著者は、このデバイスの設計の過程で有用な議論をサルマンKhetani、ジャレッドアレン、クリスFlaim、とオースティンDerfusに感謝。この作品は、全米科学財団学部初期のキャリア開発プログラム、糖尿病および消化器腎臓病の健康/国立研究所の国立研究所、そしてダビデとルシールパッカード財団によってサポートされていました。 EEHはルースL.キルシュシュタイン国立リサーチサービス賞によってサポートされていました。

培養細胞の調製： <ol><li>プラズマ処理したポリスチレンでコーティングされたシリコンのパーツで始まります。 </li><li>適切な細胞外マトリックスタンパク質とコートの部品は、所望の細胞タイプの添付ファイルをサポートする。肝細胞の場合は、37で50 g / mlのコラーゲン- 1溶液で45分間でインキュベートする。 3T3線維芽細胞の場合は、何の行列は必要ありません。 </li><li>連絡先の設定で補完的な部品と部品をロックします。 </li><li>殺菌に10分の最小値を70％エタノールに浸す。細胞培養培地でのddH 2 O、および1xの2倍すすぎます。 </li><li>適切な培養培地中に500,000個の細胞/ mlの濃度で種子の細胞。 12穴培養プレートの各ウェルに1 mlを使用してください。 </li><li>均等に再サスペンド細胞に15分毎に振り、1時間インキュベートする。 </li><li>コンフルエントな単層は、1時間後に達成されていない場合は、新鮮な懸濁液で細胞懸濁液と繰り返しシードを吸引除去する。所望の細胞密度が達成されるまで繰り返します。 </li><li>補完的な部品を取り外します。新鮮なウェルに部品を移し、細胞が付着して完全に広めるためにできるように、一晩インキュベートする。 </li><li>ギャップや連絡先の構成のいずれかに該当する部分をロックすることでフォームの共培養。構成は、培養中に任意の時点で変更される場合があります。 </li><li>メディアを変更する場合、好ましくはわずか1秒か2秒、できるかぎり短い時間のために細胞が乾燥したままにするように注意してください。片手で液体を引くとすぐにもう一方の手を使用してメディアを交換してください。 </li></ol>再利用のための処理のシリコンの部品： <ol><li>漂白剤に浸漬し、水洗して細胞を剥がします。 </li><li>部品が完全に乾いてから。ポリスチレンを除去する2時間トルエンに浸す。 </li><li> 120℃に加熱：ピラニア溶液（H 2 SO 4 1時02分、H 2 O 2）で清掃してください</li><li>のddH 2 Oの連続的な流れの下で15分間リンスすぐにポリスチレンを入金する予定がない場合は、水に部品を保管してください。 </li><li>は100 mg / mlでトルエンでポリスチレンを溶かす。約1時間、ポリプロピレンコニカルの渦、または完全に溶解するまで。溶液1 mLよりも少し毎に10部が必要です。 </li><li> 30秒2,400 rpmで、個々のシリコンの部分にスピンコートポリスチレン溶液。 </li><li> 120℃で少なくとも5時間焼く</li><li> 1分間の酸素プラズマ（200ミリトル、200 W）で扱います。 </li></ol>

<ol>
	<li>Hui, E. E., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromechanical+control+of+cell-cell+interactions.">Micromechanical control of cell-cell interactions.</a> Proceedings of the National Academy of Sciences. 104, 5722-5726 (2007).</li><li>Bhatia, S. N., Balis, U. J., Yarmush, M. L., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cell-cell+interactions+in+preservation+of+cellular+phenotype:+cocultivation+of+hepatocytes+and+nonparenchymal+cells.">Effect of cell-cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells.</a> The FASEB Journal. 13, 1883-1900 (1999).</li><li>El-Ali, J., Sorger, P. K., Jensen, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+on+Chips.">Cells on Chips.</a> Nature. 442, 403-411 (2006).</li></ol>

The authors have nothing to disclose.

このシステムは動的に細胞レベルで操作することが組織の空間的な組織を可能にするという点でユニークです。そのため、このデバイスは、そのような細胞間シグナリングのダイナミクスなどのトピック、接触を介した対水溶性シグナル伝達、細胞の運命決定、毒物学、および細胞のクロストークをスパニング、小説生物学的実験の数を有効にしている。培養基質は、標準組織培養プラスチックであるため?...

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		<th>Material Name</th>
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<td>Silicon Comb Substrates</td>
<td>Tool</td>
<td>N/A</td>
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<td>Parts are available for collaborative research projects. Please contact Elliot Hui (eehui @ alum . mit . edu) or Sangeeta Bhatia (sbhatia @ mit . edu).</td>
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silicon microchips for manipulating cell-cell interaction

細胞内微小環境の役割は、開発から悪性形質転換への事実上全ての哺乳動物の組織で細胞の運命と機能を決定する際に重要と認識されている。特に、隣接する間質との相互作用は、生物学的現象の茄多に関与されているが、従来の手法はこのような相互作用の空間的および動的な要素を調べるために能力を制限する。 マイクロメカニカルリコンフィギュラブル文化（RC）で、我々は、動的機械的な再配置を介して細胞間相互作用を制御するための可動部に微細加工シリコン基板を採用しています。以前、この方法は時間依存の相互作用および可溶性シグナル1の限られた範囲を示す、肝細胞および非実質細胞の共培養では細胞間の通信を調査するために適用されています。 ここで、我々は詳細にRCシステムの製造および使用について説明します。我々は、ギャップとの接点構成（細い80μmのギャップにより、または直接密着して分離した細胞集団）との間で作動を含めてピンセットを使用してデバイスの部品の取り扱いを示すことから始めます。次に、我々は細部文化の基質、およびコンフルエント細胞単層を得るために必要なマルチステップの細胞播種のプロセスを準備するプロセスを。ライブ顕微鏡を用いて、我々は、異なる可能性が実験的なコンフィギュレーション間の細胞のリアルタイム操作を示しています。トルエンとピラニア洗浄、ポリスチレンのコーティング、および酸素プラズマ処理：最後に、我々は再利用のためのデバイスの表面を再生するために必要な手順を示しています。

Watch this Scientific Journal Video about Silicon Microchips for Manipulating Cell-cell Interaction at JoVE.com

Silicon Microchips for Manipulating Cell-cell Interaction

この記事では、マイクロメートルスケールでの接着細胞間の細胞 - 細胞相互作用の動的な制御のための実験的アプローチを説明します。肝細胞と間質細胞との間の細胞間コミュニケーションの操作が示されている。開発プラットフォームは、開発や病因を含む生物学的過程、様々な細胞間相互作用の調査を可能にします。

細胞間相互作用を操作するためのシリコンマイクロチップ

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from ...

silicon-microchips-for-manipulating-cell-cell-interaction

Research

JoVE Journal

Biology

10.7K ビュー.  MIT - Massachusetts Institute of Technology. この記事では、マイクロメートルスケールでの接着細胞間の細胞 - 細胞相互作用の動的な制御のための実験的アプローチを説明します。肝細胞と間質細胞との間の細胞間コミュニケーションの操作が示されている。開発プラットフォームは、開発や病因を含む生物学的過程、様々な細胞間相互作用の調査を可能にします。

ビデオ: Silicon Microchips for Manipulating Cell-cell Interaction

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult retinal neurons in vitro. Procedures for preparing retinal cell cultures start with the dissection, digestion and trituration of the retina, and end with the plating of isolated retinal cells on dishes made especially for use with laser tweezers. These dishes are divided into a cell adhesive half and a cell repellant half. The cell adhesive side is coated with a layer of Sal-1 antibodies, which provide a substrate upon which our cells grow. Other adhesive substrates could be used for other cell types. The cell repellant side is coated with a thin layer of poly-HEMA. The cells plated on the poly-HEMA side of the dish are trapped with the laser tweezers, transported and then placed adjacent to a cell on the Sal-1 side to create a pair. Formation of cell groups of any size should be possible with this technique. "Laser-tweezers-controlled micromanipulation" means that the investigator can choose which cells to move, and the desired distance between the cells can be standardized. Because the laser beam goes through transparent surfaces of the culture dish, cell selection and placement are done in an enclosed, sterile environment. Cells can be monitored by video time-lapse and used with any cell biological technique required. This technique may help investigations of cell-cell interactions.

This video describes the manipulation of cultured neurons using laser tweezers in vitro.

体外で分離された神経細胞を操作するためのレーザーピンセットを使用して

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult ...

生物学

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

タンパク質の精製で使用するための自​​動化された疎水性相互作用クロマトグラフィーカラムの選択

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

タンパク質 - タンパク質相互作用を研究するために磁気ビーズを用いて液相アフィニティキャプチャーアッセイ：ポリオ-nanobodyの例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

マウス涙腺を操作する

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

ベンサミアーナ タバコ葉蛋白質蛋白質の相互作用ダイナミクスの一過性の決定のための試金補完ルシフェラーゼ イメージング

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

免疫蛍光によるDNA修復タンパク質相互作用の可視化

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA (known as BioID) have proven successful in identifying protein-protein interactions (PPIs). APEX, BioID, and TurboID, a revised version of BioID have some restrictions in addition to being valuable technologies. The recently developed AirID, a novel version of BioID for proximity identification in protein-protein interactions, overcame these restrictions. Previously, AirID has been used in animal models, while the current study demonstrates the use of AirID in plants, and the results confirmed that AirID performs better in plant systems as compared to other PL enzymes such as BioID and TurboID for protein labeling that are proximal to the target proteins. Here is a step-by-step protocol for identifying protein interaction partners using AT4G18020 (APRR2) protein as a model. The methods describe the construction of vector, the transformation of construct through agroinfiltration, biotin transformation, extraction of proteins, and enrichment of biotin-labeled proteins through affinity purification technique. The results conclude that AirID is a novel and ideal enzyme for analyzing PPIs in plants. The method can be applied to study other proteins in plants.

Here, we present a step-by-step protocol for performing the proximity labeling (PL) experiment in cucumber (Cucumis sativus L.) using AT4G18020 (APRR2)-AirID protein as a model. The method describes the construction of a vector, the transformation of a construct through agroinfiltration, biotin infiltration, protein extraction, and purification of biotin-labeled proteins through affinity purification technique.

植物におけるタンパク質間相互作用のためのAirIDベースの近接標識

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA ...

酵母ツーハイブリッド法による生体分子間相互作用研究 

Isolating Interaction-Null&#47;Impaired Mutants Using the Yeast Two-Hybrid Assay

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the role(s) and function(s) of a specific protein-protein interaction is to compare the phenotype of the wild-type (with the relevant protein-protein interaction) and those of mutants that lack the relevant interaction. Therefore, if such mutants can be isolated, they will help to elucidate the related biological processes. The yeast two-hybrid (Y2H) procedure is a powerful approach not only to detect protein-protein interactions but also to isolate interaction-null/impaired mutants. In this article, a protocol is presented to isolate interaction-null/impaired mutants using Y2H technology. First, a mutation library is constructed by combining the polymerase chain reaction and efficient seamless cloning technology, which efficiently excludes the empty vector from the library. Second, interaction-null/impaired mutants are screened by the Y2H assay. Because of a trick in the Y2H vector, undesired mutants, such as those with frameshift and nonsense mutations, are efficiently eliminated from the screening process. This strategy is simple and can, therefore, be applied to any combination of proteins whose interaction can be detected by the two-hybrid system.

著者スポットライト:酵母におけるInteraction-null変異体の分離の強化

The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired mutants.

Yeast Two-Hybrid Assayを用いたInteraction-Null/Impaired Mutantsの単離

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the ...