1。のCoCl 2ソリューションにより誘発される低酸素症塩化コバルト（II）六水和物（のCoCl 2•6H 2 O、MW = 237.9）は、低酸素誘導因子- 1.3 8の化学誘導物質です。この製品は、透明な、赤色溶液を得た、水（100 mg / ml）を可溶である。 <ol><li>滅菌DDの水に25mMのストック溶液を準備し、（使用直前に準備する） </li><li>低酸素症を誘発するために、通常の細胞培養培地中の100μMの最終濃度でのCoCl 2を使用してください。 </li><li>あなたの細胞へのCoCl 2を含む培地を加え、従来のインキュベーターで24時間のために文化をインキュベート（37℃、5％C0 2）。 </li></ol>上記の濃度は、我々がテストしたが、各細胞株の薬剤関連毒性を制限し、アッセイを最適化するために様々なインキュベーションの時間にだけでなく、種々の濃度（用量依存曲線を確立する）で試験されるべき細胞株に対して機能します。 2。モジュラーインキュベーターチャンバーに誘導される低酸素症<ol><li>少なくとも二つの同一の（ツイン文化）の培養細胞を準備します。細胞培養は、フラスコ、プレートまたは培養皿にすることができます。チャンバーを開くには、最初の部屋に取り付けられたチューブ（注入/低酸素ガスをパージするために使用されるチューブ）にある2つの白色プラスチック製のクランプを開き、ゆっくりとスチールリングクランプを開きます。 </li><li>クランプを離します。蓋とトレーが削除することができます。 </li><li>低酸素室での細胞培養を置きます。また、文化の十分な加湿を提供するために、チャンバー内に滅菌水を含むペトリ皿を置きます。 </li><li>コントロールとして正常酸素状態で&quot;双子&quot;細胞培養を置きます。 </li><li>あなたのトレイを固定し、動かないし、その蓋とチャンバーを閉じていることを確認してください。正しくチャンバーの気密閉鎖性を確保し、それを閉じるために鋼製リングクランプを配置します。 </li><li>低酸素症を作成するには、1％O 2ガスの混合物を含む&quot;低酸素タンク&quot;にチューブを取り付けます。あなたのタンクに接続されている流量計を持っていて、チャンバーは、直接（ガスタンク - 流量計 - 室）に接続されます。私たちは、レギュレータに内蔵された流量計を使用してください。 </li><li>それは大部分を削除しない場合はチャンバー内で、メディア内のすべての酸素の存在、7-10分間分あたり20リットルの流量でガスのタンクを開いて、チャンバーをフラッシュそうすることが重要です。その後すぐにオフにするガスの流れと完全に白のクランプの両方を閉じることにより、チャンバーを閉じてください。 </li><li>所望の期間については、従来のインキュベーターにチャンバーを戻す。 2時間とは、flush繰り返す - あなたが大規模な文化を使用する場合は、1の脱ガスへの文化の媒体ができます。 上記の手順は、コンストラクタの勧告&quot;（ビラップス - ローゼンバーグ、株式会社）に基づいています。このガスのタンクが正しく、すべての時点で固定されていることを確認してください。 </li></ol>注意事項： <ul><li>それは1 - 3時間後にチャンバー一度の再ガスにアドバイスされているメディアに含まれるO 2を除去するために。 </li><li> 48時間より長いインキュベーション時間のためにそれはまた、再ガス室することをお勧めします。 </li><li> O 2の測定値は、セル/チャンバー内に含まれる許容酸素センサ（電極/マノメーター）室/細胞内O 2が含まれる）のより正確な測定を提供します。 </li><li>時間曲線を作るための様々な時間の長さでの培養細胞は、特定の細胞におけるHIF -1αの最適な時間の表現を決定するに役立つだろう。 </li></ul> 3。低酸素の評価<ol><li> ウェスタンブロットによるHIF - 1αの検出。 <ol><li>セクション2.2で説明されているようにチャンバー再度開いて、すぐに氷上に自分の文化を置きます。 </li><li>プレートの5％SDS溶液を用いて低酸素処理と非処理細胞を溶解するし、管に超音波処理、スピンダウン細胞の破片をセルlyzateを転送し、上清を収集する。 </li><li> BCAキットを用いて測定するのタンパク質濃度は、5分間95℃ローディング緩衝液と加熱° Cを追加することで、サンプルを準備する。 </li><li> 8％SDS -ポリアクリルアミドゲルとニトロセルロース膜への転送に関する電気泳動タンパク質。 </li><li> 1時間またはシェーカー上で4℃で一晩、室温で5％脱脂粉乳を含むPBSで細胞膜と0.1％Tween20をブロックする。 </li><li> 4で同緩衝液中の抗HIF -1α一次モノクローナル抗体（1 / 600）で一晩イムノ℃に</li><li> 45分間、PBS + 0.1％ツイーン20でHRP -二次抗体（1 / 2000）で5分、室温でTween 20を各時間インキュベートを0.1％含有するPBSで3回洗浄する。 </li><li> PBSで室温、5分/一度にTween20を0.1％で3回洗浄する。 </li><li>タンパク質バンドを表示するにはピアースECLキットとX線フィルムを使用</li></ol></li><li> 低酸素応答配列（HRE）。 HIF -1αは低酸素規制エレメント（HRE）と呼ばれる配列に結合することにより、多数の遺伝子（すなわちVEGF、EPO）を調節する。私は、マーカー遺伝子に関連するHREを使用してtは9 HIFの活性を検出することが可能である。この方法を使用するには、まず、あなたの細胞に適したトランスフェクション法を用いてHRE -ルシフェラーゼプラスミドを使用して細胞をトランスフェクトする必要があります。例えば、我々は、リポフェクトアミン2000を使用。細胞が低酸素症および正常酸素状態でインキュベートする前に、少なくとも4時間をトランスフェクションする必要があります。この時間は、この最初のステップは、低酸素の影響を受けないように、DNAが細胞に侵入することができます。ルシフェラーゼシグナルは、ルシフェラーゼアッセイキットを用いて検出される。 <ol><li>モジュラーチャンバーで低酸素状態でHRE -トランスフェクトした細胞をインキュベートするかのCoCl 2でインキュベートHRE -トランスフェクトされた細胞を使用して、正常酸素コントロールが含まれています。 </li><li>希望の潜伏期間の後、細胞から増殖培地を取り除く。 </li><li> PBSで細胞をリンス、PBSを除去。 </li><li>培養皿に400μl1X溶解試薬を分配し、付着細胞をこすり、その後、マイクロチューブに移す。 </li><li>短時間の遠心分離によってペレットの破片。 </li><li>細胞のミックス20μlのは、ルシフェラーゼアッセイ試薬100μlの上清ライセートとルミノメーターを使用して発光を測定する。 </li></ol></li></ol> 4。代表的な結果： K562細胞（ヒト白血病細胞株）で正常酸素と比較して低酸素下で培養した2日間、低酸素症は、ウェスタンブロット（図1）によって検出されたHIF -1αタンパク質の増加を誘発する。 低酸素状態で培養されたHRE - ルシフェラーゼ改変293細胞（胚性腎臓細胞株）、HIF -1αの活性の増加著しいの使用は、低酸素細胞（図2）で検出された。 <img src="/files/ftp_upload/2899/2899fig1.jpg" alt="図1"/> 図1：低酸素細胞でのHIF -1α増加 。 K562細胞は、48時間、正常酸素と低酸素（低酸素室）における文化だったとHIF -1αに特異的な抗体を用いたウエスタンブロットにより分析した。アクチンに特異的な抗体は、ローディングコントロールのために使用されていました。 <img src="/files/ftp_upload/2899/2899fig2.jpg" alt="図2"/> 図2：HIF -1αの活性 。 HRE -ルシフェラーゼ改変293細胞を48時間低酸素状態と酸素正常状態で培養し、ルシフェラーゼアッセイキットおよびルミノメーターを用いてルシフェラーゼシグナルを検出するために溶解させた。結果は相対発光ユニット（RLU）として表現さ​​れています。

<ol>
	<li>Swinson, D. E., O'Byrne, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+hypoxia+and+epidermal+growth+factor+receptor+in+non-small-cell+lung+cancer.">Interactions between hypoxia and epidermal growth factor receptor in non-small-cell lung cancer.</a> Clin Lung Cancer. 7, 250-256 (2006).</li><li>van Laarhoven, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+in+relation+to+vasculature+and+proliferation+in+liver+metastases+in+patients+with+colorectal+cancer.">Hypoxia in relation to vasculature and proliferation in liver metastases in patients with colorectal cancer.</a> Int J Radiat Oncol Biol Phys. 64, 473-482 (2006).</li><li>Hockel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+pO2+predicts+survival+in+advanced+cancer+of+the+uterine+cervix.">Intratumoral pO2 predicts survival in advanced cancer of the uterine cervix.</a> Radiother Oncol. 26, 45-50 (1993).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1+and+human+disease:+one+highly+involved+factor.">HIF-1 and human disease: one highly involved factor.</a> Genes Dev. 14, 1983-1991 (2000).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor+1:+master+regulator+of+O2+homeostasis.">Hypoxia-inducible factor 1: master regulator of O2 homeostasis.</a> Curr Opin Genet Dev. 8, 588-594 (1998).</li><li>Tatum, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia:+importance+in+tumor+biology,+noninvasive+measurement+by+imaging,+and+value+of+its+measurement+in+the+management+of+cancer+therapy.">Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy.</a> Int J Radiat Biol. 82, 699-757 (2006).</li><li>Brahimi-Horn, M. C., Pouyssegur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF+at+a+glance.">HIF at a glance.</a> J Cell Sci. 122, 1055-1057 (2009).</li><li>Piret, J. P., Mottet, D., Raes, M., Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CoCl2,+a+chemical+inducer+of+hypoxia-inducible+factor-1,+and+hypoxia+reduce+apoptotic+cell+death+in+hepatoma+cell+line+HepG2.">CoCl2, a chemical inducer of hypoxia-inducible factor-1, and hypoxia reduce apoptotic cell death in hepatoma cell line HepG2.</a> Ann N Y Acad Sci. 973, 443-447 (2002).</li><li>Emerling, B. M., Weinberg, F., Liu, J. L., Mak, T. W., Chandel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTEN+regulates+p300-dependent+hypoxia-inducible+factor+1+transcriptional+activity+through+Forkhead+transcription+factor+3a+(FOXO3a).">PTEN regulates p300-dependent hypoxia-inducible factor 1 transcriptional activity through Forkhead transcription factor 3a (FOXO3a).</a> Proc Natl Acad Sci U S A. 105, 2622-2627 (2008).</li></ol>

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低酸素条件下における細胞増殖と生存率は細胞の種類によって大きく異なります。したがって、細胞数またはあなたがあなたの実験のために十分な細胞/タンパク質を持っていることを確認するで始まる文化プレートの数を調整する必要があります。 塩化コバルトの方法は、安価で高速であるという利点を持っています。この製品はHIF-1/3α誘導することによって低酸素症模倣するだけでなく、他の遺伝子を調節することができる、この製品は他の効果はそれが独立して&quot;の特定の細胞の機能と表現型に与えることができるものをチェックして、プロジェクトに適応されていることを確認模倣 - 低酸素&quot;の効果。また、低酸素症を模倣するために使用される別の薬剤には、デフェロキサミンメシル酸（DFO、100μMの最終濃度で使用）です。これらの薬物の使用はexperimentatorは&quot;低酸素状態を&quot;影響を与えることなく、培養プレート/ディッシュ/フラスコを何回も開くことができます。 低酸素状態の値は組織や細胞の種類によって異なるとガスの混合物に含まれる酸素のレベルは、実験や細胞の種類によって異なる場合があります。実際、いくつかのセルは、O 2、他の必要性は1％未満O 2低酸素であることが5％に低酸素です。 5％CO 2培養のpHを安定させるためにガス混合物に添加され、ガスの残りの部分は通常、窒素です。 低酸素室は独立して酸素張力のセルの動作を交互にできる薬を使用しないという利点があります。酸素は、それぞれの開口部のチャンバーを再入力するため、低酸素症を軽減しかし、実験のすべての種類が行うことができます。あなたの実験ではこの要因を考慮する必要があります。低酸素/再酸素化は、いくつかの細胞型に影響を与えることができる特定の条件です。代わりに、低酸素ワークステーション（プレミックスガスのタンクにまたはガス混合システムに接続されている）や実験、メディアを変更し、連続的な低酸素環境下で細胞を操作するより規模が大きい低酸素インキュベーター（グローブボックスと低酸素処理チャンバ）を使用することです。様々な低酸素室は、過去10年間に実用化され、あなたの研究室では使用され、プロジェクト、スペース、大きさ、そして予算に応じて選択する必要があります。常に正確な培養条件を保証するために、チャンバーの有効利用と条件を確認してください。チャンバーを用いて、一般的に、低酸素のレベルは様々なガスのミックス（すなわち1％、5％10％酸素）を選択することで変調することができます。 HIF -1αの検出に関しては、それは正常酸素状態で、一部の癌細胞ラインエクスプレスはHIF -1αを知ることが重要です。したがって、これらの細胞株におけるHIFの基礎レベルを決定するために正常酸素コントロールを使用することが重要です。 HIF -1αは、細胞の刺激やストレスは、次の非悪性細胞では正常酸素状態で存在することができる。これらの細胞が飢餓状態にされている場合にも発生する可能性がある、あなたの細胞培養が適切に供給し、維持されていることを確認してください。

<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>抗HIF -1α </td><td>インビトロジェン</td><td> 458400 </td></tr><tr><td>塩化コバルト（II） </td><td>シグマ</td><td> C8661 - 25G </td></tr><tr><td>インキュベーター商工会議所</td><td>ビラップス - ローゼンバーグ</td><td> MIC - 101 </td></tr><tr><td> HRE -ルシフェラーゼプラスミド</td><td> Adgene </td><td>プラスミド26731 </td></tr></tbody></table>

induction and testing of hypoxia in cell culture

低酸素症は臓器、組織、または細胞内の酸素の減少または欠如として定義されています。酸素張力のこの減少は、酸素の供給削減（原因は不十分な血管のネットワーク、欠陥のある血管、および貧血を含む）または電源（突然のより高い細胞増殖率に起因する）に対する相対的な酸素の消費の増加が原因である場合もあります。低酸素症は、そのような固形癌1-3、関節リウマチ、アテローム性動脈硬化症などのような生理学的または病理学することができます...それぞれの組織や細胞は、この新しい条件に適応するためのさまざまな能力を持っている。低酸素時には、低酸素誘導因子α（HIF）は安定化とそのような酸素4の血管新生や輸送に関与しているような種々の遺伝子を調節しています。この蛋白質の安定化は、したがって、HIFが日常的に低酸素症5月7日のスクリーニングに使用されている検出、低酸素症の特徴です。 この記事では、我々は哺乳動物細胞培養とこれらの細胞の低酸素状態を評価するための簡単​​なテストでは低酸素を誘導するために2つの単純なメソッドを提案する。

Watch this Scientific Journal Video about Induction and Testing of Hypoxia in Cell Culture at JoVE.com

Induction and Testing of Hypoxia in Cell Culture

ここでは、細胞培養や文化の低酸素状態を評価するための簡単​​なテストでは低酸素を誘導するための簡単​​な方法を提案する。

細胞培養で誘導し、低酸素のテスト

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

induction-and-testing-of-hypoxia-in-cell-culture

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

84.5K ビュー.  Baylor College of Medicine. ここでは、細胞培養や文化の低酸素状態を評価するための簡単​​なテストでは低酸素を誘導するための簡単​​な方法を提案する。

ビデオ: Induction and Testing of Hypoxia in Cell Culture

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

マウス神経幹細胞の前駆体の文化

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

生物学

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

ラットにおける養子遅延型過敏症の誘導とモニタリング

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

初代培養およびコルチのマウス臓器のプラスミドエレクトロ。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

ラット肝細胞の単離及び初代培養

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

外植片培養での歯胚の開発に続いてのためのスライス培養法

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

筋衛星細胞の単離、培養、および移植

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

低酸素症後単一細胞レベルでのグローバルのRNA合成の分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

C-FOSタンパク質免疫組織学的検出：特定の生理反応に関与する中央経路のマーカーとして有用ツール<em&gt;インビボ</em&gt;と<em&gt; ex vivoで</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological substances or characterization of specific biological activities. However, due to the lack of universally applicable cell culture media and protocols, well described cell culture methods for marine organisms are still limited. Meanwhile, the commonly occurring microbial contamination and polytropic properties of marine invertebrate cells further impede the establishment of an effective cell culture strategy for marine invertebrates. Here, we describe an easy-to-handle method for culturing intestinal cells from sea cucumber Apostichopus japonicus; additionally, we provide an example of in vitro apoptosis induction and detection in primary cultured intestinal cells. Moreover, this experiment provides details about the appropriate culture medium and cell collection method. The described protocol is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea, and it can provide sufficient cells for multiple in vitro experimental applications. This technique would enable researchers to efficiently manipulate primary cell cultures from marine invertebrates and to facilitate the functional evaluation of targeted biological materials on cells.

This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

ナマコ腸細胞の一次培養におけるアポトーシス誘導と検出

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological ...