私たちは、批判的なコメントのためにと日常的にトラブルシューティング退色実験のため、特にMeshorer研究室のメンバーは、シャイMelcer、アディAlajem、Edupugantiラグーラム、バディスリランカSailaja、アンナMattoutとアルバBiranに感謝。 EMは、ジョセフH.とベルR.ブラウンライフサイエンスの上級講師であり、イスラエル科学財団（ISF 943/09）、保健イスラエル省（6007）欧州連合（IRG - 206872および238176）でサポートされていますイスラエル癌研究財団、ヘブライ大学、イスラエルの精神生物学研究所の内部実用的な医療補助。

1。 ES細胞をめっき T = 0時間 MEFのメッキ<ol><li>コー​​トライブイメージングμ-スライド8ウェル（ibidi、ミュンヘン、ドイツ）ゼラチンまたはチャンバーカバーガラスで（ラボ- Tek社、ロチェスター、ニューヨーク州）とやガラス底培養皿で（マテック、アッシュランド、マサチューセッツ州）。 5〜30分放置し、遊離ゼラチンを離れて吸引除去する。 </li><li>シード22000 MEFを/ウェルのDMEMを250μlの総容量で[10％ウシ胎児血清（FBS）を添加した]。細胞は組織培養インキュベーター（37℃、5％CO2）で成長することができます。 </li></ol> T = 6時間 ES細胞のメッキ<ol start="3"><li> DMEMを吸引除去する。 </li><li> ES細胞の培地250μlの総体積で15,000 R1細胞/ウェルでうまく被覆種子各MEFの[10％ESC -グレードウシ胎児血清（FBS）、1mMピルビン酸ナトリウム、0.1mMの非必​​須アミノ酸、0.1mMのβ-を添加したメルカプトエタノール、および1000 U / mlの白血病抑制因子（LIF）]、翌日30％〜50％コンフルエントに取得する。 </li></ol> 2。 ES細胞のトランスフェクション T = 24時間 一過性トランスフェクション<ol><li> 250μL/ウェル、新鮮なES細胞のメディアのES細胞の培地を交換してください。 </li><li> 1.5 mlの滅菌試験管に、100μlの無血清培地[のOpti - MEM（Gibco社）]、その後、直接無血清培地に10μlのトランジット- LT1トランスフェクション試薬（Mirus）を追加を追加。穏やかなピペッティングにより混和し、5〜20分間室温でインキュベートする。 </li><li>希薄化後のトランジット- LT1試薬に1.5μgのGFP融合プラスミドDNA（H1E、H1oまたはHP1）を追加します。穏やかなピペッティングにより混和し、15〜30分間室温でインキュベートする。 </li><li>トランスフェクション混合物の13.5μl/ウェルを追加。さらに分散性を確保するためにμ-スライド8ウェル渦巻き。 24時間後には、新鮮なES細胞の培地を250μlと古いのES細胞培地を交換してください。 </li></ol> 3。 FRAPやFLIPの実行 T = 48〜72時間<ol><li>実験は、任意の共焦点レーザー走査顕微鏡（CLSM）で実行することができますが、通常のFRAP / FLIP実験で以来、多くの連続した​​画像が取得され、それが買収の速度を可能にしないし、その何を確実に​​回転するディスク共焦点顕微鏡を、使用することをお勧めします。漂白望ましくないサンプルは、最初の意図的な白化現象は、次の発生します。ここで、我々は、横河電機CSU - X回転するディスクのヘッドを、ディスク共焦点システム（www.Andor.comを）回転革命を使用することをお勧めします。このシステムは、ポイントスキャンシステムで特殊なFRAPPAモジュールを使用して、そして急速に回転するディスクを使用して画像を収集するためにバックライトを切り替えるには光退色に二重の能力を持っています。実験の光退色に使用する3最も一般的な蛍光タンパク質はGFP、YFPとチェリーです。 GFPまたはYFPが使用されている場合は、〜488 nmのレーザーが必要です。チェリーの場合は、〜560nmのレーザーを使用してください。すべてのケースでは、我々は固体レーザを使用することをお勧めします。自動ステージを持つことは有用ですが、必須ではありません。生きた細胞が撮像されているので、それは酸素、湿度、CO 2と温度を制御する、（私たちはLIS、スイスからいずれかを使用）環境室を使用することが不可欠です。 FRAPは、イメージングが（通常10％の面積で、蛍光レベルが十分なとき）に必要な最小限のレーザーパワーで行われている間、最大レーザー強度を使用して実行されます。 適切な波長の蛍光灯の光で細胞を観察し、60X油浸レンズを用いてGFPを発現する細胞を選択する。正しい細胞内分布を確認してください。時折、発現レベルが高すぎるときに、タンパク質の局在化は、核小体などの他の区画に&quot;スピル&quot;があります。このような細胞が選択されるべきではない。 </li><li>今イメージングプロトコルを設定します：ユークロマチンまたはヘテロクロマチン（要約GFP巣として表示）にし、退色前に退色3から5フレームを収集し、250から1000ミリ秒の間隔で、光退色後の90から120フレームを収集してください。H1E - GFP、1000 MS、H1oとHP1 - GFP、250ミリ秒（非常に動的な蛋白質は、短い間隔の時間を必要とする蛋白質のダイナミクスに応じてインターバル時間の変更、）。我々は通常、20〜40μseconds（1-2の繰り返し）のレーザーパルスで光退色に対して80〜100％のレーザー強度を使用しますが、これらの数値は、分析タンパク質と発現レベルに応じて変更することができます。退色が適切な場合、あなたはGFPの蛍光で&quot;ブラックホール&quot;を観察する必要があります。ブラックホールは徐々に蛍光次の回復と再充填されている。回転するディスクは1秒間に60前後の画像（良い蛍光強度を持つとする場合、単一のセルにズームイン）まで得ることができますが、我々は低画質と潜在的な増加光毒性に起因するような高速でシステムを使用することはお勧めしません。 </li><li> FLIP実験では、別の撮像プロトコルを設定する：画像を収集中に同じ場所に漂白を繰り返し開始し、漂白の前に3から5フレームを収集する。 H1o - GFP、漂白のためのH1E - GFP、漂白剤ごとに5秒、毎2秒のため、とHP1 - GFP漂白剤毎に1秒のため。漂白剤D実験全体を通して何度も画像を収集する。 </li><li>どちらの手法の場合、20〜30細胞にプロセスを繰り返します。統計上の目的のために、できれば別の日に、実験を3回以上繰り返す。均質な集団と正しい設定では、標準偏差は通常低い（&lt;5％）。 FRAPやFLIPの両方に対して、漂白領域の大きさや形状は、回復のダイナミクスに影響を与え、実験内で一定でなければなりません。また、つのセルを比較する場合、同一のプロトコルを使用する必要がありますし、細胞をパワーレーザーと他の条件は変動することがありますし、実験の結果に影響を及ぼす可能性があるため、同じ日に連続して分析する必要があります。 </li></ol> 4。 FRAPやFLIPデータ解析<ol><li>すべてのFRAPフレームが収集さで、ROI（投資収益率B =漂白エリア）、背景領域（ROI BG）で蛍光強度を測定し、前と漂白後の時間の関数としての無漂白の核領域（ROI NB）。漂白地域はごくわずかであるときに全体の核は、正規化の目的のために選択することができます。 </li><li> /（ROI NB - ROI BG） - （ROI BG ROI B）/（pbROI B - pbROI BG）/（pbROI NB - pbROI BG）、鉛は、事前に漂白意味：すべての時間ポイントの場合は、公式にしたがってデータを正規化する。事前に漂白剤の画像の場合は約1の値を取得する必要があります。漂白剤の後の最初のイメージは、ブリーチの深さが示されます。実際のブリーチ深さ値1から値を引く。すべてのセルと各実験から平均20から30のセルに対して繰り返します。 </li><li>収集されたすべてのFLIPのフレームで、（ROI NB =無漂白面積、ROI BG =バックグラウンド）無漂白核領域での蛍光強度、および背景領域を測定する。 FLIPデータの計算は、FRAPの曲線に似てのみ分析したROI（投資収益率nbは ）計算に使用されていない実際の漂白地域、異なっている必要があります：（ROI NB - ROI BG）/（pbROI NB - pbROI BG） 。それは正規化の目的のために隣接セル（ROI N =隣接セル）を使用することも可能です：（ROI NB - ROI BG）/（ROI N - ROI BG）/（pbROI NB - pbROI BG）/（pbROI N - pbROI BG ）。 データの収集に続いて、それはコンピュータシミュレーションに実験データをフィットすることが可能です。これは良い近接、モバイル分数、不動小数と半値で、計算することができます。ここでは、FRAP解析の数学的および統計的側面 ​​を議論し、他の優れた出版物4-9を参照されたいことはありません。漂白剤の深さが事前に漂白剤（100％）信号と漂白剤の後の最初のイメージとの間の距離（y軸上）を指し、モバイル割合は、漂白剤の深さとの間の距離（y軸上）を指し反応速度がプラトーに達する、と不動小数が復元された信号とプリ漂白剤（100％）の信号の間の距離（y軸上）（図1Bおよび2Bを参照）を参照している。復元された信号この分析を超えて、データに合わせて適切な数学モデルがあります。単一指数、方程式のための <img src="/files/ftp_upload/2696/eq0.jpg" alt="式" width="80" /> tは時間であるときは、1モバイル割合であり不動割合であり、kはオフ解離定数であり、データに合わせて使用することができ、結合のオフ率の直接推定値（K オフ ）することができます得だけでなく、会合速度を計算するために使用できるパラメータ、のために。 </li></ol> 5。代表的な結果： <img src="/files/ftp_upload/2696/2696fig1.jpg" alt="図1" /> 図1及びBは、R1 ES細胞では、HP1（左）の代表的なFRAP曲線を示すH1o（真ん中）とH1E（右）。簡単でわかりやすく図の場合は1Aは、いかなる正規化し、計算の前に単セルの生データを示しています。黄色の曲線は、漂白の領域に対応する、紫の曲線は、無漂白の核領域に相当する（漂白地域は核全体が正規化の目的のために選択することができる無視できる場合）、および赤の線は、バックグラウンドの蛍光に対応し、されているこの場合の最小限の。垂直方向の矢印は、漂白時間を表します。正規化と平均化されたデータは、図1Bに示されています。 HP1（赤）と比較してH1（青）の遅い回復に注意してください。また、H1Eバリアント（ダークブルー）はH1oバリアント（ライトブルー）よりも遅くなります。モバイルと不動の分画とブリーチの深さは、HP1のために示されている。 <img src="/files/ftp_upload/2696/2696fig2.jpg" alt="図2" /> 図2及びBは、ユークロマチンを比較する代表的なFRAP曲線を示す（R1 ES細胞におけるHP1のヘテロクロマチン（右）と）左。同様に図1を、図2Aは、単一セルの生データを示し、黄色の曲線は、漂白の領域に対応する、紫の曲線は、無漂白の核領域に相当し、赤の線は、バックグラウンドの蛍光に対応しています。垂直方向の矢印は、漂白時間を表します。正規化と平均化されたデータは、図2Bに示されています。ユークロマチン（明るい赤）と比較してヘテロクロマチン（暗赤色）の遅い回復に注意してください。モバイルと不動の分画とブリーチの深さは、ユークロマチンのために示されている。 <img src="/files/ftp_upload/2696/2696fig3.jpg" alt="図3" /> 図3。。 H1o R1 ES細胞の典型的なFLIPの実験は、図3A（生、非正規化データ）とB（正規化と平均化されたデータ）に示されています。この実験では紫色の曲線は、無漂白の核領域に対応する、緑色の線は隣接する細胞核に相当し、赤線は、バックグラウンドの蛍光に対応しています。

<ol>
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利害の衝突は宣言されません。

細胞集団または固定化細胞、FRAP実験から精製されたクロマチンを含むほとんどの利用できる技術とは異なり、生きた細胞のクロマチン蛋白質のダイナミクスの変化に従ってください。我々はクロマチンの蛋白質のダイナミクスは、クロマチン可塑性の良い指標であることが判明。しかし、それは融合のGFPと目的の遺伝子を必要とするため、蛍光タグを追加するには、タンパク質の機能を妨げ...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td> DMEM </td><td>シグマ</td><td> D5671 </td></tr><tr><td>ゼラチン</td><td>メルク</td><td> 1.04078 </td></tr><tr><td>のOpti - MEM </td><td>ギブコ</td><td> 31985 </td></tr><tr><td>トランジット- LT1 </td><td> Mirus </td><td> MIR2300 </td></tr><tr><td> μ-スライド8ウェル</td><td> ibidi </td><td> 80826 </td></tr></tbody></table>

photobleaching assays (frap &amp; flip) to measure chromatin protein dynamics in living embryonic stem cells

光退色後蛍光回復（FRAP）や光退色で蛍光損失（FLIP）が良好な空間分解能と時間分解能を持つ生体細胞内の蛋白質のダイナミクスの研究を可能にします。ここでは、FRAP及びマウス胚性幹（ES）細胞のH1とHP1、を含む、クロマチンタンパク質のFLIPアッセイを、実行する方法について説明します。 FRAP実験で、細胞は緑色蛍光タンパク質（GFP）またはその誘導体（YFP、CFP、チェリー、など）を融合させた目的のタンパク質で、どちらか一過性または安定的に、トランスフェクトされています。トランスフェクト、蛍光細胞では、強烈な焦点レーザービームは、興味の比較的小さい領域（ROI）を漂白剤。レーザーの波長は、融合に使用する蛍光蛋白質に応じて選択されます。レーザー光が不可逆的にROI内の分子の蛍光シグナルを漂白剤と、すぐに漂白以下、漂白地域における蛍光シグナルの回復 - 無漂白の分子と漂白分子の交換によって媒介は - タイムラプスイメージングを使用して監視されます。生成された蛍光回復曲線は、タンパク質の流動性に関する情報を提供しています。蛍光分子が不動であれば、何蛍光回復は観察されません。補完的なアプローチでは、光退色における蛍光損失（FLIP）は、レーザービームは、繰り返し同じ場所を漂白し、信号強度は、他の場所で蛍光を発する細胞で測定されます。 FLIPの実験では、したがって、信号の減衰ではなく、蛍光の回復を測定し、タンパク質の移動度と同様に細胞区画を行き来し蛋白質を決定するのに便利です。一時的な結合は、クロマチン関連タンパク質の一般的なプロパティです。各クロマチン蛋白質の主要な割合が定常状態での任意の時点でクロマチンにバインドされていますが、バインディングは一時的であり、ほとんどのクロマチンタンパク質は、秒の順番で滞留時間で、クロマチン上で高い離職率を持っている。これらのプロパティは、ゲノムの式1に高い可塑性を発生させるために重要です。退色実験は、特にクロマチンタンパク質の動的な交換が（ヘテロクロマチンタンパク質1（HP1）、リンカーヒストンH1とコアヒストンを含む）以上であるES細胞、で、それゆえクロマチン構造蛋白質のGFP融合バージョンを使用してクロマチン可塑性を決定するために特に有用です。 2,3分化細胞に比べて。

Watch this Scientific Journal Video about Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells at JoVE.com

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

我々は、光退色後蛍光回復（FRAP）や胚性幹（ES）細胞におけるクロマチン蛋白質の動態を監視する（FLIP）光退色で蛍光の損失を含む光退色の方法を説明します。クロマチン可塑性を研究する手段の一つであると考えられているクロマチン蛋白質のダイナミクスは、多能性細胞で強化されています。

胚性幹細胞を生体内でクロマチン蛋白質のダイナミクスを測定するアッセイ（FRAP＆FLIP）を光退色

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

photobleaching-assays-frap-flip-to-measure-chromatin-protein-dynamics

Research

JoVE Journal

Biology

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

27.1K ビュー.  The Hebrew University of Jerusalem. 我々は、光退色後蛍光回復（FRAP）や胚性幹（ES）細胞におけるクロマチン蛋白質の動態を監視する（FLIP）光退色で蛍光の損失を含む光退色の方法を説明します。クロマチン可塑性を研究する手段の一つであると考えられているクロマチン蛋白質のダイナミクスは、多能性細胞で強化されています。

ビデオ: Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

ヒト胚性幹（ES）細胞の増殖

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

マウス胚性幹細胞から造血幹細胞の導出

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

バイオフリップチップを用いた胚性幹細胞のパターニング

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

ヒト胚性幹細胞のミトコンドリア複合体活性の状態調査

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

ヒト胚性幹細胞からクロマチン免疫沈降

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

ヒト胚性幹細胞を凍結融解

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

胚におけるプログラム細胞死を検出するためのLysoTrackerの使用は、胚性幹細胞を分化

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

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 ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

定量的RT-PCRによって個々のヒト胚性幹細胞のプロファイリング

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

で微小管ダイナミクスを測定するためにplusTipTrackerソフトウェアを使用した<em&gt;アフリカツメガエル</em&gt;成長円錐

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...