我々は彼女の親切な助けのためにリッパウォーレンに感謝します。

1。境界アッセイ： <ol><li>基本的な準備：チャンバースライドは、ポリ- D -リジン一晩滅菌ガラススライドでコーティングされている実験を開始する前に用意されています。培地は10％ウシ胎児血清（FCS）、および1％ペニシリン - ストレプトマイシン- Fungizon（PSF）を添加したダルベッコ改変イーグル培地（DMEM）を用いて調製される。また、カルシウム/マグネシウムフリーハンクス平衡塩溶液（HBSS）のボトルが用意されています。 </li><li>フラスコで培養したラット初代シュワン細胞は、0.1％トリプシンを用いて3分間トリプシン処理されています。初代ラットアストロサイトの培養は、0.1％トリプシンを用いて5分間トリプシン処理しています。 トリプシンは、DMEMを加えると10％FCS、1％PSFを補充によってinactivedている。 </li><li>トリプシン処理シュワン細胞と星状膠細胞を15mLファルコンチューブを分離するために移し、5分間300Gで遠心分離されています。 </li><li>シュワン細胞とアストロサイトは2 × 10 6（細胞密度の変動のための代表的な結果のセクションを参照）の密度で再懸濁する。 </li><li>シュワン細胞懸濁液50μLの低下は、PDLコーティングチャンバースライドの井戸の一端に配置されます。ガラスのストリップは、チャンバの中心に向かって降下はストレートエッジを生成するためにスメアするために使用された。 （ガラスカッターを使用して、長方形のガラス製カバースリップは、それが境界のアッセイに使用されるチャンバースライドの幅に収まるだろうと縦方向にようにカットされます） </li><li>アストロサイトの懸濁液50μLの番目のドロップは同じ井戸の反対側の端に置かれ、それらの間の小さな0.2ミリメートルのギャップを持つ最初のドロップ塗抹標本に平行なエッジを作成するために中心に向かって塗抹した。 </li><li>チャンバーは、ドロップが2時間37℃のインキュベーター内に置かれている含まれているスライド。 </li><li> 2時間後、培養物を、非添付の細胞残渣およびDMEM / 10％PSFが追加さFCS / 1％を含む新鮮な培地を除去するDMEMで洗浄した。 注：DMEM / 10％FCS / 1％PSFは、シュワン細胞の増殖を刺激するフォルスコリン（2μmの）とウシ下垂体抽出物（BPE）（10μgの/ mL）で補足することができます。 BPEは、長期的なシュワン細胞の培養を可能にするために必要な成長因子を供給し、フォルスコリンは、細胞増殖を誘導するシュワン細胞内サイクリックアデノシン一リン酸のレベルを発生させます</li><li>細胞を37 ° C、7％CO 2インキュベーターで約8-10日間培養されています。培地は3日ごとに変更する必要があります。つのセルの前線は、最終的にお互いに達し、鋭い境界は、2種類の細胞型との間で形成されます。この段階での実験は、境界形成に関与する因子を分析を行うことができます。 </li><li>共培養は4％ホルムアルデヒドとシュワン細胞を用いて固定することができる共培養中の細胞を識別するために、抗GFAP抗体と抗P75抗体およびアストロサイトを用いて免疫染色することができます。 P75抗体は、アストロサイトは欠いているシュワン細胞上の低親和性NGF受容体を認識する。抗GFAP抗体は、アストロサイトの細胞質に局在するグリアfirbrillary酸性タンパク質を認識する。 （代表的な境界については、図1Aを参照してください） </li></ol> 2。マイグレーションアッセイ： <ol><li> 4ウェルプレートには、（15mm厚井戸）アストロサイト単分子膜を作成するためのPDLコーティング晩と準備です。 </li><li>一次星状膠細胞培養は、0.1％トリプシンを用いて5分間トリプシン処理しています。トリプシンは、DMEM、10％FCS、1％PSFと5分間300Gで遠心分離し細胞を添加した添加することにより不活化される。 （図2を参照） </li><li>アストロサイトは、1 × 10 5細胞/ mLで再懸濁する。この溶液1mlは、4ウェルプレートの各ウェルPDLコートされたに追加されます。単分子層が完全にコンフルエントになるまで、アストロサイトは、24〜48時間培養する。 </li><li>アストロサイト単分子層の作成と平行状細胞単層上のシュワン細胞の遊走を、評価するために、円形のPDLコートされたガラス製カバースリップを50 mLのチューブに入れ、ガラスの小さな断片を作成するためにプラスチック製のピペットを用いて粉砕されています。 </li><li>ガラスの破片を6ウェルプレートの皿にウェルに移し、ピンセット、適切なサイズのフラグメントを使用して選択し、他のウェルに配置されている。 5ガラスの破片を6ウェルプレートの各ウェルに配置されます。 </li><li>フラスコで培養した主なシュワン細胞は、0.1％トリプシンを用いて3分間トリプシン処理されています。トリプシン処理シュワン細胞を15 mlのFalconチューブを分離するために移し、5分間300gで遠心分離されています。シュワン細胞は、DMEM/10％FCS / 1％PSFで2倍に10 6細胞/ mLに再懸濁されている。 </li><li>シュワン細胞懸濁液20μL滴を6ウェルプレートとセルに入れ、各5カバーガラスの破片で37インキュベートしている° C、7％CO 2で2時間かけて添加される。 </li><li> 2時間後、シュワン細胞は、カバーガラスの破片とDMEM/10％FCS / 1％PSF（BPEのフォルスコリン及び10μgの/ mLの2μmのがそれぞれに追加されるもので、断片が完全に培地に覆われていることを補足した）に接続されている。 </li><li>ガラスの破片が完全にCONになるまでの断片を含む6ウェルプレートは、24〜48時間インキュベーター内に配置されていますシュワン細胞と流暢。 </li><li>一度フラグメントはシュワン細胞でコンフルエントの、各断片は、慎重に鋭いピンセットを使って拾い、アストロサイトの単分子層への面を下に反転してアタッチされていない細胞を除去し、HBSS浸漬される。 </li><li>各ウェルは、FCS / 1％PSFとシュワン細胞は24から48までの期間のために移行するために許可されているDMEM/10％1mLで覆われている。この期間中にそのような増殖因子として実験的な試薬は、シュワン細胞の遊走の数と距離に与える影響を評価するために追加することができます。 </li><li>移行期間に続いて、ガラスの破片は30分間、4％パラホルムアルデヒド（PFA）を追加することにより、各ウェルの底部に固定されています。固定に続いて、P75（低親和性NGF受容体を認識する）ためのシュワン細胞を免疫染色し、それらを光学顕微鏡下でシュワン細胞の可視化を可能にするためにABCストレパビジンキット - ジアミノベンジジン（DAB）を、使用して染色。 </li></ol> 3。代表的な結果： 8-10日以下のシュワン-アストロサイト共培養は、2種類の細胞型との間の鋭い境界を示す。シュワン細胞の増殖が増強されるフォルスコリンおよびBPEは、培養に使用されている場合、この境界はより顕著です。 は境界が10日間の期間中に観察されていない場合の境界が確立されるまで、文化の時間がさらに増加することができます。細胞密度の比を変更すると、境界形成を増加させる別の方法です。 3時01分シュワンの比率：細胞懸濁液8,18を準備する際にアストロサイトの細胞密度を使用することができます。 ラインを混ざりセルを評価するために、シュワン細胞やアストロサイトを識別するには、以下の免疫染色は、境界が確立されており、アストロサイトの領域に移行するシュワン細胞の数を蛍光顕微鏡下でカウントすることができますどこに描画することができます。 下の画像取得の境界、整形式のシュワン細胞とアストロサイトの分離（図1A）と細胞が悪い手法（図1B）のためにも別の地域に分離していないものと、持たないものの2つの例を示します。 <img src="/files/ftp_upload/2214/2214fig2.jpg" alt="図1" /> 図1境界アッセイ：シュワン-アストロサイト相互作用。シュワン細胞の増殖を刺激するフォルスコリンとBPEの存在下でA）10日間共培養アッセイは、2種類の細胞（赤= P75、緑= GFAP）との間の鋭い境界を示しています。 B）境界が形成されていない10日間共培養アッセイおよび二つの細胞の前線が混在。 （赤= P75、緑= GFAP）の境界の準備中に隣同士に星状膠細胞とシュワン細胞の液滴を配置しながら、これは2つの液滴の混合することによるものです。 <img src="/files/ftp_upload/2214/2214fig1.jpg" alt="図2" /> 図2。コンフルエントアストロ単層の代表画像マイグレーションアッセイは、別の細胞型の表面上で1つの細胞型の動きを評価するため、境界の形成とは異なる現象を評価する。 練習は単分子膜上にカバースリップが鋭い鉗子によるシュワン細胞に覆われたガラスの破片やアストロサイト単層に損傷が発生するしない反転確保するために必要とされる。シュワン細胞の遊走を評価するために、移行の最大距離とカバースリップの端から移行する細胞の数を光学顕微鏡下で測定することができます。 下記のアストロサイト単分子層の表面（図3）にDAB染色を用いてP75抗体で染色されたシュワン細胞のイメージがあります。 <img src="/files/ftp_upload/2214/2214fig3.jpg" alt="図3" /> 逆カバースリップのアッセイを用いて図3。移行アッセイ。 A）シュワン細胞（茶色、P75抗体で免疫染色）カバースリップの端から離れて移行。離れてカバースリップから細胞移動の方向に矢印が。

<ol>
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利害の衝突は宣言されません。

上記のアッセイは、シュワン-アストロサイトとアストロサイトの環境に限定されたシュワン細胞の遊走との間の境界形成に関与する複数の要因の役割を示す様々な研究で使われてきました。 それは戦略の開発を最適化できると移植後のシュワン細胞移植の統合を強化し、そうすることで可能に宿主組織に移植片の背面からの再生軸索の出口を促進するとして、これらの?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41966-029</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14170-088</td>
<td>&nbsp;</td>
<tr>
<td>FCS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10091-148</td>
<td>&nbsp;</td>
<tr>
<td>PSF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240-062</td>
<td>&nbsp;</td>
<tr>
<td>BPE</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13028-014</td>
<td>&nbsp;</td>
<tr>
<td>Forskolin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>344273</td>
<td>&nbsp;</td>
<tr>
<td>Coverlips</td>
<td>&nbsp;</td>
<td>VWR-International</td>
<td>631-0149</td>
<td>&nbsp;</td>
<tr>
<td>Coverlips(rectangle)</td>
<td>&nbsp;</td>
<td>MENZEL-GLASER (Supplied by Thermo Scientifics)</td>
<td>BB022050A1</td>
<td>&nbsp;</td>
<tr>
<td>Chamber slides</td>
<td>&nbsp;</td>
<td>Nunc-LabTek </td>
<td>177380</td>
<td>&nbsp;</td>
<tr>
<td>4 well plates, 6 well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>&nbsp;</td>
<td>4 well plates 
6 well plates</td>
</tr>
<tr>
<td>rat p75 antibody</td>
<td>&nbsp;</td>
<td>Millipore, Temecula,California</td>
<td>MAB365</td>
<td>&nbsp;</td>
<tr>
<td>GFAP antibody</td>
<td>&nbsp;</td>
<td>Dako</td>
<td>Z0334</td>
<td>&nbsp;</td>
<tr>
<td>Secondary antibodies (Alexa-conjugated)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-11004 A-11034</td>
<td>Goat anti mouse 568 
Goat anti Rabbit 488</td>
</tr>
<tr>
<td>Secondary biotinylated </td>
<td>&nbsp;</td>
<td>Vector</td>
<td>&nbsp;</td>
<td>Goat anti mouse</td>
</tr>
<tr>
<td>DAB tablets </td>
<td>&nbsp;</td>
<td>Sigma-aldrich</td>
<td>D4418</td>
<td>&nbsp;</td>
<tr>
<td>ABC elite kit</td>
<td>&nbsp;</td>
<td>Vector</td>
<td>PK-6100</td>
<td>&nbsp;</td>
<tr>
<td>Fine Forceps </td>
<td>&nbsp;</td>
<td>FST</td>
<td>11295-10</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P6168</td>
<td>&nbsp;</td>
<tr>
<td>Fluorosave</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>345789</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

analysis of schwann-astrocyte interactions using in vitro assays

シュワン細胞は、脊髄損傷、以下の修復方法で一般的に使用される細胞の一つです。シュワン細胞は軸索再生をサポートしていると分泌成長因子の1,2により、発芽と接着分子3と細胞外マトリックス分子4を成長促進を提供することが可能です。さらに、彼らは怪我5部位で脱髄軸索をmyelinate。 しかし移植後、シュワン細胞は、インプラントのサイトから移行しないと、ホストアストロ6,7と混合しないでください。シュワン細胞とアストロサイトとの間の鋭い境界の形成にこの結果、近位および遠位宿主組織に移植バックを終了しようとして成長する軸索のための障害物を作成する。シュワン細胞と接触するアストロサイトはまた、肥大とアップレギュレーション阻害分子8-13を受ける。 in vitroアッセイモデルシュワン細胞-アストロサイト相互作用するために使用されており、細胞挙動の根底にあるメカニズムを理解する上で重要な役割を演じてきた。 in vitroアッセイこれらは、共培養は、2つのセルの前線を分離するだけの小さなギャップと異なる地域を占める各セルのタイプと二つの異なる細胞を用いて作られている境界のアッセイを、含まれています。細胞が分裂と移行するように、2つの細胞の前線が近づくごとに、他に取得し、最終的に衝突する。これは2つの細胞集団の振る舞い​​が境界で分析することができます。同技術のもう一つの変化は、文化の2つの細胞集団を混合し、時間をかけて2種類の細胞型は、シュワン細胞と分離するとともに、複数のシュワン-アストロサイトの境界を作成するアストロサイトとの間の島々として一緒に凝集することです。 2種類の細胞型の相互作用を研究に使用される第二のアッセイは、細胞の動きが他の細胞型単層14,15の表面に追跡することができます遊走アッセイです。このアッセイは、一般的に反転したカバースリップのアッセイとして知られています。シュワン細胞は、小さなガラスの破片上で培養され、それらは、アストロサイト単分子膜の表面に顔を下に反転していると、移行はカバースリップの端から評価される。 両方のアッセイは、細胞の排除と境界形成に関与する根本的なメカニズムの研究に尽力されている。これらの手法を用いて同定された分子の一部はN -カドヘリン15、コンドロイチン硫酸プロテオグリカン（CSPGs）16,17、FGF /ヘパリン18、エペソ/エフリン19を含む。 この記事では、段階的に境界の分析および移行のアッセイを記述すると発生する可能性のある技術的な問題を解明していきます。

Watch this Scientific Journal Video about Analysis of Schwann-astrocyte Interactions Using In Vitro Assays at JoVE.com

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

この記事では、段階的に記述しようとする、一般的に使用される<em&gt; in vitroで</emシュワン細胞asrtocyte相互作用を研究に使用される&gt;アッセイ。

使用シュワン-アストロサイト相互作用の解析インビトロアッセイ

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

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Biology

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

12.1K ビュー.  University of Cambridge. この記事では、段階的に記述しようとする、一般的に使用される in vitroでアッセイ。

ビデオ: Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

磁気変調バイオセンシングシステムを用いた生物学的アッセイの迅速な均質な検出

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

生物学

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

PDZ-依存性のCFTRの高分子シグナル伝達複合体のin vitroでの解析で

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

インビトロでの細胞遊走および浸潤アッセイにおける

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3&#039; End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

事前のmRNA基質の3 &#39;末端切断：無細胞系を用いたRNAプロセシング反応の解析<em&gt; in vitroで</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

高リン酸化タウタンパク質を用いたインビトロ凝集アッセイにおいて

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

SPRi、ナノのSPRiおよびELISAアッセイを用いて血清中のヒト成長ホルモンの比較分析

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

細胞外のフラックスを用いた Explanted 網膜組織のエネルギー代謝の測定

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

酵母におけるポリグルタミン毒性の試金を使用して融合パートナーの蛍光タンパク質の効果

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

2段階PCRと次世代16S rRNA遺伝子シーケンシングを用いての微生物叢解析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

イン・ヴィトロ E3ユビキチンリガーゼ関数の解析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...