我々は、原稿上の議論、技術的なヘルプと編集支援のための治大野耕作篠田ルイシャープ、恵美友田、とローレンルイズに感謝します。この作品は、コペンハーゲンとEU FP7プロジェクトDIABAT（健康F2の大学から博士号SHAREのフェローシップでサポートされていた画期的な生物医学研究のためのプログラムからとアスビオファーマ株式会社からSKULAには、NIH（DK087853）からの補助金によって支えられてリセマドセンとKarstenクリステンセンに-2011-278373）。またDERCセンター助成（NIH P30 DK063720）を認める。

1。消化培地を調製組織当たり5匹（約1ml / 1グラムの脂肪組織）ごとに5ミリリットルを加えます。 <ol><li>消化酵素で重量を量る： - Collaginase D：1.5 U / mlの（1108874103、1グラム、ロシュ、70334223） - ディスパーゼⅡ：2.4 U / mlの（04942078001、0980ミリグラム/ LYO、ロシュ、11466200） </li><li> 25ミリリットルのPBSを加え、溶解させるためによく混ぜる</li><li> 10mMの最終濃度で組織の消化直前にCaCl 2を追加</li></ol> 2。マウスから脂肪組織を解剖<ol><li>切開は慎重に行うべきであり、すぐにマウスの後（6-8週齢）に屠殺されています。それを開く前に、マウスの毛皮の上に噴霧し、エタノールで滅菌作業。クリーン、PBS、別甲骨間BATおよび皮下鼠径WATで直接組織を置きます。 </li><li>肩甲骨間BATの場合：最大直面背にして配置されたマウスを持って、背中や首へのすべての道に沿って切り開く。茶色脂肪組織は右肩（肩甲骨間）の間の皮膚の下に見つけることができます。 BATは2葉、蝶の形として見ることができます。 BATをカバーする白色脂肪の薄い層を注意深く除去する必要があります。 </li><li>皮下WAT（鼠径WAT）の場合：皮膚を取り除きます。鼠径WATは睾丸に向かって太ももと下の内側、ほぼ背面に始まると一緒に行く、両側の皮膚の直下にあります。 </li></ol> 3。脂肪組織を切り取ってダイジェスト<ol><li>素早く髪、骨格筋および組織切片から結合組織のように、すべての汚染物質を除去。 </li><li> PBSで切開培地を希釈し、乾板の上に置いたりしないように紙の上で素早く乾いたティッシュ。 </li><li>消化液（消化前にCaCl 2を添加した）と小さな断片に組織をミンチを追加します。 5 mlのピ​​ペットでピペッティングによって非常によく混ぜる。消化メートルの残りの部分との50mlチューブでミンチ組織を移すediumは、上下にピペッティングして、さらに混ぜる。 </li><li>ダイジェスト37℃·40から50分間150rpmで一定に撹拌しながら。消化がうまくいっていることを確認するとオーバー消化を防止するために15分間隔でチェックします。 </li></ol> 注：正しい消化媒体と時間が重要である。組織は、消化をよくする必要がありますが、過剰消化が細胞を損傷することができる。 4。フィルタ細胞懸濁液<ol><li> 5ミリリットル完全培地（DMEM/F12含む10％のFPSとP / S）を添加することによって消化を停止します。細胞がほぼ完全に均一である必要があります。ピペッティングでよく混ぜる。 </li><li> 10分間、700×gで遠心分離します。 </li><li> SVFは今やチューブの底に茶色がかったペレットとして見ることができます。トップと液体層の最も上に油性の成熟脂肪細胞層を吸引 - ペレットを維持し、10mlの完全培地中でそれを溶かす。よく混ぜる。 </li><li>新しい50の上にセルストレーナー（50〜70μmの直径）を配置<html> mlチューブに細胞懸濁液をろ過する。 </li></ol> 5。細胞をプレート<ol><li> 10分間、700×gで15 mlのチューブと遠心分離機に細胞懸濁液を移す。 </li><li> 10mlの完全培地で培地を吸引除去し、再懸濁ペレット。ピペット、よく混ぜる。 </li><li>必要とされているどのように多くのプレートを見積もる。これはこれとは異なり、ペレットの大きさに依存するかもしれません。一般的なルールは、鼠径WATのために5匹とBATのための1 10cmプレートから10cmプレートの2です。 </li><li>完全培地中でコラーゲンコートディッシュ（R＆D）に細胞をプレート</li><li> 1-2時間細胞、培地を吸引めっき後、PBSで2回洗浄し、新鮮な培地を追加します。これは赤血球、免疫細胞やその他の汚染物質を除去することができるため、この手順は重要です。 </li></ol> 6。細胞を分化<ol><li>保守と誘導媒体を作る。 </li></ol>維持培地付加した培地を完了して下さい： ENT &quot;&gt;インスリン、最終濃度5μg/ mlの（5 mg / mlストック、酸性化した水のpHが2.5を準備するために10 mlのH 2 O中の酢酸***100μlの。酸性水でインスリンを溶かす。店舗在庫で-20℃） 3,3 &#39;、5 - トリヨード-L-チロニン（T3）は、最終濃度。 1nMの（10μMストック、***は1N NaOHに溶解し、T3は10μMストックを作るために媒体を追加します。シグマ猫＃T-2877） 4℃で保存、一週間のために良い誘導培地以下の化合物を含む維持培地： インドメタシン、最終濃度。 125μMの（エタノール中の0.125 Mのストック、シグマの猫＃I-7378）。インドメタシンを60℃に加熱しなければならない°C溶解させる。 デキサメタゾン、最終濃度。 2μg/ mlの（エタノール中の2 mg / mlの靴下、シグマ猫＃D-1756） 3 - イソブチル-1 - メチルキサンチン（IBMX）、最終濃度。 0.5mMの（DMSO中0.25Mの株式、シグマ猫＃I-5879） ロシグリタゾンは、最終濃度。 0.5μMで（DMSO中10mMストック、シグマ猫＃R-2408） 注：毎回新鮮な培地誘導を行います。 <ol start="2"><li>完全培地中で95から97パーセントのコンフルエンス（コンフルエントが、​​あまりにも梱包されていない）に一次脂肪細胞を成長させる</li><li>誘導培地（0日目）との定期的な完全培地を変更</li><li> 48時間（2日目）の後、グリタゾン（0.5μM）で維持培地に培地を変更</li><li>さらに48時間（4日目）の後に、さらに2〜3日間のロシグリタゾンと新鮮な維持培地（1μM）に変更します。 6-7日誘導培地を添加した後、細胞を完全に脂肪細胞を成熟させる差別化され、油滴で満たされている。液滴は、誘導後3-4日周囲に表示されます。 </li></ol> 注：細胞は完全に分化されるまで2〜3日毎に培地を変更します。 細胞は、今では収穫し、UCP1とパーソナルプラグインのmRNA発現することができますrは褐色脂肪細胞特異的遺伝子は、qRT-PCRにより測定することができます。ウエスタンブロット法を用いて、タンパク質を検出するために使用されます。

<ol>
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著者らは、彼らが競合する経済的利益を持っていないことを宣言します。

ここで我々は、マウスの初代培養脂肪細胞におけるベージュ/ブライト細胞の発達を研究するために信頼性の高い携帯電話システムを提案する。など、いくつかの利用可能な不死化細胞株と比較して、このシステムは、in vivoでの白色脂肪の褐変に強化された関連性を提供することがございます。 これらの主要な脂肪細胞の研究はいくつかの利点を提供していますが...

<html>肥満が劇的に世界的に増加しており、今では公衆衛生1に最も深刻な懸念の一つと考えられている。この条件は、支出にエネルギー摂取量の相対でmisbalanceと白色脂肪組織（WAT）における脂質として格納されている余分なエネルギーの結果に関連しています。褐色脂肪組織は熱を生成するために余分なエネルギーを放散する能力を持っていながら、拡大されたWATは、増加した体重と体重に関連付けられています。したがって、BATは、寒さと肥満2,3の両方に対する保護として機能することができます。これは、タンパク質1（UCP1）を脱共役することによってミトコンドリアにおける電子伝達の脱共役することによって達成される。このタンパク質は、BAT 3で熱発生をnonshiveringための顕著な特徴と考えられている。近年のいくつかの研究では、成人の人間が機能BAT 4-8を持っていることを明らかにし、その結果、ヒトにおけるBATの操作は、肥満とそれに関連する疾患との闘いにおいて、潜在的な治療介入することができます。 jove_contentは &quot;&gt;現在の証拠は褐色脂肪細胞の2種類がげっ歯類に存在することを示し、&quot;古典的な &quot;または&quot;既存の &quot;褐色脂肪は、出生前の段階で開発し、肩甲間部やその他の末梢組織における専用の褐色脂肪デポを形成している一方。 、褐色脂肪（いわゆるブライトやベージュ細胞と呼ばれる）の &quot;誘導&quot;フォームは、出生後の段階で開発し、白色脂肪組織中に散在して表示されます。褐色脂肪細胞の2つのタイプは、異なる発達の起源で区切られています。既存ながら褐色脂肪細胞はmyoblastsicライクMyf5前駆体から生じる、WATに点在誘導BRITE /ベージュ細胞は非Myf5系統9,10から生じる。さらに、この細胞型の調節経路はMyf5由来の茶色とは異なる可能性が高い脂肪11。ベージュ細胞（白色脂肪のすなわち &quot;褐変&quot;）の開発が慢性寒冷暴露へとに応答して活性化することができますβ3-アドレナリン受容体アゴニスト、大人の12月14日でのPPARγアゴニスト。ベージュ/ BRITE細胞は全体のエネルギーバランスを操作するための有望な治療標的となる可能性がある、潜在的肥満治療の一部になることができますので、環境への手がかりはベージュの開発を制御する正確な分子機構とシグナル伝達経路を理解することが重要です細胞。 前駆脂肪細胞の分化ではなく非同期に行われ、それがその場で 15 で細胞を検出することは困難であるとして、白色脂肪の褐変の分子制御を理解するために、in vitroの実験で最も適している。脂肪細胞の開発に関する研究はこれまでそのような、3T3-L1、3T3-F442AまたはHIB1などの細胞株では、主に行われてきたが、これらの細胞株はベージュ細胞の分子署名を欠いているように見える。一方、皮下WATから分離された主要な脂肪細胞はプロセッを再現する可能性が最も高い細胞自律的に白色脂肪の褐変のだ。ここでは、脂肪組織から間質血管画分の効果的な分離のためとPPARγアゴニストに応答して、白色脂肪の褐変を誘導するためのプロトコルを提供する。ロシグリタゾンは、これらの細胞における褐変の特に有効なメディエーターであることが示されている。以前に16を示唆したように、この細胞系は、ベージュ/ブライト細胞の発達を研究するために信頼性の高い携帯電話システムを提供するために使用することができます。

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 試薬 </td></tr><tr><td> Collaginase D </td><td>ロッシュ</td><td> 11088874103 </td><td></td></tr><tr><td>ディスパーゼII </td><td>ロッシュ</td><td> 04942078001 </td><td></td></tr><tr><td> CaCl 2を </td><td></td><td></td><td></td></tr><tr><td> DMEM培地</td><td>フィッシャー</td><td> 10017-CV </td><td>ピルビン酸ナトリウムなし2,5 g / lのグルコースとL-グルタミン</td></tr><tr><td>インスリン</td><td></td><td></td><td></td></tr><tr><td> T3（3,3 &#39;、5 - トリヨード-L-チロニン） </td><td>シグマ</td><td> T-2877 </td><td></td></tr><tr><td>インドメタシン</td><td>シグマ</td><td> I-7378 </td><td></td></tr><tr><td>デキサメタゾン</td><td>シグマ</td><td> D-1756 </td><td></td></tr><tr><td> IBMX </td><td>シグマ</td><td> I-5879 </td><td></td></tr><tr><td>ロシ</td><td>シグマ</td><td> R-2408 </td><td></td></tr><tr><td></td><td></td><td></td><td> 機器 </td></tr><tr><td>コラーゲンコートディッシュ</td><td> BD </td><td> 354450 </td><td> 10cmプレート</td></tr><tr><td> 70μmのフィルター</td><td> BDファルコン</td><td> 352350 </td><td>セルストレーナー、70μmのナイロン1/ea </td></tr></tbody></table>

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

褐色脂肪細胞は、ミトコンドリアの呼吸鎖を切り離すと熱などの化学的エネルギーを放散する能力を持っています。白色脂肪組織中のUCP1陽性褐色脂肪細胞（いわゆるベージュまたはBRITE細胞と呼ばれる）の開発が強く、したがって、そのような慢性の寒冷暴露などの環境合図の様々な方法によって、またはPPARγアゴニストにより誘導され、この細胞型は、治療標的としての可能性を秘めている肥満治療。最も不死脂肪線は文化の中で白色脂肪の &quot;褐色化&quot;の過程を再現することはできませんが、皮下の白色脂肪組織における間質血管画分（WAT）から単離された主要な脂肪細胞はBRITE /ベージュ細胞分化の分子制御を研究するために信頼性の高い携帯電話システムを提供。ここでは、主要な脂肪細胞を効果的に分離するための、文化の中でブライトベージュ/細胞への分化を誘導するためのプロトコルを記述します。褐変効果は褐色脂肪選択的マークの発現によって評価することができるそのようなUCP1としてERS。

主要な脂肪細胞の褐変は、qRT-PCRによりmRNAのUCP1の発現と他の褐色脂肪特異的または選択的な遺伝子を測定することによってアクセスすることができます。 図1に示さ鼠径WAT-由来の初代脂肪細胞における遺伝子発現データです。細胞はそれぞれ、50nmおよび500nmにおけるロシグリタゾンの二つの異なる用量の存在下で分化するように誘導された。細胞のサブセットは、収穫前?...

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

マウスにおける白色脂肪組織から単離された脂肪細胞は、一次白ベージュ/ブライト細胞に分化させることができる。白色脂肪の &quot;焦げ目&quot;の分子調節を研究するために信頼性の高い細胞モデルシステムは、ここに提示されます。

ベージュ/ブライト細胞間質血管細胞の単離と分化

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

38.4K ビュー.  University of California, San Francisco. マウスにおける白色脂肪組織から単離された脂肪細胞は、一次白ベージュ/ブライト細胞に分化させることができる。白色脂肪の "焦げ目"の分子調節を研究するために信頼性の高い細胞モデルシステムは、ここに提示されます。

ビデオ: 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.

ヒト臍帯由来間葉系幹細胞（MSCs）と血管内皮コロニー形成前駆細胞（ECFCs）の単離および動物血清自由膨張

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

生物学

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.

間葉転換を誘導し、上皮の分析

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.

マウス大動脈内皮細胞の単離および初代培養

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の単離<sup&gt; +</supラット肺から&gt;常駐肺間葉系幹細胞

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.

マウス冠動脈血管平滑筋細胞の単離

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.

ゼブラフィッシュにおける腎毒素マイクロインジェクションは急性腎障害をモデルに

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.

動的細胞突起におけるソフトウェア支援トラッキングのためのグラフィカルユーザインタフェース

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.

分離と性質、大人マウス心臓から心臓の幹細胞の分化

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.

アカゲザル脂肪由来幹細胞の単離・増殖・分化

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+過渡現象とL型カルシウム電流の同時測定のための高品質マウス心房および心室筋細胞の分離

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