1. Üç boyutlu Co-kültür oluşturulması <ol><li> Co-kültürler kuran bir gün önce, bir gecede 4 ° C&#39;de buzdolabında buz üzerinde dondurucu ve çözülme gelen Matrigel çıkarın. </li><li> % 10 bovin serumu, 5 ug / mL insülin ile DME/F12 orta: deney gününde, kullanılmak üzere epitelyal hücreleri için orta (bu örnekte HMEC orta karşılık gelen ortak kültürü için orta hazırlanması , 1 ug / mL hidrokortizon ve 1 ng / mL EGF). Önceden Matrigel ile karıştırılarak buz en az 1 saat orta tutun. </li><li> Çözülmüş Matrigel sulandırmak için, Matrigel toplam istenilen miktar belirlemek: orta (300 uL başına da 24-iyi plakalar için) ve buz üzerinde steril bir tüpe buz HMEC orta yarısı bu birimde ekleyin. Daha sonra bir 1:1 seyreltme elde etmek için tüpüne çözülmüş Matrigel aynı yarı-toplam hacim ekleyin. Buz tüpü tutun. </li><li> Yeri 50 Matrigel arasında uL: 24-kuyulu plakanın belirlenmiş kuyuların orta-iyi orta karışımı venemli hava ve% 5 CO2 içeren bir 37 ° C inkübatöründe 10 dakika süreyle inkübe edilir. </li><li> Kuyuları, orta ve başka bir karışımı 60 dakika süreyle inkübe edilir: Matrigel ilave bir 450 uL ekleyin. </li><li> Kuluçka sırasında, 0.5 x 10 arasında derişimde fibroblast (burada GFP ifade s-tert-ölümsüzleştirilmiş İndirgeme Meme fibroblastlar) ve epitel hücreleri (burada RFP ifade s-tert-ölümsüzleştirilmiş insan meme epitel hücre çizgisi) 1:1 lik bir süspansiyon hazırlamak 5 hücreleri HMEC orta 0.5 ml her. </li><li> 60 dakikalık inkübasyon adım sonra, yavaşça katılaşmış jel üstüne hücre süspansiyonu bırakın ve inkübatör kültür dönün. </li><li> 2 gün boyunca kültürlerin rahatsız etmeyin. Bundan sonra, orta nazikçe iyi ve yavaşça taze HMEC orta yerine bir taraftan orta aspire edilerek 2-3 gün her değiştirilebilir. </li><li> Mikroskobu altında günlük sfero oluşumu izleyin. Kültürler 10-14 gün sonra genellikle olgun. Görüntü olarakışık veya floresan mikroskobu kullanarak uygun. Sfero boyutunu ve projeksiyon uzunluk ışık mikroskobu ile ölçülebilir. </li></ol> 2. Co-kültürler Sfero gelen Hücre İzolasyonu <ol><li> Steril 2 ml&#39;lik cam pipet ile çok nazikçe, kültürünü Pipeti ve aşağı 10x. Alternatif olarak, kültür Pipeti için% 70 etanol içinde otoklav veya daldırma ile steril makas kullanırken, 1000 mikrolitre plastik pipet ucunu kesti. Cam pipet veya steril bir 15 ml&#39;lik bir santrifüj tüpüne kesme plastik pipet ucu ve oda sıcaklığında 2000 rpm&#39;de 5 dakika boyunca santrifüj ile ya kültürü transfer edin. Hücresel sferoidler tortu altında olacak. </li><li> Yavaşça, santrifüj tüpünün üst kesintiye Matrigel kaldırmak için steril bir Pastör pipet kullanın. </li><li> Pelet sferoidler 1 ml HMEC orta ekle, yukarı ve aşağı pipetleme 2-3 kez steril 2 ml&#39;lik cam pipet ile, ve 24-iyi bir kuyuya kültürü aktarmakdoku kültürü plakası. </li><li> Sferoidler orta değişen önce en az 48 saat çanak eklemek için izin ver. Zamanla hücreler küresel yapılar terk edecek ve mono tabakaları gibi büyür. </li><li> Hücrelerinin% 50 izdiham ulaşmak Pasajı hücre kültürleri. İlk 100 mm çanak (yaklaşık 3-5 gün) sonra bir 35 mm çanak (yaklaşık 2-4 gün) kültür genişletin ve. Her geçiş adımı hemen önce, elle mümkün olduğunca çok sayıda kalıntı &quot;hayalet sferoidler&quot; olarak kaldırmak için steril bir Pastör pipet kullanın. </li><li> Floresans hücre sıralama gerekirse, uygun hücre yüzey belirteçleri ve daha fazla ayrı ayrı hücre popülasyonu kullanarak nüfus saflık için akım sitometri ile hücrelerin analiz edin. Istediğiniz gibi izole hücreler daha fazla analiz için şimdi hazır. </li></ol> 3. Temsilcisi Sonuçlar Co-kültürler üç boyutlu sfero olarak, mühendislik Tiam1 susturulması ile fibroblastlar ma içine arttı işgali nedenmeme epitel hücreleri tarafından trix (Şekil 1, D karşılaştırmak, E, A, F, B, C). Ile fibroblast Tiam1 aşırı ekspresyonu meme kanseri hücre hattı SUM159 (Şekil 1, G, H, I J, K, L karşılaştırın) azalarak matris işgaline neden mühendislik. Şekil 2 de gösterildiği gibi bir kez co-kültüre sferoidler kurulmuş, hücresi grupları, iki boyutlu bir bağlamda yeniden kaplanması ile sferoidler izole edilebilir. Test edilen hücrelerin nispi büyüme oranları bağlı olarak, saf popülasyonlarının seri kanalı yoluyla ortaya çıkabilir (Şekil 3 içinde gösterildiği gibi) veya uygun bir hücre belirteçler kullanılarak sıralanabilir. Ko-kültür 3D izolasyon sonra, epitel hücreleri analiz için yeterli fırsat sağlayan, uzun süre üzerinde fibroblastlar ile ko-kültür öngördüğü özelliklerini korurlar. Bu örnekte, Tiam1 eksikliği fibroblastlar maruz hafta süreyle devam co-kültürlü HMECs artan göç indüklenen bir3D ko-kültür arası fter izolasyonu (Şekil 4). <img alt="Şekil 1" src="/files/ftp_upload/3760/3760fig1.jpg" /> Şekil 1. Işık ve floresan mikroskobu altında ortak kültürlerin kurulan sfero Görüntüleme. Sfero kültürleri meme epitel ve fibroblast hücre hatları ile kurulan ve 10-14 gün boyunca kültür büyümeye izin verilir. AF: epitel = HMEC-mCherry hücreler. GL: epitel hücreleri = SUM159-mCherry hücreler. AC, GI: fibroblast = kontrolü RMF-GFP hücreler. DF: fibroblast = RMF-GFP Tiam1 susturulması ile. JL: fibroblast = RMF-GFP Tiam1 fazla ifade ile. Oklar matris içine işgalci epitel projeksiyonları göstermektedir. <img alt="Şekil 2" src="/files/ftp_upload/3760/3760fig2.jpg" /> Şekil 2. Matrigel kültürden izolasyonu sonra ışık ve floresan mikroskobu altında kültürlerin Seri görüntüleme. Gün 0 hemen sonra izole görüntüleme temsil eder; günde 1, 2 ve5 izolasyon gün sonra göstermektedir. Zamanla sfero artar bırakarak hücrelerin korona. Sonunda çok az sayıda hücre ile bir &quot;hayalet&quot; sfero (ok) kalır. Çok az sferoidler hayalet kaldırılması ve ilk geçişini sonra kalır. <img alt="Şekil 3" src="/files/ftp_upload/3760/3760fig3.jpg" /> Matrigel co-kültürden sonra izole hücrelerin Şekil 3. Akış sitometrisi analizi. Co-kültürler steoid olgunlaşmış sonra, hücreler pipetleme ile eş-kültürü 3D ve pasajlanmasını seri izole edilir. Örnek alikotları akım sitometri ile analiz edilir (tanımlamak için yeşil floresan ve kırmızı floresan kullanarak bu örnekte GFP-ifade sırasıyla fibroblast ve mCherry-salgılayan epitel hücreleri) epitel ve fibroblast hücrelerin göreceli nüfusu belirlemek için. <img alt="Şekil 4" src="/files/ftp_upload/3760/3760fig4.jpg" /> Şekil 4. HMECs arasında Transwell göç isolati sonra devamMatrigel ko-kültür itibaren. Saf popülasyonlarının kültür veya floresans hücre ayırma ile elde edilmiş bir kez istenildiği gibi, hücreler analiz edilebilir. İşte HMECs ya kontrol (C) veya delikli transwells arasında Tiam1 eksikliği (SHT) fibroblastlar ile ortak kültür göçü ko-kültür izolasyonu sonrası 2, 4 ve 8. haftalarda değerlendirildi.

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Çıkar çatışması ilan etti.

Burada anlatılan yöntem tümör hücreleri dahil olmak üzere, epitel hücreler, üzerine stromal fibroblastlann etkileri analiz için basit bir yaklaşım temsil eder. Daha fazla analiz için matristen hücrelerin kolaylıkla ekstraksiyon sağlarken Bu, hücre dışı bazal membran matris tarafından desteklenen bir üç-boyutlu bağlamında doğrudan hücre-hücre etkileşim için olanak sağlar. Bu fibroblast hatları tercih ve epiteliyal hatları Şekil 1 &#39;de gösterildiği gibi, invaziv potan...

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> BD Matrigel </td><td> BD Biosciences </td><td> 356237 </td><td> Ücretsiz Fenol-kırmızı </td></tr><tr><td> Hyclone Klasik Sıvı Orta: DMEM F12 </td><td> ThermoScientific </td><td> SH30023.01 </td><td></td></tr><tr><td> Hyclone Sığır Buzağı Serumu </td><td> ThermoScientific </td><td> SH3007303 </td><td></td></tr><tr><td> Ensülin </td><td> Merck Kimyasallar </td><td> 407709 </td><td> 0.005N HCI içinde çözülür; 0.22 uM filtresi </td></tr><tr><td> Hidrokortizon </td><td> Sigma-Aldrich </td><td> H4001 </td><td> % 50 EtOH içinde çözünmesi; 0.22 um bir filtreden </td></tr><tr><td> EGF </td><td> Sigma-Aldrich </td><td> E9644 </td><td> 10 mM asetik asit içinde çözülür; filtre 0.22 uM </td></tr></tbody></table>

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

Muazzam çabaları kolay hücre manipülasyonu için izin klasik iki boyutlu hücre kültür modelleri kullanılarak çok bu işin yapılmıştır normal ve malign hücre davranışları 1-2, yer sinyal yolları ve moleküller girmiş iken. Bu hücre içi sinyal yollarının boyutluluğu ve 3-4 hücre yüzey gerilimi gibi ekstraselüler güçleri tarafından etkilendiğini açıkça ortaya çıktı. Çoklu yaklaşımlar daha doğru biyolojik mimari doku 3 temsil ettiği üç boyutlu modeller geliştirmek için alınmıştır. Bu modeller çok boyutluluğu ve mimari gerilmeler dahil ederken, hücrelere sonuç etkileri çalışma modellerinin sınırlamaları ve sonraki analiz için hücre ayıklanması zorluk nedeniyle iki boyutlu doku kültürü daha az becerikli olduğunu. Tümörogenez ve tümör davranış tümörlerin yaklaşık mikroçevresinin önemli rol ilar giderek 4 tanınmış oldu. Tümör stromanın birden fazla hücre türleri ve hücre dışı moleküller oluşur. Tümör gelişimi sırasında tümör hücreleri ve stromal hücreleri 5 arasında çift yönlü sinyaller vardır. Tümör stromanın co-evrimi katılan bazı faktörler tespit edilmiş olmasına rağmen, sistematik olarak belirlemek ve bu sinyalleri 6 tam diziyi incelemek için basit bir teknik geliştirmek için hala gereksinim vardır. Fibroblastlar normal veya tümörle ilgili stromal dokularda en bol hücre tipi olup, birikimi ve bazal membran ve parakrin büyüme faktörleri 7 bakım katkıda bulunmaktadır. Birçok grup tümör tedavilere yanıt, bağışıklık hücrelerinin işe alma, sinyal molekülleri, proliferasyon, apoptoz, anjiyogenez ve işgali 8-15 gibi çeşitli hücresel fonksiyonları üzerinde fibroblast rolünü incelemek için üç boyutlu kültür sistemleri kullanılmıştır. Biz eşek için basit bir yöntem optimizekarışık hücre popülasyonlarının (co-kültür) 16-22 üç boyutlu kültür oluşturmak için piyasada bulunan hücre dışı matriks modeli kullanılarak meme epitel hücreleri üzerinde meme fibroblastların etkileri Essing. Devam ko-kültür ile hücrelerin iç fibroblastlar kümeleme ve büyük ölçüde parçacıklarının dış ve matriks içine çok hücreli çıkıntılar oluşturan epitel hücreleri ile sferoidler oluştururlar. Değişmiş epitel hücre invaziv yol açar fibroblastların Manipülasyon kolayca numaraları ve epitel projeksiyonları 23 uzunluğu değişiklikler ile belirlenebilir. Dahası, epitel davranışı üzerindeki fibroblast maruz etkileri analizi kolaylaştırır, üç boyutlu bir ko-kültür epitelyal hücreleri izole etmek için bir yöntem geliştirmişlerdir. Biz ortak kültürün etkileri birden fazla testleri gerçekleştirmek için yeterli zaman tanıyarak, epitel hücre izolasyonu haftalar sonra devam bulduk. Bu metod ile hücrelerin uyarlanabilirmalign potansiyeli değişen ve hiçbir özel ekipman gerektirir. Bu teknik birden koşullar altında in vitro hücre modellerinde hızlı değerlendirme için izin verir, ve karşılık gelen sonuçları in vivo hayvan doku modelleri yanı sıra, insan doku örneklerine göre olabilir.

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

Basit bir metodu ile ilişkili epitel hücreleri üzerindeki doku fibroblastlann etkileri analiz etmek için tarif edilmiştir. Bu yöntem, üç boyutlu bir doku kültürü kombinasyonu 3D izole sonra hücrelerin analizi kolaylaştırabilir. Tekniği tümör hücreleri üzerindeki tümör-stroma etkileri ile ilişkili sistematik çalışma izin veren, habis potansiyeli değişen hücreleri için de geçerlidir.

Üç boyutlu Karışık-hücre Sfero Co-kültürden Meme Epitel Hücrelerinin İzolasyonu

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

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Biology

16.0K Görüntüleme.  Tufts Medical Center. Basit bir metodu ile ilişkili epitel hücreleri üzerindeki doku fibroblastlann etkileri analiz etmek için tarif edilmiştir. Bu yöntem, üç boyutlu bir doku kültürü kombinasyonu 3D izole sonra hücrelerin analizi kolaylaştırabilir. Tekniği tümör hücreleri üzerindeki tümör-stroma etkileri ile ilişkili sistematik çalışma izin veren, habis potansiyeli değişen hücreleri için de geçerlidir.

Video: 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.

Fare Meme Epitel Hücreleri lactogenic Farklılaşma sırasında Mammospheres formu

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

Biyoloji

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.

Çözünür Sinyal aracılı Etkileşimleri Araştırılması için Epitel Hücreleri ve Bakteriler mikroakışkan Co-kültür

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.

Meme Epitel Nakli Prosedürü

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.

İnsan Cilt Yetişkin Epitel Kök Hücre İzolasyonu ve Kültür

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 Saflaştırma tarafından Kromatin İzolasyon (chirp)

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.

Mezenkimal Geçiş indüksiyon ve Epitel Analizi

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.

Yetişkin Fare insizör Diş Epitel Kök Hücreleri İzolasyon ve Kültür

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.

Üç Boyutlu Eş-Kültür Sistemi Kullanılarak Yumurtalık Kanseri Kök Benzeri Hücrelerin Anjiyogenetik Özelliklerinin Değerlendirilmesi, 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.

Üç Boyutlu Organoid ve Küresel Modellerin Boyama ve Yüksek Çözünürlüklü Görüntülenmesi

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.

Epitel Hücrelerinin İnsan Diş Foliküllerinden İzolasyonu

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