本研究では、NIHの研究資金（TIへDK090868とGM092930）によってサポートされていました。ケージラパマイシンアナログの保留中の特許があります。

1。プラスミドDNAのトランスフェクション<ol><li>プラスミドDNAを追加し、0.5μgの膜係留FRB（以下、LDRと呼ばれる）と0.5μgのFKBP-Tiam1（T-細胞リンパ腫の浸潤や転移を誘発するタンパク質1：RAC活性剤）37.5μLのdH 2 Oに蛍光タンパク質でタグ付け1μlのトランスフェHDを追加します。 </li><li> 20分間室温でボルテックスし、インキュベートします。一方、ステップ1.3から1.8に進みます。 </li><li> 50μlのポリ-D-リジンで8ウェルチャンバーの各ウェルを洗浄します。 </li><li>のdH 2 Oで各ウェルの中央を洗う</li><li> 80から85パーセントコンフルエントNIH-3T3細胞をトリプシン処理し、10 mlの培地（10％FBSを含むDMEM）に希釈する。 </li><li>懸濁液375μlを削除し、3分間1750 rpmで遠心します。 </li><li>ペレットを乱さないように注意しながら、培地を吸引除去する。 </li><li> DMEMのw / 10％FBSを再懸濁し、750μlを添加します。 </li><li>のdH 2 O / DNA /トランスフェHDソリューションに細胞懸濁液を加え、穏やかに混ぜる。 </li><li> 250を追加します。各ウェルに細胞/ DNA懸濁液の液（3ウェルにいっぱいになります）。 </li><li> 15から18時間のために°C、5％CO 2で37トランスフェクションした細胞をインキュベートします。 </li></ol> 2。 CRB-溶液の調製<ol><li>ケージラパマイシン-ビオチン付加物（CRB）を合成。詳細については、 &quot;CRBの合成スキーム&quot;を参照してください。簡潔に、ケージとビオチン部分が別々に用意されています。ケージ部分は、その後、ラパマイシン（LCラボ）、その後、CRBを与えるためにクリック反応7によってビオチン部分に結合された製品に結合している。 </li><li> DMSO中のCRBの1μMのストック溶液を準備します。 </li><li> 0.5ミリリットルエッペンドルフチュー​​ブにPBS 250μlで1mgのアビジンを溶解する。 </li><li>溶解アビジンにCRBのストック溶液を1μlを追加します。反転数回混和する。 </li><li> CRB-アビジンコンジュゲート（CRB-もたらすために室温で15分間インキュベート）ソリューションを提供します。 </li><li>バインドされていない小分子を排除し、CRB-サイズ排除カラムを用いて精製する。 </li></ol> 3。顕微鏡と細胞内レベルでのライト照明<ol><li>血清は12時間FBSを含まないDMEMでトランスフェクトした細胞を飢えさせる。 </li><li>穏やかに、細胞から培地を吸引除去すると、準備400nMのCRB-ソリューション（ステップ2.6から）250μlを加える。 </li><li>ディスク顕微鏡を回転させて、光治療の前と後細胞内分解能で生きた細胞内のタンパク質の動態を可視化する（ 図2a）を用いた。 UV照度は落射蛍光光（ 図2b）と紫外LEDの光（365 nm）のための光学部品を結合させることによって達成された。 </li><li> MetaMorphソフトウェアは、15秒ごとにトランスフェクトした細胞の蛍光画像をキャプチャするために使用された。 </li><li> UV光により励起され蛍光色​​素で被覆したガラス板を使用することによって照明スポットの座標とサイズを定義します。サイズは缶異なる倍率で対物レンズを適用することにより、異なるコアサイズの光ファイバを選択することによって変化させることができます。 </li><li>膜の波打ちのバックグラウンドレベルを決定した後、細胞の周囲に照射UV光。次の2つの手順では、ローカライズされた効果の前に対比することです。 </li><li>グローバル水銀ランプによって提供された365nmの光で細胞を照射します。 </li><li> DMSOの後、PBSでuncaged、そのため、元ラパマイシン（最後の400 nM）を溶解し、グローバルな効果を誘導するイメージング室にソリューションを追加します。 </li></ol> 4。代表的な結果代表的な結果の一例を図3に示します。ローカライズの携帯端急速に誘導され形成の近くの小さな領域での紫外線照射は、照射領域と近いだけフリル。これはWHIの両方の、我々は、その後世界的に紫外線で細胞を照射し、実際に地元の活動であった、またはバルクメディアにラパマイシンを加えていることを確認するにはchは、膜全体のグローバルフリルの形成を誘導した。定量分析のために、私たちは4つの象限に標的細胞を分割し、フリル、ローカル時の各象限の形成と同様に、コントロール（図4）を含む様々な条件の下でグローバルな刺激後の周波数を数えた。それだけで、UV照射、CRB-、地元の波打ちを誘導する適切なFKBP-FRBの構成要素の組み合わせです。 <img alt="図1" src="/files/ftp_upload/3794/3794fig1.jpg" /> 図1。ケージラパマイシン-アビジンコンジュゲート（CRB-A）と紫外光を用いて空間的に閉じ込められたタンパク質の二量体の模式図。 UV照射は、化学dimerizer（HE-ラパ 、C40-O-（2 -ヒドロキシエチル） -ラパマイシン）とその副産物のリリースでは、その結果、ラパマイシンおよびビオチン間のリンカーを切断する。リリースdimerizerは、細胞内に拡散し、FKBP-POI（興味のあるタンパク質）とプラズマMEMBの間に二量体化を誘導するRANEは、照射領域（青丸）の近くにFRBを固定。 UV照射することなく、FKBPとFRBは、それ故に何の効果も生産しない、（赤い十字）関連付けられているわけではない。 （著作権@ ACS2011）6 <img alt="図2" src="/files/ftp_upload/3794/3794fig2.jpg" /> 図2。カスタマイズされた共焦点蛍光顕微鏡の画像。共焦点顕微鏡の（a）は正面図（Axiovert 200、ツァイスを反転）。 EPI蛍光およびUV照射のためのカップリングユニット（b）のクローズアップ、トップビュー。 <img alt="図3" src="/files/ftp_upload/3794/3794fig3.jpg" /> ローカライズされたUV照射により誘導されるトランスフェクトしたNIH3T3細胞の3。空間に閉じ込めフリルの形成図 。 YFPタグの付いたFKBP-Tiam1（RACアクチベーター）とLDR（FRBを膜に固定）でトランスフェクトされたNIH3T3線維芽細胞の共焦点蛍光画像は、Lとの縁の近くに狭い領域で照射を受けた15秒は、バルクメディアのCRB-共役の存在下で210秒で起動するための365 nmでEDが点灯します。この治療は1063​​年で1秒の水銀ランプとグローバルイルミネーション秒および1560秒でラパマイシン、最終的な400nmのグローバル添加した。セルは初め（）でほとんど波打ち活性を示さなかったが、空間的にローカライズされた照明（黄色の丸）（b）の後の波打ち閉じ込められた認められた。セルは、ローカルフリル形成はRacの空間的に閉じ込められた活性化によって引き起こされたことを示す、グローバルイルミネーションおよびラパマイシン（CD）を添加した後にグローバルなフリルの形成を示した。スケールバー：10μmである。 （著作権@ ACS2011）6 <img alt="図4" src="/files/ftp_upload/3794/3794fig4.jpg" /> 図4：トランスフェクトしたNIH3T3細胞の象限のフリル形成の頻度。 CRB-と、次のグローバルUV照射は、13細胞で行われた。CRB-を使用したローカルのUV照射</strong&gt;さらに（UV照射なし）、アビジンの有無にかかわらず地元のUV照射は、それぞれ、13、5と6のセルに実装されました。観測された細胞のいずれも、UV照射、またはローカルUV照射バルクメディアのアビジンの有無にかかわらずなしCRB-とフリルの形成は認められなかった。我々は低バックグラウンドフリル形成と細胞からデータを収集しますが、これは、我々はラパマイシン（最後の400 nm）の加算の結果として、グローバルなフリルの形成を確認して、それぞれの実験の後にチェックラパマイシン誘発性フリル形成の能力を持っていた。これらの結果は、CRB-およびローカルのUV照射は、細胞内のローカルフリル形成に必要であることを示した。 （著作権@ ACS2011）6 略語： FKBP：FK506結合タンパク質 FRB：FKBP-ラパマイシン結合タンパク質 Tiam1：T-細胞リンパ腫の浸潤や転移を誘発するタンパク質1（RAC GEF） LDR：リン-DSAGリンカーFRB（リン：LynキナーゼのN末端11アミノ酸） HE-ラパ：C40-O - （2 -ヒドロキシエチル） -ラパマイシン CRB：ビオチン部分とケージラパマイシン CRB-：アビジンにコンジュゲートcrbを

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利害の衝突が宣言されません。

我々は、秒の時間スケールでの正確な細胞内の場所でのRho GTPase活性を操作するために、FKBP-FRBヘテロシステムと小説ケージド化合物を一緒に採用してテクニックを説明します。 現在のアプローチの3つの制約があります。方法は、プラズマ膜を横切ってdimerizerの拡散を防止または許可に基づいているので、まず、ターゲット細胞の位置は、原形質膜またはその付近である?...

<table border="1"><tbody><tr><td> 試薬/装置の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>ラパマイシン</td><td> Tecoland </td><td> RAPA99 </td></tr><tr><td>トランスフェHD </td><td>ロシュ社</td><td> 04709691001 </td></tr><tr><td>アビジン</td><td>シグマ</td><td> A9275-10mgの</td></tr><tr><td> DMSO </td><td>シグマ</td><td> D2650-5X5ML </td></tr><tr><td>サイズ排除カラム</td><td> GEヘルスケア</td><td>スピントラップG-25 </td></tr><tr><td>ガラス底8ウェルチャンバー</td><td>サーモフィッシャーサイエンティフィック</td><td> 12-565-47 </td></tr><tr><td>倒立蛍光顕微鏡</td><td>ツァイス</td><td> Axiovert200M </td></tr><tr><td>紫外線LED光源</td><td>ラップオプトエレクトロニクス</td><td> UVILED </td></tr><tr><td>共焦点回転するディスク</td><td>横河電機（株） </td><td> CSU10 </td></tr><tr><td> CCDカメラ</td><td>浜松ホトニクス</td><td> ORCA-ER </td></tr><tr><td> 100倍対物レンズ</td><td>ツァイス</td><td>プランアポクロマート</td></tr></tbody></table> CRBの合成スキーム（著作権ACS2011）6 <a href="/files/ftp_upload/3794/3794scheme1_lg.jpg"><img src="/files/ftp_upload/3794/3794scheme1.jpg" alt="スキーム1" /></a> 合成条件：（a）BnBr、K 2 CO 3、DMF（b）にf.HNO 3、AcOHで（c）にTFA（d）のプロパルギル-Brを、K 2 CO 3、DMF、（e）のグリセロールは、猫。 PTSA、トルエン（F）のNaBH 3 CN、TiCl 4の 、MeCN中（G）のTf 2 O、ピリジン（h）のメタノール塩酸は、（i）のLiAlH 4、THF（J）TSCL、ピリジンのCH 2 Cl 2（K）はNaN 3、DMF（L）8、2,6 -ジ-t-ブチル、CH 2 Cl 2中 （M）13日のCuSO 4、アスコルビン酸、2 -プロパノール、H 2 O、CH 2 Cl 2で 。

spatio-temporal manipulation of small gtpase activity at subcellular level and on timescale of seconds in living cells

大きな時空間精度で小さなグアノシントリホスファターゼのRhoファミリー（GTPアーゼ）の動的な制御は、様々な細胞機能やイベントの1、2のために不可欠です。その時空の動的な性質は、リアルタイム3それらの活性と局在の可視化によって明らかにされています。分子レベルでの多様な細胞機能における役割のより深い理解を得るために、次のステップは、正確な細胞内局在場所とタイミングでタンパク質の活動の摂動である必要があります。 （1）ラパマイシン誘発性FKBP-FRBのヘテロおよびラパマイシン（2）写真ケージング方法：この目標を達成するために、我々は、光誘起、二つの手法を組み合わせることにより、低分子量GTPaseの時空間、時間的に制御されたアクティベーションの方法を開発した。ラパマイシン媒介FKBP-FRBのヘテロを使用すると、我々は急速に低分子量GTPase includiの活性化または不活性化を誘導するための方法を開発しましたngのRAC 4、ラパマイシンは、FRBが固定されている細胞膜に、FKBP-融合GTPアーゼ、またはそれらの活性化の移行を誘導するCdc42の4、RhoAの4とRasの5。このヘテロシステムと結合するために、我々はまた、ラパマイシン類似体の写真ケージシステムを開発した。写真ケージド化合物は、その活動ケージング基として知られている光開裂性保護基で抑制されている小分子である。完全にヘテロの活性を抑制するために、我々はセル6内の化学dimerizerとして事実上のバックグラウンド·アクティビティにつながる、結果として大規模な複合体が細胞膜を通過できないような高分子につながれているケージラパマイシンを設計しました。 図1は、我々のスキームを示していますシステム。これら二つのシステムの組み合わせにより、我々は局所的に数秒の時間スケールで形質膜へのRac活性化を採用し、細胞内レフで光誘起Racの活性化を達成しましたエル6。

Watch this Scientific Journal Video about Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells at JoVE.com

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

光による小さなGTPase活性の時空間制御の方法が記載されている。このメソッドは、ラパマイシン誘発性FKBP-FRBのヘテロと写真ケージシステムに基づいています。光照射の最適化は、細胞内レベルでの低分子量GTPaseの時空間時間的制御の活性化を可能にします。

生きた細胞の細胞内レベルでの秒のタイムスケールの上に小さなGTPase活性の時空間操作

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

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10.7K ビュー.  Johns Hopkins University. 光による小さなGTPase活性の時空間制御の方法が記載されている。このメソッドは、ラパマイシン誘発性FKBP-FRBのヘテロと写真ケージシステムに基づいています。光照射の最適化は、細胞内レベルでの低分子量GTPaseの時空間時間的制御の活性化を可能にします。

ビデオ: Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

生細胞における分子ローターの蛍光寿命イメージング

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

生物工学

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

原子間力顕微鏡を用いた生細胞の機械的特性を測定する

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and KATP channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo tissues.

Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological ...

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a F&ouml;rster resonance energy transfer (FRET) probe in which the &epsilon; subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the &epsilon; subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.

We describe the use of two ratiometric, genetically encoded biosensors, which are based on GFP, to monitor mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells.

リビング酵母細胞におけるミトコンドリア酸化還元状態とATPを測定レシオメトリックバイオセンサー

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

外科的組織からの初代ヒト結腸腫瘍細胞の単離およびトラクションサイトメトリーのためのソフト弾性基板上にそれらを直接培養

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

単一細胞分泌物の時空間イメージング用ラベルフリー技術

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

生細胞共役ポリマーにより電気的活動の光制御

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

ガットオンチップマイクロ流体デバイスにおけるマイクロ加工ヒト腸絨毛と生活Microbiomeの共培養

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

人間歯槽骨膜から骨膜由来細胞の骨形成促進活性に及ぼすビタミン D の間葉系幹細胞の分離

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

蛍光技術の組み合わせを用いた生細胞内力感受性タンパク質ダイナミクスの測定

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...