Biz tartışma, teknik yardım ve el yazması üzerinde editoryal yardım için Haruya Ohno, Kosaku Shinoda, Louis Sharp, Emi Tomoda ve Lauren Ruiz ederim. Bu çalışma Atılım Biyomedikal Araştırma ve Asubio Ecz Inc için Programı SKULA için NIH (DK087853), hibe Kopenhag ve AB 7.ÇP Proje Diabat (SAĞLIK-F2 Üniversitesi&#39;nden HİSSE doktora bursları ile desteklenmiştir tarafından desteklenmiştir Lise Madsen ve Karsten Kristiansen için -2.011-278373). Biz de DERC merkezi hibe (NIH P30 DK063720) kabul etmektesiniz.

1. Sindirim Orta hazırlayın Doku başına 5 fare (yaklaşık 1 ml / 1 g yağ dokusu) başına 5 ml olun. <ol><li> Sindirim enzimleri tartılır: - Collaginase D: 1.5 u / ml (1108874103, 1 g, Roche, 70334223) - Dispase II: 2.4 u / ml (04942078001, 0,980 mg / lyo, Roche, 11466200) </li><li> 25 ml PBS ekleyin ve çözünmesi için iyice karıştırın </li><li> 10 mM nihai bir konsantrasyonda doku sindirim hemen önce CaCI2 eklemek </li></ol> 2. Fare dan Yağ Dokusu teşrih <ol><li> Diseksiyon çok dikkatli yapılmalıdır, ve hemen fareler sonra (6-8 haftalık) kurban edilir. Açmadan önce fare kürk üzerine püskürtülür etanol ile steril çalışın. Temiz PBS, ayrı interskapular BAT ve subkutan kasık WAT doğrudan doku yerleştirin. </li><li> Interskapular BAT için: kadar karşı karşıya arka yerleştirilen fare sırt ve boyun için tüm yol boyunca açık kesti. Kahverengiadipoz doku doğru omuzlar (interskapular) arasındaki deri altında bulunabilir. BAT iki lobu, şeklindeki kelebek olarak görülebilir. BAT kapsayan beyaz yağ ince bir tabaka dikkatlice çıkarılması gerekmektedir. </li><li> Subkutan WAT (inguinal WAT) için: cilt kaldırmak. Kasık WAT içinde testis karşı uyluk ve aşağı, hemen hemen arkasında ve yanında başlangıç ​​olacak, her iki tarafta da deri altına direkt olarak bulunur. </li></ol> 3. Yağ Dokusu Kes ve Digest <ol><li> Çabuk saç, iskelet kası ve doku kesitlerinde gelen bağ dokusu gibi, tüm kontaminasyonu temizlemek. </li><li> PBS ile diseksiyon orta sulandırmak ve kuru bir tabağa yerleştirin değil kağıt üzerinde hızla doku kurulayın. </li><li> Sindirim orta (ön sindirime CaCI2 eklenir) ve küçük parçalar halinde doku kıyma ekleyin. 5 ml&#39;lik pipet ve aşağı yukarı pipetleme tarafından çok iyi karıştırın. Sindirim m geri kalanı ile 50 ml tüp içinde kıyılmış doku aktarınedium, aşağı yukarı pipetleme ve daha da karıştırın. </li><li> Digest 37 ° C 40-50 dakika için 150 rpm&#39;de sabit ajitasyon. Sindirim iyi gidiyor ve aşırı-sindirim önlemek için emin olmak için her 10-15 dakika kontrol edin. </li></ol> Not: Doğru sindirim, orta ve zaman önemlidir. Doku de sindirilmesi gerekir, ama aşırı sindirim hücrelerine zarar verebilir. 4. Filtre Hücre Süspansiyon <ol><li> 5 ml tam orta (DMEM/F12 içeren% 10 FPS ve P / S) ekleyerek sindirim durdurun. Hücreler hemen hemen tamamen homojen olması gerekir. Pipetleme ile iyice karıştırın. </li><li> 10 dakika boyunca 700 xg&#39;de santrifüjleyin. </li><li> SVF şu anda, tüpün altına bir kahverengimsi bir pelet olarak görülebilir. Üst ve sıvı tabaka çoğu üzerinde yağlı olgun adiposit tabaka aspire - pelet tutmak ve 10 ml tam bir ortam içinde çözülür. İyice karıştırın. </li><li> Yeni bir 50 üzerinde bir hücre süzgeç (50-70 mikron çaplı) yerleştirin<html> ml tüp ve hücre süspansiyonu filtre. </li></ol> 5. Plaka Hücreler <ol><li> 10 dakika boyunca 700 xg&#39;de 15 ml tüp ve santrifüj hücre süspansiyonu aktarın. </li><li> 10 ml tam bir ortam içinde ortam aspire ve yeniden askıya pelet. Pipet ve iyi karıştırın. </li><li> Ihtiyaç kaç tabak tahmin. Bu değişiklik ve topak boyutuna bağlı olarak değişebilir. Genel bir kural inguinal WAT için 5 fareler ve BAT için bir 10 cm plaka 10 cm tabak 2&#39;dir. </li><li> Tam orta kollajen kaplı yemekleri Tabak hücreleri (Ar-Ge) </li><li> 1-2 saat hücreleri, aspirat orta kaplama sonra, iki kez PBS ile yıkayın ve taze orta ekleyin. Bu kırmızı kan hücreleri, bağışıklık hücreleri ve diğer kirleticiler kaldırmak bu adım önemlidir. </li></ol> 6. Hücreler farklılaştırın <ol><li> Bakım ve indüksiyon ortamlar olun. </li></ol> Bakım orta Eklenen orta tamamlayın: ent &quot;&gt; İnsülin, nihai kons. 5 ug / ml (5 mg / ml stok, pH 2.5 asitlendirilmiş su hazırlamak için 10 ml H2O içindeki asetik asit *** 100 ul. asitlendirilmiş su içinde insülin çözülür. deposu stok itibariyle -20 ° C) 3,3 &#39;,5 triiodo-L-thyronine (T3), son kons. 1 nM (10 uM stok, *** 1N NaOH T3 çözülür ve 10 uM stok yapmak için orta ekleyin. Sigma kedi # T-2877) 4 ° C arasında, bir hafta için iyi İndüksiyon orta Aşağıdaki bileşikler ihtiva eden bakım ortamı: İndometazin, son kons. 125 uM (etanol 0.125 M stok, Sigma kedi # I-7378). Indometasin 60 ısıtılması gerekir ° C&#39;de eritildi gerekir. Deksametazon, son kons. 2 ug / ml (etanol içinde 2 mg / ml çorap, Sigma cat # D-1756) 3-izobütil-1-metilksantin (IBMX), nihai kons. 0.5 mM (DMSO 0.25 M stok, Sigma kedi # I-5879) Rosiglitazon, son kons. 0.5 uM(DMSO içinde 10 mM stok, Sigma kedi # R-2408) Not: Her zaman orta taze indüksiyon olun. <ol start="2"><li> Tam orta 95-97% izdiham (konfluent ama çok dolu değil) birincil adipositler büyümek </li><li> Indüksiyon orta (gün 0) düzenli komple orta değiştirme </li><li> 48 saat (2 gün) sonra, rosiglitazon (0.5 uM) ile bakım ve orta orta değiştirin </li><li> Ek 48 saat (günde 4) sonra ek 2-3 gün rosiglitazon ile taze bakım ortamı (1 uM) değiştirebilirsiniz. 6-7 gün indüksiyon orta ekledikten sonra, hücreler tamamen yağ hücrelerinin olgunlaşması için farklılaştığı ve yağ damlacıkları ile dolu. Damlacıklar indüksiyon 3-4 gün sonra etrafında görünecektir. </li></ol> Not: Değişim orta hücreleri tamamen farklılaşmış olan 2-3 gün kadar. Hücreler artık hasat ve Ucp1 ve Othe mRNA edilebilirr kahverengi adiposit-özgü genler qRT-PCR yöntemi ile ölçülebilir. Western blot proteinleri tespit etmek için kullanılacaktır.

<ol>
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Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Burada farelerde primer kültürlerinde adipositlerde bej / brite hücrelerinin gelişimini incelemek için güvenilir bir hücresel sistem sunuyoruz. Gibi çeşitli mevcut ölümsüzleştirilmiş hücre soyları ile karşılaştırıldığında, bu sistem, in vivo beyaz yağ kahverengileşme ile geliştirilmiş İlgiye sunmak için olasıdır. Bu birincil adipositlerin çalışmanın bazı avantajlar sunuyor olsa da, orada da düşünülmesi gereken önemli bazı sınırlamalar ve ...

<html> Obezite ölçüde dünya çapında artan ve şu anda halk sağlığı 1 için en ciddi kaygılarından biri olarak kabul edilir. Bu durum bir harcama enerji alımı göreli misbalance ve beyaz yağ dokusu (WAT) lipid olarak depolanan fazla enerji sonuçları ile ilgilidir. Kahverengi adipoz dokuda ısı üretmek için fazla enerjiyi dağıtmak için yeteneğine sahipken Büyümüş WAT, artan vücut kütlesi ve ağırlığı ile ilişkilidir. Bu nedenle BAT soğuk ve obezite 2,3 hem de karşı koruma olarak işlev görebilir. Bu protein 1 (UCP1) ile ayrılmasının mitokondri içinde elektron nakil bölgesinin ayrılmasının elde edilir. Bu protein, BAT 3 termojenez nonshivering için ayırt edici bir özelliği olarak kabul edilir. Son yıllarda çeşitli çalışmalar erişkin insanlarda fonksiyonel BAT 4-8 var ve dolayısıyla insanlarda BAT manipülasyon obezite ve ilgili hastalıklara karşı savaşta potansiyel bir terapötik girişim olabileceğini ortaya koymuştur. jove_content &quot;&gt; Mevcut kanıtlar kahverengi adiposit iki tip kemirgenlerde bulunduğunu gösterir,&quot; klasik &quot;ya da&quot; önceden mevcut olan &quot;kahverengi yağ doğum öncesi aşama sırasında ortaya çıkar ve interskapular ve diğer periferik dokularda adanmış kahverengi yağ deposu oluşturan diğer taraftan. , kahverengi yağ (yani brite veya bej hücreler denir) bir &quot;indüklenebilir&quot; şeklinde doğum sonrası aşamasında gelişir ve beyaz adipoz dokularında serpiştirilmiş görünür. kahverengi adipositlerin iki tip de farklı gelişimsel kökenleri ile ayrılır. önceden mevcut iken kahverengi adipositler Myf5 öncüleri myoblastsic gibi ortaya çıkar, WAT olarak serpiştirilmiş indüklenebilir bej / brite hücre dışı bir Myf5 soy 9,10 ortaya çıkar. Buna ek olarak, bu tip hücre düzenleyici yollar Myf5 türevi kahverengi farklı olması muhtemeldir adipositler 11. bej hücreleri (beyaz yağ yani &quot;esmerleşme&quot;) gelişimi kronik soğuk maruziyeti ve yanıt olarak aktive edilebilirβ3-adrenoseptör agonistler veya yetişkin 12-14 yılında PPARy agonistleri. Bej / brite hücreler genel enerji dengesi manipülasyonu için umut verici bir terapötik hedef olması muhtemel ve potansiyel obezite tedavisinin bir parçası haline gelebilir, bu nedenle, çevresel ipuçları bej gelişimini kontrol hangi hassas moleküler mekanizmaları ve sinyal yolları anlamak önemlidir hücreler. Beyaz yağ esmerleşme moleküler kontrolünü anlamak için, in vitro deneylerde iyi preadipositlerden oldukça uyumsuz gerçekleşir farklılaşması olarak uygun ve yerinde 15 hücreleri tespit etmek zordur. Adiposit geliştirme çalışmaları, şimdiye kadar bu tür 3T3-L1, 3T3-F442A veya HIB1 gibi hücre soyları üzerine esas olarak gösterilmiş olsa da, bu hücre hatları bej hücre moleküler imza eksikliği görülmektedir. Diğer taraftan, derialtı WAT izole birincil adipositler süreç yeniden özetlemek için büyük olasılıklaBir hücre özerk moda beyaz yağ esmerleşme s. Burada yağ dokularından stromal-vasküler fraksiyonun etkili izolasyonu için ve PPARy agonistleri yanıt olarak beyaz yağ kahverengileşme indüklenmesi için bir protokol sağlar. Rosiglitazon, bu hücrelerde esmerleşmenin özellikle etkili bir aracı olduğu gösterilmiştir. 16. Daha önce de belirtildiği gibi, bu hücresel sistem bej / brite hücrelerinin gelişimini incelemek için güvenilir bir hücresel sistem hizmet etmek için de kullanılabilir.

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Reaktif </td></tr><tr><td> Collaginase D </td><td> Roche </td><td> 11088874103 </td><td></td></tr><tr><td> Dispase II </td><td> Roche </td><td> 04942078001 </td><td></td></tr><tr><td> CaCl2 </td><td></td><td></td><td></td></tr><tr><td> DMEM ortamı </td><td> Balıkçı </td><td> 10017-CV </td><td> Sodyum piruvat olmadan 2,5 g / l glukoz ve L-glutamin ile </td></tr><tr><td> Ensülin </td><td></td><td></td><td></td></tr><tr><td> T3 (3,3 &#39;,5-triiodo-L-thyronine) </td><td> Sigma </td><td> T-2877 </td><td></td></tr><tr><td> İndometazin </td><td> Sigma </td><td> I-7378 </td><td></td></tr><tr><td> Deksametazon </td><td> Sigma </td><td> D-1756 </td><td></td></tr><tr><td> IBMX </td><td> Sigma </td><td> I-5879 </td><td></td></tr><tr><td> Rosiglitazon </td><td> Sigma </td><td> R-2408 </td><td></td></tr><tr><td></td><td></td><td></td><td> Ekipman </td></tr><tr><td> Kollajen kaplı yemekleri </td><td> BD </td><td> 354450 </td><td> 10 cm tabaklar </td></tr><tr><td> 70 um bir filtreden </td><td> BD Falcon </td><td> 352350 </td><td> Hücre süzgeç, 70 mikron naylon 1/ea </td></tr></tbody></table>

isolation and differentiation of stromal vascular cells to beige/brite cells

Kahverengi adipositler mitokondrilerde solunum zinciri ayırınız ve ısı gibi kimyasal enerjiyi için yeteneği var. Beyaz adipoz doku (yani bej veya brite hücreler olarak adlandırılır) UCP1 pozitif kahverengi adipositlerin Kalkınma yüksek kronik soğuğa maruz kalma ya da PPARy agonistleri tarafından çevresel işaretlerin çeşitli neden, bu nedenle, bu hücre tipi için bir terapötik hedef olarak potansiyele sahip obezite tedavisinde. Çoğu ölümsüzleştirdi adiposit hatları kültüründe beyaz yağ &quot;esmerleşme&quot; süreci özetleyen edemesek de, birincil adipositler brite / bej hücre gelişiminin moleküler kontrolü çalışmak için güvenilir hücresel sistem sağlamak subkütan beyaz adipoz dokuda stromal vasküler fraksiyon (WAT) izole . Burada primer preadipositlerden etkin izolasyonu ve kültür bej / brite hücrelerine farklılaşma inducing için bir protokol açıklar. Esmerleşme efekti kahverengi yağ seçici işareti ifade tarafından tespit edilebilirBöyle UCP1 gibi ers.

Birincil adipositlerin Browning mRNA Ucp1 ifade ve qRT-PCR ile diğer kahverengi yağ-spesifik veya seçici genler ölçerek erişilebilir. Şekil 1&#39;de Sunan inguinal WAT-kökenli birincil adipositlerde gen ekspresyonu veridir. Hücreler, sırasıyla 50 nM ve 500 nM de rosiglitazon iki farklı dozda varlığında ayırt etmek için oluşturuldu. Hücrelerinin bir alt kümesi, hasattan önce 4 saat boyunca 10 uM forskolin ile de muamele edilmiştir. Bu hücrelerde siklik-AMP (cAMP) teşvik v...

Watch this Scientific Journal Video about Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells at JoVE.com

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Farelerde beyaz adipoz dokulardan izole İlköğretim beyaz preadipositlerden bej / brite hücrelerine ayırt edilebilir. Burada sunulan beyaz yağ &quot;esmerleşme&quot; moleküler düzenlenmesi okumak için güvenilir hücresel modeli sistemidir.

Bej / Brite Hücreler Stromal Vasküler Hücre İzolasyonu ve Farklılaşma

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

isolation-differentiation-stromal-vascular-cells-to-beigebrite

Research

JoVE Journal

Biology

38.5K Görüntüleme.  University of California, San Francisco. Farelerde beyaz adipoz dokulardan izole İlköğretim beyaz preadipositlerden bej / brite hücrelerine ayırt edilebilir. Burada sunulan beyaz yağ "esmerleşme" moleküler düzenlenmesi okumak için güvenilir hücresel modeli sistemidir.

Video: Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

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

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

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

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

İnsan Göbek kordonu İzolasyon ve Hayvan Serum Genişleme Mezenkimal stromal hücreler (MKH) ve endotel Koloni Oluşturan Progenitör Hücreler (ECFCs) Türetilmiş

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

Biyoloji

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 Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

İzolasyon ve Fare aort endotel hücreleri İlköğretim Kültür

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

CD146 İzolasyonu<sup&gt; +</supSıçan Akcigerlerinden&gt; Resident Akciğer Mezenkimal Stromal hücreler

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

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

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

Murin Koroner Damar düz kas hücrelerinin izolasyonu

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

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Zebra balığı Nephrotoxin Mikroenjeksiyon Akut Böbrek Yaralanması Model

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Dinamik Hücresel Protrüzyonlarda Protein Konsantrasyonunun Yazılım Destekli Takibi için Grafiksel Bir Kullanıcı Arayüzü

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Yalıtım, karakterizasyonu ve yetişkin fare kalp kalp kök hücre farklılaşma

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

Rhesus Macaque Adipoz Türevi Kök Hücrelerin İzolasyonu, Proliferasyonu ve Farklılaşması

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+ geçici ve L-tipi kalsiyum akımının eşzamanlı ölçümleri için yüksek kaliteli murin atriyal ve ventriküler miyositlerin izolasyonu

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...