この作品は、ウェルカムトラスト助成番号[077108]によってサポートされていましたGJWに授与されました。

<ol>
	<li>Fagerberg, L., Jonasson, K., von Heijne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+the+human+membrane+proteome.">Prediction of the human membrane proteome.</a> Proteomics. 10, 1141-1141 (2010).</li><li>Wright, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+initiation+in+biological+systems:+the+properties+and+detection+of+transient+extracellular+protein+interactions.">Signal initiation in biological systems: the properties and detection of transient extracellular protein interactions.</a> Molecular bioSystems. 5, 1405-1405 (2009).</li><li>Bushell, K. M., Sollner, C., Schuster-Boeckler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+screening+for+novel+low-affinity+extracellular+protein+interactions.">Large-scale screening for novel low-affinity extracellular protein interactions.</a> Genome research. 18, 622-622 (2008).</li><li>Durocher, Y., Perret, S., Kamen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-level+and+high-throughput+recombinant+protein+production+by+transient+transfection+of+suspension-growing+human+293-EBNA1+cells.">High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells.</a> Nucleic acids research. 30, e9-e9 (2002).</li><li>Bendtsen, J. D., Nielsen, H., von Heijne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Prediction+of+Signal+Peptides:+SignalP+3.0.">Improved Prediction of Signal Peptides: SignalP 3.0.</a> Journal of molecular biology. 340, 783-783 (2004).</li><li>Krogh, A., Larsson, B., von Heijne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+transmembrane+protein+topology+with+a+hidden+markov+model:+application+to+complete+genomes.">Predicting transmembrane protein topology with a hidden markov model: application to complete genomes.</a> Journal of molecular biology. 305, 567-567 (2001).</li><li>Brown, M. H., Barclay, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+immunoglobulin+and+scavenger+receptor+superfamily+domains+as+chimeric+proteins+with+domains+3+and+4+of+CD4+for+ligand+analysis.">Expression of immunoglobulin and scavenger receptor superfamily domains as chimeric proteins with domains 3 and 4 of CD4 for ligand analysis.</a> Protein engineering. 7, 515-515 (1994).</li><li>Wright, G. J., Puklavec, M. J., Willis, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoid/neuronal+cell+surface+OX2+glycoprotein+recognizes+a+novel+receptor+on+macrophages+implicated+in+the+control+of+their+function.">Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function.</a> Immunity. 13, 233-23 (2000).</li><li>Dustin, M. L., Golan, D. E., Zhu, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+affinity+interaction+of+human+or+rat+T+cell+adhesion+molecule+CD2+with+its+ligand+aligns+adhering+membranes+to+achieve+high+physiological+affinity.">Low affinity interaction of human or rat T cell adhesion molecule CD2 with its ligand aligns adhering membranes to achieve high physiological affinity.</a> The Journal of biological chemistry. 272, (1997).</li><li>Wright, G. J., Martin, S., Bushell, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+identification+of+transient+extracellular+protein+interactions.">High-throughput identification of transient extracellular protein interactions.</a> Biochemical Society transactions. 38, (2010).</li><li>de Wildt, R. M., Tomlinson, I. M., Ong, 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=Isolation+of+receptor-ligand+pairs+by+capture+of+long-lived+multivalent+interaction+complexes.">Isolation of receptor-ligand pairs by capture of long-lived multivalent interaction complexes.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, (2002).</li><li>Jiang, L., Barclay, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+leucocyte+surface+protein+interactions+by+high-throughput+screening+with+multivalent+reagents.">Identification of leucocyte surface protein interactions by high-throughput screening with multivalent reagents.</a> Immunology. 129, 55-55 (2010).</li><li>Urech, D. M., Lichtlen, P., Barberis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+growth+selection+system+to+detect+extracellular+and+transmembrane+protein+interactions.">Cell growth selection system to detect extracellular and transmembrane protein interactions.</a> Biochimica et biophysica acta. 1622, 117-117 (2003).</li><li>Voulgaraki, D., Mitnacht-Kraus, R., Letarte, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivalent+recombinant+proteins+for+probing+functions+of+leucocyte+surface+proteins+such+as+the+CD200+receptor.">Multivalent recombinant proteins for probing functions of leucocyte surface proteins such as the CD200 receptor.</a> Immunology. 115, 337-337 (2005).</li><li>Wojtowicz, W. M., Wu, W., Andre, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vast+repertoire+of+Dscam+binding+specificities+arises+from+modular+interactions+of+variable+Ig+domains.">A vast repertoire of Dscam binding specificities arises from modular interactions of variable Ig domains.</a> Cell. 130, 1134-1134 (2007).</li><li>Martin, S., Sollner, C., Charoensawan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+Large+Extracellular+Protein+Interaction+Network+and+Its+Resolution+by+Spatiotemporal+Expression+Profiling.">Construction of a Large Extracellular Protein Interaction Network and Its Resolution by Spatiotemporal Expression Profiling.</a> Mol. Cell. Proteomics. 9, 2654-2654 (2010).</li><li>Crosnier, C., Bustamante, L. Y., Bartholdson, S. J., Bei, A. K., Theron, M., Uchikawa, M., Mboup, S., Ndir, O., Kwiatkowski, D. P., Duraisingh, M. T., Rayner, J. C., Wright, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basigin+is+a+receptor+essential+for+erythrocyte+invasion+by+Plasmodium+falciparum.">Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum.</a> Nature. 480, 534-537 (2011).</li><li>Sollner, C., Wright, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell+surface+interaction+network+of+neural+leucine-rich+repeat+receptors.">A cell surface interaction network of neural leucine-rich repeat receptors.</a> Genome biology. 10, R99-R99 (2009).</li></ol>

著者らは、開示することは何もありません。

生きている細胞はin vivoで観察された場合、その細胞膜は非常に動的な挙動を示す：彼らが連絡して、彼らの隣人2と通信する際の拡張と後退膜プロセス。これらの接点の多くを仲介する細胞膜に埋め込まれた受容体タンパク質間の物理的相互作用は、この非常に運動性の動作を許可するように非常に弱いように進化してきました。それは50μMとして弱いように単量体の相互作用の強さは非常に2,10に挑戦する新たな相互作用を検出できます生理的受容体密度が9で自発的な相互作用を駆動するのに十分であることと推定されている。低い偽陽性率でスケーラブルな方法で実装することができるタンパク質の相互作用：ここで説明しAVEXISメソッドは一時的な細胞外タンパク質を検出する感度の高い方法を提供します。他のスケーラブルな方法は、11月15日細胞の相互作用を検出するために開発されているが、AVEXISの一つの利点は、そのような餌と捕食活動などの実験パラメータが低い偽陽性率3を維持しながらも（T 1/2≤0.1秒）の相互作用の弱いを検出するために定量化されていることです。さらに、合理化された堅牢な試料調製手順は、他のアッセイよりもはるかに大規模に実装するために、このアッセイを有効にしていると我々は最近〜16500潜在的な相互作用16歳以上の合計体系的相互作用の画面を説明してきました。 アッセイは、可溶性の組換えタンパク質断片を発現させることが可能となっている任意のタンパク質を実行することができます。したがって、これはそのようなI型、GPI-結合及び分泌タンパク質のN末端シグナルペプチドを含むタンパク質が含まれています。我々は最近、今私たちが餌17とタイプIIのタンパク質を発現することができますベクトルのスイートを設計しました。アッセイは、一般的に、イオントランスポーターとして、マルチパスの膜タンパク質には適していませんsまたはポンプが正常に細胞膜のコンテキストの外側で折られることはほとんどありませんので。我々は、異なる様々なシステムにこれを適用していると正常ゼブラフィッシュ3,16,18、ヒト、マウス、およびPから細胞外タンパク質を表明している対話画面での熱帯熱マラリア 。 AVEXIS画面を設定するために必要なリソースの面で、我々は重大なボトルネックが選定、建設、発現プラスミドの完全なシーケンスであることを見出した。メソッドのこの側面は、〜100餌と獲物の構造を作るために年に取ることができますが、遺伝子合成の減少コストで、我々は現在、市販プロジェクトのこの側面をアウトソーシング好む。大揺れ組織培養インキュベーター（我々はINFORS HT Multitronセルを使用してください）​​と一緒に哺乳動物発現系は発現し、約100餌と獲物タンパク質月までに正規化する単一の研究者を可能にします。タンパク質が生成し、s、正規化されたら、相互作用のためにcreeningする便利な一日あたり処理されている12までの96ウェルプレートで迅速です。我々は、タンパク質のような大規模な数の生産を容易にするために開発されていることを唯一のオーダーメイドの装置が並列に0.22μmフィルターを通して5上清に渡すために使用されている圧縮空気駆動型ピストンです。 これらの相互作用のいくつかは3を検出することができますが、文献（偽陰性）から予想される相互作用に比べ見逃されるかもしれない相互作用のメインクラスは、同種の相互作用があります。予想される相互作用が検出されない場合には、私たちは、これがその後固定化した餌のタンパク質との相互作用を防ぐことができ、さらに餌タンパク質による同種の相互作用の形成（獲物 &quot;マスキング&quot;効果）によるものであると信じています。相互作用が検出された周波数は、スクリーニングされている蛋白質ライブラリのコンテンツに依存します。読者へのガイド&gt; 30の画面として、ゼブラフィッシュ免疫グロブリンスーパーファミリー内の000の相互作用は、188陽性の結果- 0.6パーセント3,16,18の周波数。つまり、結合タンパク質のいずれかの餌として存在するかどうかにかかわらず、同じ相互作用を検出することができます。任意の正のヒットを確認するために実行することができます非常に迅速なチェックでは、相互作用は、両方の餌 - 捕食の方向で検出することができるかどうかを判断することですまたは獲物。我々はこの往復動作を観察したときは、バインディングが続いて表面プラズモン共鳴を用いたタンパク質の精製直接飽和結合を示すことにより、特定であることが示されている。それは我々が人工的に餌のタンパク質をpentamerisingによって結合親和性を高めるために、我々は定量的に相互作用強度を比較するための適切なアッセイを考慮していないことを強調する必要があります。相互作用の生物物理学的結合パラメータを取得するために我々は、単量体蛋白質と表面プラズモン共鳴を使用しています。最後に、かつての相互作用が同定された、ハイアッセイのGHスループットの性質は、そのような抗体または相互作用をブロックする小分子などの試薬をスクリーニングするアッセイ法を適応することが比較的容易になります。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> 猫。数 </td><td> コメント </td></tr><tr><td>フリースタイルメディア</td><td>インビトロジェン</td><td> 12338018 </td><td></td></tr><tr><td>ニトロセフィン</td><td>カルビオケム</td><td> 484400 </td><td></td></tr><tr><td> SAコーティングされたマイクロタイタープレート</td><td>ヌンク</td><td> 734-1284 </td><td></td></tr><tr><td> OX68抗体</td><td> AbDSerotec </td><td> MCA1022R </td><td></td></tr><tr><td>透析チューブ</td><td>ピアース</td><td> PN68100 </td><td></td></tr><tr><td>ポリミキシンB </td><td>シグマ</td><td> P4932 </td><td></td></tr><tr><td> D-ビオチン</td><td>シグマ</td><td> B4639-5G </td><td></td></tr><tr><td>基板-104 </td><td>シグマ</td><td> 1040-506 </td><td></td></tr><tr><td> Vivaspin-20遠心コンセントレータ</td><td>縫工筋</td><td> VS2002 </td><td></td></tr><tr><td> 0.22μmのフィルター</td><td>ナルゲン</td><td> 190-2520 </td><td></td></tr></tbody></table>

avidity-based extracellular interaction screening (avexis) for the scalable detection of low-affinity extracellular receptor-ligand interactions

細胞外タンパク質の分泌や膜繋留タンパク質間のタンパク質相互作用は、細胞間の通信を開始し、多細胞生物の中で凝集を確保するため、両方のために重要である。細胞の相互作用を形成すると予測タンパク質はヒトの遺伝子1の約4分の1でエンコードされていませんが、その重要性と豊かさにもかかわらず、これらのタンパク質の大部分は結合パートナーをまとめました。主に、これは彼らの生化学的な難しによるものです。膜に埋め込まれたタンパク質は、それらのネイティブなコンフォメーションに可溶化することが困難であり、構造的に重要な翻訳後修飾が含まれています。また、受容体タンパク質との相互作用の親和性は、しばしば多くの一般的に使用される高スループットの方法2でそれらの検出を排除し、極めて低い相互作用の強さ（半減期&lt;1秒）によって特徴付けられる。 ここでは、アッセイ、AVEXIS（親和性ベースの細胞外を記述する低い偽陽性率は3で、非常に弱いタンパク質相互作用の検出（T 1/2≤0.1秒）を有効にするこれらの技術課題を克服LARインタラクション画面）。アッセイは、通常、便利なマイクロタイタープレートフォーマットの相互作用（図1）の何千の体系的なスクリーニングを有効にするには、ハイスループット形式で実装されています。それは相互作用をスクリーニングするためにその中に細胞表面の受容体または分泌タンパク質の細胞外ドメインのフラグメントを含む可溶性組換えタンパク質ライブラリーの生産に依存しているため、このアプローチでは、タイプに適しているI、II型、GPIアンカー型細胞表面受容体と分泌このようなイオンチャネルやトランスポーターとして、マルチパスの膜タンパク質のタンパク質ではなく。 組換えタンパク質のライブラリは、そのようなglycosylaなどの重要な翻訳後修飾を確保するために、便利な高レベルの哺乳類発現系4を使用して生成されまするとジスルフィド結合が追加されます。発現した組換えタンパク質が培地中に分泌され、二つの形式で生成されます。スクリーニングに適したストレプトアビジンでコーティングされた固相上に捕捉し、pentamerised酵素タグを付けることができ、ビオチン化餌（β-ラクタマーゼ）餌食。餌と獲物タンパク質は、従来のELISA（図1）に似て、それらの間の直接的な相互作用を検出するためにバイナリ形式でお互いに提示されます。捕食中のタンパク質のpentamerisationは軟骨オリゴマーマトリックスタンパク質（COMP）からペプチド配列によって達成し、それにより検出されるにも非常に一時的な相互作用を有効にするには、重要な親和性の向上を提供する外部ドメインの局所濃度を増加させています。餌とスクリーニングの前に所定のレベルに獲物の両方の活動を正規化することによって、我々は0.1秒の単量体の半減期を持つ相互作用が低い偽陽性率3で ​​検出できることが示されている。

Watch this Scientific Journal Video about Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions at JoVE.com

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXISは、細胞認識のプロセスに関与する新規細胞外受容体 - リガンドペアの体系的に画面に開発した高スループット·タンパク質相互作用アッセイである。これは特に、他の高スループットアプローチを用いて識別することが困難である一時的なタンパク質相互作用を検出するように設計されています。

低親和性細胞外受容体 - リガンド相互作用の検出のためのスケーラブルな親和性ベースの細胞外相互作用のスクリーニング（AVEXIS）

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

avidity-based-extracellular-interaction-screening-avexis-for-scalable

Research

JoVE Journal

Biology

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

21.4K ビュー.  Wellcome Trust Sanger Institute. AVEXISは、細胞認識のプロセスに関与する新規細胞外受容体 - リガンドペアの体系的に画面に開発した高スループット·タンパク質相互作用アッセイである。これは特に、他の高スループットアプローチを用いて識別することが困難である一時的なタンパク質相互作用を検出するように設計されています。

ビデオ: Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

細胞外多糖のオリゴマスプロファイリング（オリンプ）

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

生物学

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

フローサイトメトリーを用いて細胞外小胞の分析のための技術

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

イオン選択自己参照微小電極法を用いた細胞外イオンフラックスの測定

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

効率的な哺乳動物細胞発現および結晶化のための細胞外糖タンパク質のシングルステップの精製

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

細胞外マトリックスを視覚化するために浸軟組織の電子顕微鏡をスキャンします

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

細胞由来の細胞外マトリックスの急速な、単離、機能性研究のためのスケーラブルな方法、および分析

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

酸性細胞外 pH 培養系の確立

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

A BW Reporter System for Studying Receptor-Ligand Interactions

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native receptor and the ligand are challenging since the ligand of a specific receptor may be unknown and experimental procedures with the native ligand can be technically complicated. To address these obstacles, we describe a reporter system to detect the binding and activation of a specific receptor by a ligand of interest. In this reporter system, the extracellular domain of a specific receptor is conjugated to mouse CD3&#950; and this chimeric protein is then expressed in mouse BW cells. These transfected BW cells can then be incubated with different targets (e.g., cells or antibodies). Activation of a transfected receptor leads to the secretion of mouse interleukin-2 (mIL-2) which can be detected by enzyme-linked immunosorbent assay (ELISA). This reporter system has the advantages of being sensitive and specific to a single receptor. In addition, the activation level of a specific receptor can easily be quantified and can be used even in cases where the ligand of the receptor is unknown. This system has been implemented successfully in many of our studies to characterize receptor-ligand interactions. We have recently employed this system to study the activation of human Fc&#947; receptors (Fc&#947;Rs) by different monoclonal anti-CD20 antibodies in clinical use.

This protocol describes how to establish a reporter system that can be used to identify and quantify receptor-ligand interactions.

受容体-リガンド相互作用を研究するための BW レポーター システム

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native ...

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

細胞外小胞の定量とサイズ決定のためのナノ粒子追跡解析

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular communication in various research fields; cancer biology, neuroscience, and drug delivery. Many EV uptake assays have been reported in the literature; however, there is a lack of practical, detailed experimental methodology. EV uptake can be assessed by fluorescently labeling EVs to detect their location within cells. Distinguishing between internalized EVs in cells and the superficial EVs on cells is difficult, yet critical, to accurately determine the EV uptake. Therefore, an assay that efficiently quantifies EV uptake through three-dimensional (3D) fluorescence confocal microscopy is proposed in this work. Fluorescently labeled EVs were prepared using a nano-filtration-based microfluidic device, visualized by 3D confocal microscopy, and then analyzed through advanced image-processing software. The protocol provides a robust methodology for analyzing EVs on a cellular level and a practical approach for efficient analysis.

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Extracellular vesicles (EVs) contribute to cellular biology and intercellular communications. There is a need for practical assays to visualize and quantify EVs uptake by the cells. The current protocol proposes the EV uptake assay by utilizing three-dimensional fluorescence imaging via confocal microscopy, following EV isolation by a nano-filtration-based microfluidic device.

共焦点顕微鏡イメージング解析による細胞外小胞取り込みアッセイ

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular ...