의 RNA를 수득하면서 페놀 오염을 제거하기 위해 위상 잠금 젤을 이용한다. 또한, 상기 분리 단계에서 모든 오염 물질을 제거하는 추가 단계를 소개한다. 이 프로토콜은 혈청에서 최대 100 NG / μL의 총 RNA의 수율을 분리하는데 매우 효과적이지만 다른 생물 조직에 적용 할 수 있습니다.

그림 1은 혈청에서 분리 된 RNA의 일반적인 UV / 비스 스펙트럼을 나타냅니다. 이 프로필에서 우리는 각각 모두 270 ㎚, 230 ㎚에서의 페놀 및 유기 오염 물질을 280 nm에서 단백질의 오염을 지적했다. 잔류 구 아니 디늄 티오 시아 네이트는 260 nm에서 관찰되었다. 오염물을 줄이기 위해 최적화 일련의 단계는 표준 트라이 시약 RT-LS 절차로 만들어졌다. 우리는 총 RNA의 수율을 모두 증가하고 ?...

Watch this Scientific Journal Video about Isolation of Small Noncoding RNAs from Human Serum at JoVE.com

Isolation of Small Noncoding RNAs from Human Serum

이 프로토콜은 인간 혈청에서 작은 RNA를 추출하는 방법을 설명합니다. 우리는 DNA 배열에 사용하기 위해 암 혈청에서 마이크로 RNA를 분리하고 또한 singleplex 정량 PCR이 방법​​을 사용했습니다. 프로토콜은 고품질의 RNA를 수득 수정하여 페놀 및 아니 디늄 티오 시아 네이트 시약을 이용한다.

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 ...

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18.0K 조회수.  University of Technology, Sydney.  이 프로토콜은 인간 혈청에서 작은 RNA를 추출하는 방법을 설명합니다. 우리는 DNA 배열에 사용하기 위해 암 혈청에서 마이크로 RNA를 분리하고 또한 singleplex 정량 PCR이 방법​​을 사용했습니다. 프로토콜은 고품질의 RNA를 수득 수정하여 페놀 및 아니 디늄 티오 시아 네이트 시약을 이용한다. 

비디오: Isolation of Small Noncoding RNAs from Human Serum

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

인간의 사후 뇌 조직에서 핵의 연결을 격리

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

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생물학

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

인간 췌장암의 Xenografts에서 줄기 세포의 분리

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

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 ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포로부터 인간 심장 엽 성의 전구 물질과 Cardiomyocytes의 효율 유도

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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 Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

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, ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

진동절단 심근슬라이스에서 인간 심실 심근세포의 분리

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Isolation of Human Neutrophils from Whole Blood and Buffy Coats

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells recruited at the site of inflammation via rapid extravasation through vessels. There, neutrophils perform an array of functions to kill invading pathogens and mediate immune signaling. Freshly purified neutrophils from human blood are the model of choice for study, as no cell line fully replicates PMN functions and biology. However, neutrophils are short-lived, terminally differentiated cells and are highly susceptible to activation in response to physical (temperature, centrifugation speed) and biological (endotoxin, chemo- and cytokines) stimuli. Therefore, it is crucial to follow a standardized, reliable, and fast method to obtain pure and non-activated cells. This protocol presents an updated protocol combining density gradient centrifugation, red blood cell (RBC) sedimentation, and RBC lysis to obtain high PMN purity and minimize cell activation. Furthermore, methods to assess neutrophil isolation quality, viability, and purity are also discussed.

This protocol details a method for the isolation of neutrophils from whole blood, buffy coats, or leukapheresis membranes, achieving good yield, high purity, and minimal cell activation. We utilize gradient purification, red blood cell (RBC) sedimentation, and RBC lysis to obtain a high-quality/purity neutrophil preparation.

전신 혈액과 버피 코트에서 인간의 호중구의 격리

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells ...

Isolation of Epithelial Cells from Human Dental Follicle

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while &#945;-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells.
The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

The dental follicle contains an epithelial population and mesenchymal cells. The epithelial population was selected from the heterogeneous dental follicle cell population by providing a distinct culture medium. Epithelial cells survived and formed colonies in a serum-free medium.

인간 치과 여포에서 상피 세포의 격리

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was ...