1. 입체 공동 문화 구축 <ol><li> 공동 문화를 확립하기 전에 하루, 하룻밤은 4 ° C 냉장고에서 얼음 냉동실과 해동에서 Matrigel를 제거합니다. </li><li> 10% 소 송아지 혈청, 5 μg / ML 인슐린과 DME/F12 매체 : 실험 당일 사용되는 상피 세포에 대한 매체 (이 예에서는 HMEC 중간에 해당하는 공동 문화를 위해 매체를 준비 , 1 μg / ML의 하이드로코티손, 1 NG / ML EGF). 이전 Matrigel과 믹싱까지 얼음으로 최소한 1 시간마다 매체를 보관하십시오. </li><li> 해동 Matrigel을 희석하기 위해 Matrigel의 총 원하는 수량 결정 : 매체 (300 μL마다 잘 24 - 잘 접시) 및 얼음에 멸균 튜브에 얼음처럼 차가운 HMEC 매체의 절반 볼륨을 추가합니다. 그렇다면 1:1 희석을 달성하기 위해 튜브에 해동 Matrigel의 같은​​ 반 전체 볼륨을 추가합니다. 얼음 튜브를 유지. </li><li> 장소 50 Matrigel의 μL : 24 - 잘 접시의 지정된 우물의 중간 중간 잘 혼합하고humidified 공기 5 % CO 2와 37 ° C 배양기에서 10 분 동안 품어. </li><li> 우물에 매체를 혼합하여 다른 60 분 품어 : Matrigel의 추가 450 μL를 추가합니다. </li><li> 인큐베이션 기간 동안, 0.5 × 10의 농도에서 섬유아 세포 (여기서 GFP를 표현 H-TERT-불후의 감소 Mammary 섬유아 세포)과 상피 세포 (여기서 RFP을 표현 H-TERT-불후의 인간 Mammary 상피 세포주)의 1:1 정지를 준비 5 세포 HMEC 매체의 0.5 ML 각. </li><li> 60 분 인큐베이션 단계 후에 부드럽게 경화 젤 상단에 세포 현탁액을 떨어뜨, 그리고 보육에 문화를 반환합니다. </li><li> 2 일간 문화를 방해하지 마십시오. 이렇게하면 중간 아주 부드럽게 잘 부드럽게 신선한 HMEC 매체와 교체의 측면에서 매체를 aspirating하여 2~3일마다 변경할 수 있습니다. </li><li> 현미경 하에서 일상 회전 타원체 형성을 모니터링합니다. 문화는 10-14일 후 일반적으로 성숙입니다. 이미지로빛 또는 형광 현미경을 사용하여 적절한. 회전 타원체의 크기와 투영 길이 광 현미경 하에서 측정할 수 있습니다. </li></ol> 2. 공동 문화권 회전 타원체로부터 세포의 분리 <ol><li> 멸균 2-ML 유리 피펫을 사용하여 매우 부드럽게, 문화를 피펫 아래 10X. 또는 문화를 피펫하기 위하여 70 % 에탄올에 autoclaving 또는 침지 소독 가위를 사용하여 1000 microliter 플라스틱 피펫 팁의 끝부분을 잘라. 유리 피펫 또는 멸균 15 ML의 원심 관에 컷 플라스틱 피펫 팁 및 실온에서 2,000 rpm으로 5 분 원심 중과 문화를 전송합니다. 세포 spheroids은 침전 하단 것입니다. </li><li> 부드럽게 centrifuged 튜브의 상단에서 중단 Matrigel을 제거 멸균 파스퇴르 피펫 팁을 사용합니다. </li><li> pelleted spheroids 1 ML HMEC 매체를 추가, 최대 피펫 아래로 3 배를 멸균이 ML 유리 피펫으로, 그리고 24 우물에 잘으로 문화를 전송조직 배양 플레이트. </li><li> spheroids이 매체를 변경하기 전에 최소 48 시간 동안 접시에 연결하도록 허용합니다. 시간이지나면서 세포가 회전 타원체의 구조를 떠날 것이며 monolayers로 성장. </li><li> 세포가 50 % 합류점에 도달 통과 세포 문화. 첫 100 밀리미터 접시 (약 3~5일)에 나서 35 밀리미터 접시 (약 2-4일)에 문화를 확장합니다. 각 통로 단계로 바로 전에 수동으로 가능한 많은 잔류 &#39;유령 spheroids &quot;로 제거하는 멸균 파스퇴르 피펫을 사용합니다. </li><li> 형광 셀 정렬 필요한 경우가 적절한 세포 표면 마커 더 나아가 별도의 서로 다른 세포 인구를 사용하는 인구의 순도를위한 유동세포계측법으로 세포를 분석합니다. 원하는대로 분리된 세포는 추가 분석을 위해 지금 준비가되어 있습니다. </li></ol> 3. 대표 결과 공동 문화 입체 회전 타원체에서는 공학 Tiam1 입을와 섬유아 세포는 mA로 증가 침공을 유발mammary 상피 세포에 의해 trix (그림 1, D를 비교, E에 F, B, C). 와 섬유아 세포는 Tiam1 이상의 표현이 유방암 세포주 SUM159 (그림 1, G, H, I로 J, K, L을 비교)에 의해 감소 매트릭스 침공을 유도 설계. 그림 2와 같이 일단 공동 교양 spheroids가 설립되어, 세포 인구는 2 차원 맥락에서 다시 도금에 의한 spheroids으로부터 격리 수 있습니다. 테스트 세포의 상대적인 성장 속도에 따라 순수 인구는 시리얼 통로를 통해 등장할 수있다 (그림 3 참조) 또는 적절한 세포 마커를 사용하여 정렬할 수 있습니다. 공동의 문화 차원에서 격리 후, 상피 세포 분석을위한 충분한 기회를있게 장시간 동안 섬유아 세포와 공동으로 문화 수여 속성을 유지합니다. 이 예제에서는 Tiam1 - 결함 섬유아 세포에 노출이 주 동안 지속된 공동 교양 HMECs 증가 마이 그 레이션을 유도3 차원의 공동 문화의 fter 절연 (그림 4). <img alt="그림 1" src="/files/ftp_upload/3760/3760fig1.jpg" /> 그림 1. 빛과 형광 현미경 하에서 공동 문화 확립 회전 타원체의 이미징. 회전 타원체 문화가 mammary 상피와 fibroblast 세포 라인과 함께 설립하여 10-14일 위해 문화에 성장할 수있게됩니다. AF : 상피 = HMEC-mCherry 전지. GL : 상피 세포 = SUM159-mCherry 전지. AC, GI : fibroblast = 컨트롤 RMF-GFP 세포. DF : fibroblast = RMF-GFP Tiam1 입을 가진. JL : fibroblast = RMF-GFP Tiam1 넘는 표현으로. 화살표는 매트릭스에 침입하는 상피 전망을 나타냅니다. <img alt="그림 2" src="/files/ftp_upload/3760/3760fig2.jpg" /> 그림 2. Matrigel 문화로부터 격리 후 빛과 형광 현미경 하에서 문화 시리얼 이미징. 데이 0 즉시 격리 후 이미징 나타내고 일 1, 2 및5 고립 후 일을 나타냅니다. 시간이 지남에 따라 회전 타원체 증가를 떠나는 세포의 코로나. 결국 거의 세포와 &quot;귀신&quot;회전 타원체는 (화살표) 남아 있습니다. 거의 spheroids 귀신 제거하고 처음 통과 후 남아 있습니다. <img alt="그림 3" src="/files/ftp_upload/3760/3760fig3.jpg" /> Matrigel 공동의 문화로부터 고립 후 세포의 그림 3. 유동세포계측법 분석. 공동 문화권 회전 타원체가 성숙되면 세포는 pipetting과 공동 문화 3D 및 passaging 시리얼으로부터 격리됩니다. 샘플 aliquots가 유동세포계측법에 의해 분석된다 (식별하는 녹색 형광과 붉은 형광을 사용하여이 예제에서 GFP를 - 표현하는 각각 섬유아 세포와 mCherry-표현 상피 세포) 상피와 fibroblast 세포의 상대 인구를 결정합니다. <img alt="그림 4" src="/files/ftp_upload/3760/3760fig4.jpg" /> 그림 4. HMECs의 Transwell 마이 그 레이션 isolati 후에도Matrigel 공동 문화부터. 순수 인구가 문화 또는 형광 셀 정렬을 통해 얻은되고 나면 원하는대로, 세포 분석 할 수 있습니다. 여기 HMECs 중 제어 (C) 또는 천공 transwells 걸쳐 Tiam1 결함 (shT) 섬유아 세포와 공동 교양의 마이 그 레이션은 공동의 문화로부터 고립 후 2, 4 및 8 주가되면 평가되었다.

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관심의 어떠한 충돌 선언 없습니다.

여기에 제시된 방법은 종양 세포를 포함한 상피 세포에 stromal 섬유아 세포의 효과를 분석에 대한 간단한 접근 방식을 나타냅니다. 추가 분석을 위해 모체의 세포는 쉽게 추출을 가능하게하면서 그것은 세포외 지하 멤브레인 매트릭스에서 지원하는 입체적인 맥락 내에서 직접적인 세포 - 세포 상호 작용을 허용합니다. 이것은 fibroblast 라인은 선택과 상피 라인은 그림 1에서와 같이 침?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> BD Matrigel </td><td> BD Biosciences </td><td> 356,237 </td><td> 무료 페놀 - 레드 </td></tr><tr><td> Hyclone 고전 액체 매체 : DMEM F12 </td><td> ThermoScientific </td><td> SH30023.01 </td><td></td></tr><tr><td> Hyclone 소의 송아지 고기 세럼 </td><td> ThermoScientific </td><td> SH3007303 </td><td></td></tr><tr><td> 인슐린 </td><td> EMD 화학 </td><td> 407,709 </td><td> 0.005N HCL에 디졸브, 0.22 μm의 필터 </td></tr><tr><td> 하이드로코티손 </td><td> 시그마 - 올드 리치 </td><td> H4001 </td><td> 50% EtOH에 디졸브, 0.22 μm의 필터 </td></tr><tr><td> EGF </td><td> 시그마 - 올드 리치 </td><td> E9644 </td><td> 10mM 초산에 용해, 필터는 0.22 μm의를 </td></tr></tbody></table>

isolation of mammary epithelial cells from three-dimensional mixed-cell spheroid co-culture

거대한 노력은 쉬운 세포 조작을 허용 고전적인 2 차원 세포 배양 모델을 사용하여 많은이 작품의이 완료되어 정상 및 악성 세포 행동 1-2,,에 관련된 신호 전달 경로와 분자를 식별하고 있진 않지만. 그것은 세포 내 신호 전달 경로는 차원 및 3-4 세포 표면 장력을 포함한 세포외 세력에 의해 영향을받는 것이 분명되었다. 다중 접근 방식은 더 정확하게 생물 학적 조직 구조 3 대표하는 입체적인 모델을 개발하기 위해 촬영되었습니다. 이러한 모델은 다중 차원 건축 스트레스를 통합할 수 있지만, 세포의 논리상 필연의 효과 연구 모형의 한계 및 후속 분석을 위해 세포를 추출의 어려움으로 인해 2 차원 조직 배양에보다 쉽게​​ 이해되는 것입니다. tumorigenesis 및 종양 행동 종양 주위 microenvironment의 중요한 역할 I의 점점 4 인정되고. 종양 기질은 여러 세포 유형 및 세포 분자로 구성되어 있습니다. 종양이 개발 중에 종양 세포 stromal 세포 5 사이의 양방향 신호가 없습니다. 종양 - 기질 공동 진화에 참여하는 몇 가지 요소가 확인되어 있지만, 체계적으로 파악하고 이러한 신호 6 전체 배열을 공부하는 간단한 기술을 개발할 필요가 여전히있다. 섬유아 세포는 정상이나 종양 - 관련 stromal 조직에서 가장 풍부한 셀 타입이며, 증착 및 지하 막과 paracrine 성장 요인 7의 유지에 기여한다. 많은 그룹은 종양 치료에 대한 반응, 면역 세포의 채용, 신호 전달 분자, 증식, apoptosis, angiogenesis, 그리고 침공 8-15 등 다양한 세포 기능에 섬유아 세포의 역할을 공부하고 입체 문화 시스템을 사용해 왔습니다. 우리는 엉덩이에 대한 간단한 방법을 최적화했습니다혼합 세포 인구 (공동 문화) 16-22의 입체 문화를 만드는 상용 세포외 매트릭스 모델을 사용 mammary 상피 세포에 mammary 섬유아 세포의 효과를 essing. 지속적인 공동 문화와 세포 내부에있는 섬유아 세포 클러스터링과 크게 spheroids의 외관에와 매트릭스로 멀티 셀룰러 전망을 형성 상피 세포와 spheroids를 형성하고 있습니다. 변경된 상피 세포 invasiveness로 연결 섬유아 세포의 조작이 쉽게 숫자와 상피 전망 23 길이의 변화에 의해 계량하실 수 있습니다. 더욱이, 우리는 상피 행위에 대한 fibroblast 노출의 효과 분석을 용이하게 입체 공동 문화 상피 세포를 분리하는 방법을 고안했습니다. 우리는 공동 문화의 영향이 여러 assays를 수행하기 위해 충분한 시간을 허용, 상피 세포 격리 후 몇 주 동안 지속 것으로 나타났습니다. 이 방법의 세포에 대한 적응악성 잠재력을 변화하고하는 것은 전문적인 장비를 필요하지 않습니다. 이 기술은 여러 조건 하에서 체외 세포 모델의 급속한 평가를 허용하고, 해당 결과는 생체내 동물 조직 모델뿐만 아니라 인간의 조직 샘플에서 비교할 수 있습니다.

Watch this Scientific Journal Video about Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture at JoVE.com

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

간단한 방법은 관련 상피 세포에서 조직 섬유아 세포의 효과를 분석하는 설명되어 있습니다. 이 방법과 3 차원 조직 배양의 결합 차원에서 고립 후 세포의 분석을 용이하게하실 수 있습니다. 기술은 종양 세포에서 종양 - 관련 기질의 효과를 체계적으로 연구를 허용, 악성 잠재력을 변화의 세포에 적용됩니다.

세 가지 차원 혼합 셀 회전 타원체 공동 문화에서 Mammary 상피 세포의 분리

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

isolation-mammary-epithelial-cells-from-three-dimensional-mixed-cell

Research

JoVE Journal

Biology

16.0K 조회수.  Tufts Medical Center. 간단한 방법은 관련 상피 세포에서 조직 섬유아 세포의 효과를 분석하는 설명되어 있습니다. 이 방법과 3 차원 조직 배양의 결합 차원에서 고립 후 세포의 분석을 용이하게하실 수 있습니다. 기술은 종양 세포에서 종양 - 관련 기질의 효과를 체계적으로 연구를 허용, 악성 잠재력을 변화의 세포에 적용됩니다.

비디오: Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

마우스 내유 상피 세포는 Lactogenic 차별 동안 Mammospheres를 형성

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

연구

생물학

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Microfluidic 가용성식이 중재 신호의 상호 작용을 조사하는 상피 세포 박테리아의 공동 문화

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

내유 상피 이식 절차

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

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 ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA 정제에 의한 염색질 절연 (처프)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

간엽 전환을 유도하고 상피의 분석

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

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 ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

3차원 공동배양시스템을 이용한 난소암 줄기유사세포의 혈관신생적 특성 평가 NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and disease modeling, drug discovery, and regenerative medicine. To fully exploit these models, it is crucial to study them at cellular and subcellular levels. However, characterizing such in vitro 3D cell culture models can be technically challenging and requires specific expertise to perform effective analyses. Here, this paper provides detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed in vitro 3D cell culture models ranging from 100 &#181;m to several millimeters. These protocols are applicable to a wide variety of organoids and spheroids that differ in their cell-of-origin, morphology, and culture conditions. From 3D structure harvesting to image analysis, these protocols can be completed within 4-5 days. Briefly, 3D structures are collected, fixed, and can then be processed either through paraffin-embedding and histological/immunohistochemical staining, or directly immunolabeled and prepared for optical clearing and 3D reconstruction (200 &#181;m depth) by confocal microscopy.

Here, we provide detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed three-dimensional cell culture models ranging from 100 &#181;m to several millimeters, thus enabling the visualization of their morphology, cell-type composition, and interactions.

3차원 오가노이드 및 스페로이드 모델의 염색 및 고해상도 이미징

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and ...

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 ...