我々は、博士に感謝する。ジョンハンコック、レイグリル、アンドリューモリスとその支援のためのオルガChumakova。 FlexStation 3システムはUTHSC Cytodynamicイメージング施設の一部です。我々はまた、博士に感謝します。スクリップス研究所とHEK293 - hTRPA1安定した細胞株を提供するためのノバルティス研究財団（GNF）のゲノム研究所からArdem PatapoutianとマイケルBandell。研究は、NIH（MXZにGM081658）とテキサスメディカルセンター消化器病センター（HH）およびミッション接続/ TIRR財団（HHまで）からの助成金によって部分的にサポートされています。

1。 96ウェルプレートでの細胞の調製品 一時的にcDNAをヒトのTRPA1とtrasfected HEK293細胞用 <ol><li> HEK293細胞は、10％熱不活化ウシ胎児血清（Invitrogen社、16000から044）を添加した4.5 mg / mlのグルコースを（サーモサイエンティフィック、SH3002201）を含むダ​​ルベッコの最小必須培地（DMEM）、で1〜2日間、50ユニットのために栽培されている/ mlペニシリン、50μg/ mlのストレプトマイシン（Invitrogen社、15140から163）。トランスフェクション当日に、細胞密度が70〜90パーセントに到達する必要があります。 </li><li>コー​​ト96ウェルの各ウェルに50μlのポリ- L -オルニチン（シグマP3655、滅菌蒸留水で20μg/ ml）をピペッティングし、37℃でインキュベートすることによりプレート℃で少なくとも15分間。のMg 2 +とCa 2 +（サーモサイエンティフィック、SH3002803）なし（DPBS）緩衝生理食塩水ダルベッコのリン酸で一回の各ウェルをリンス。プレートは、数時間のための細胞培養フードのままになることがあります。 </li><li> 96ウェルプレートの各ウェルについて、1.5 mlのエッペンドルフチュー​​ブに25μlのOPTI - MEM（Invitrogen社、31985から070）のcDNAは0.2μgで希釈する。同じcDNAでトランスフェクトされる井戸の数に応じて比例的にスケールアップ。また、別の1.5 mlエッペンドルフチュー​​ブにトランスフェクトされる各ウェルに25μlのOPTI - MEM 0.4μlをリポフェクトアミン2000（インビトロジェン社、11668から019）で希釈する。このアップトランスフェクトされる井戸の総数に応じて拡大縮小します。 </li><li>簡潔に渦希釈したcDNAとリポフェクトアミン2000を、それらが約5分間室温で放置します。 </li><li>希釈cDNAを、渦を含むチューブに希釈リポフェクタミン2000の等量を転送し、混合物を15分〜2時間室温で放置します。この時間の間に、49μL/ウェルポリ- L -オルニチン被覆プレートへのcDNA /リポフェクタミン2000混合物を転送する。 </li><li> HEK293細胞を（ステップ1.1）トリプシン、血球計算盤または伯爵自動細胞計数装置（Invitrogen社、C10227）を用いて細胞密度をカウントし、15 mlの滅菌に（一般的には約〜125,000-150,000細胞/ウェル）の細胞の所望量を転送する円錐底遠沈管。 5分間1000 × rpmで遠心。 10％熱不活性化ウシ胎児血清を添加したDMEM培地で〜1,250,00-1,500,000細胞/ ml、、で、抗生物質なしで再懸濁し、細胞。 </li><li>マルチチャンネルピペッターまたはMicroFillのマイクロプレートディスペンサー（バイオテック）を用いてcDNA -リポフェクタミン2000混合物を含む各ウェルに98μlの細胞懸濁液を分注する。プレートは加湿細胞培養インキュベーターにそれを配置する前に少なくとも30分間フードに座ってみましょう。 37℃、20〜44時間、5％のCO 2を 。 </li></ol> 安定的にTRPA1を発現するHEK293細胞について <ol start="8"><li>コー​​ト96ウェルプレートにステップ1.2に従ってください。 </li><li>誘導HEK293 - hTRPA1細胞株は、寛大に（Invitrogen社、博士A. Patapoutian（​​スクリプス研究所）によって提供され、野生型HEK293細胞（ステップ1.1）と同じ培地中で維持が、5μg/ mlのブラストサイジンを添加したA1113902）及び50μg/ mlのハイグロマイシンB（Invitrogen社、10687010）。細胞密度が約90％に達すると、細胞をトリプシン、細胞密度をカウントし、5分間1000 × RPMでの細胞の所望量を遠心してください。ドキシサイクリン（フィッシャー、BP265​​35、0.25μg/ ml）を補充した同じ培地で〜1,000,000セル/ mlの密度で細胞を再懸濁します。 </li><li> /ウェルマルチチャンネルピペッターまたはMicroFill（バイオテック）を用いて100℃、ポリ- L -オルニチン被覆されたプレートに細胞懸濁液を分注する。プレートは加湿細胞培養インキュベーターにそれを配置する前に少なくとも30分間フードに座ってみましょう。 37℃16℃、5％CO 2 - 24時間を。 </li></ol> 2。ソリューション<ol><li>ハンクのバッファ塩溶液（HBSS）は細胞外液として使用されます。それは（mMで）含まれています：137のNaCl、5.4 KClを、0.25のNa 2 HPO 4、0.44 KH 2 PO 4、1.3のCaCl 2、1.0のMgSO 4、1.0を1N NaOHを用いて7.4に調整pHを持つのMgCl 2、10グルコース、10 HEPES、）。 </li><li>であるFluo - 4アッセイ用緩衝液は、HBSSでプロベネシド2mMのが含まれています。 0.5 N NaOH中の250mM株式プロベネシド（Sigma社、P8761）を作る。 1:125の割合でHBSSに希釈する。 1 N HClを用いて7.4のpHを再調整してください。 </li></ol> 3。であるFluo - 4 AMで細胞ローディング<ol><li> 1mMのストック溶液を作るために912μlの無水DMSOには1mgであるFluo - 4 AM（Invitrogen社、F14202）を溶かす。であるFluo - 4原液が少なくとも一ヶ月は-20℃で保存できる回答。 </li><li> 5ミリリットルにみたFluo - 4アッセイバッファー（ステップ2.2）に全体の内容を転送して10μlのプルロニックF - 127（Invitrogen社、P3000MP、DMSOの20％（w / v）溶液）でAMおよび10μlの1mMのであるFluo - 4をミックス（Sigma社、A9418）0.1％ウシ血清アルブミンを補充した。 </li><li> 80μlの/ウェルマイクロプレートウォッシャーを（そのようなバイオテックからELx405TMなど）を使用して2回で、HBSSで96ウェルプレート（ステップ1.7または1.10）で培養した細胞を洗浄する。 </li><li>フルオを追加-4 /ウェルマルチチャンネルピペッターまたはMicroFill（バイオテック）を使用して、37でプレートをインキュベート50℃（ステップ3.2）ソリューションをロードした° Cで1​​時間。 </li><li> ELx405TMマイクロプレートウォッシャーを使用してみたFluo - 4アッセイバッファー（ステップ2.2。）で細胞を3回洗浄。最後の洗浄の後、各ウェルにみたFluo - 4アッセイ用緩衝液80μlを残す。 </li></ol> 4。化合物のプレートの準備<ol><li> 1時間のセルローディング期間中に、みたFluo - 4アッセイ緩​​衝液中の所望の最終濃度の3倍のアゴニスト（または拮抗薬）の3倍希釈系列を用意する。 </li><li> 96ウェル化合物のプレートに希釈化合物溶液のピペット250〜300μL。 FlexStation 3が一度に1つの列を読み取るために、可能な限り複合板の同じ列にある化合物と対応するポジティブおよびネガティブコントロールの希釈系列を持つように手配する。 </li></ol> 5。 FlexStation 3のプレートの読み取り<ol><li>システムの電源を入れます（FlexStation 3、コンピュータ、およびソフトマックスプロ5.2ソフトウェア） </li><li>新しいファイル名で実験を保存します。セットアップでは、フレックスモードを選択し、次の値を入力します。 </li></ol> プロトコール＃1、アゴニストの効果 <img src="/files/ftp_upload/3149/3149table1.jpg" alt="表1" /> プロトコル＃2、アンタゴニスト作用 <img src="/files/ftp_upload/3149/3149table2.jpg" alt="表2" /><ol start="3"><li> FlexStation 3の適切なトレイに新しいヒントボックス、化合物のプレートと読んでプレートを置き、ボタンを&quot;読み&quot;をクリックします。 FlexStation 3は、一度に1列を読んで、読書を中断することなく、あらかじめプログラムされた時点での化合物を追加します。これは、1つの列とマシンがすべての列が読み取られるまで、ステップ5.2のユーザーによってプログラムが実行化合物の追加と次の列を読み取るために引き続きを読み終えるには約3分（議定書＃2の5分）かかります。 </li></ol> 6。データの分析<ol><li>実験データは、Microsoft Excelなどの、任意のスプレッドシートプログラムに直接ソフトマックスプロ5.2ソフトウェアを使用して処理するかコピー＆ペーストすることができます。 TRPA1アゴニストフルフェナム酸（FFA）の濃度 - 反応曲線は、次の最小二乗フィッティングルーチンを使用して構築されます。 <img src="/files/ftp_upload/3149/3149eq1.jpg" alt="式（1）" /> 、Yは観測された応答である場合、Y maxは、正規化最大の応答である、Cは対応する薬剤の濃度であり、EC 50は最大値の半分の応答を誘発濃度であり、n Hは見かけヒル係数である。 （100μM）FFAの一定濃度を使用して、そして徐々に次のように当てはめ、最小二乗により計算される化合物の化合物AのIC 50値の濃度を増加させることにより得られる推定TRPA1アンタゴニスト化合物の濃度-阻害曲線方程式。 <img src="/files/ftp_upload/3149/3149eq2.jpg" alt="式（2）" /> 、Yは観測された応答であり、Y maxは正規化ピーク応答である、[Antは]対応する化合物の濃度である、IC 50は FFA -誘発反応の最大阻害の半分を生産する拮抗薬の濃度であり、そしてnはHが明らかヒルです係数。 </li></ol> 7。代表的な結果： TRPA1は、Ca 2 +感受性色素、フルオ- 4（1-2）によって検出することができるカルシウムの流入につながる活性化、そのうちのCa 2 +透過性陽イオンチャネルである。 HEK293 - hTRPA1安定した細胞株は、TRPA1アゴニストおよびアンタゴニストの濃度 - 反応関係を研究するために使用されていました。細胞は、みたFluo - 4 AMをロードアッセイバッファー80μlの内に置かれ、FlexStation 3を使って読んでいた。 30秒のベースラインの蛍光を記録した後、TRPA1アゴニスト、FFA、所望の最終濃度の3倍で、それぞれの連続希釈の40μlをマルチチャンネルピペッターを介して細胞に添加した流体のモジュールの一部として含まれFlexStation 3。蛍光の変化は、さらに180秒間モニターした相対的にみたFluo - 4各ウェルの蛍光の変化はΔF=として表現された（F - F 0）、FとF 0はそれぞれ、興味や読書の始まりの時の蛍光値です。 FFA濃度の一連の反応の蛍光変化に時間のコースの代表的なトレースが図に示されています。 1。最大応答レベルは似ていますが、活性化速度論が低いFFAの濃度でより高いで明らかに速いですが、最後の4つのFFA濃度のために注意してください。したがって、これらの違いを区別するために、濃度 - 反応関係は、FFAの様々な濃度（図2）の添加により蛍光増の初速度を（傾き）を測定することによって構築した。半最大効果FFAのIVEの濃度（EC 50）が 55.4μM（N = 3）であると決定された。推定TRPA1アンタゴニストをテストするには、化合物、我々はプロトコル＃2（ステップ2）を使用して同様の実験を行った。このケースでは、拮抗薬の希釈系列を30秒（3倍の所望の最終濃度の40μl）で添加し、FFAは（100μMの最終濃度は300μMで60μl）を180秒後に追加されました。化合物Aは用量依存的に4.3μMの半最大阻害濃度（IC 50）（n = 3）を（図2）とFFAにTRPA1の細胞の応答を減少させる。 <img src="/files/ftp_upload/3149/3149fig1.jpg" alt="図1"/> 図1濃度に依存するFFAによるヒトTRPA1の活性化。トレースは、時間の経過とともに蛍光のFFA誘発増加（最初の60秒でだけ応答が良くFFA -誘発反応の速い反応速度を示すために掲載されています）です。高濃度でFFAがピーク蛍光レベルが類似していたにもかかわらず、はるかに高速に運動低濃度でよりと蛍光の増加を誘発ことに注意してください。 <img src="/files/ftp_upload/3149/3149fig2.jpg" alt="図2"/> 図2集中- 。FFA誘起蛍光の変化と化合物安定HEK293細胞で発現されたヒトTRPA1のFFAの応答の誘発抑制のための反応関係。 Δ、濃度 - FFAの反応関係は、FFAの濃度を変化させる添加時に正規化された蛍光増の初速度を（傾き）を測定することによって決定した。 O、推定されるTRPA1拮抗薬によってFFA誘発カルシウム応答の阻害の濃度 - 反応関係、化合物Aの

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利害の衝突は宣言されません。

細胞内Ca 2 +などの神経伝達物質放出（3）、心臓の活動電位（4）、筋細胞の収縮（5）、細胞のシグナル伝達（6-7）、と興奮細胞死（のような多くの生理学的および病理学的条件下において重要な役割を果たしている8）。細胞内Ca 2 +濃度の測定は、機能+透過チャネルが細胞膜や細胞内小器官のCa 2 +の変化との寄与を解明するのに役立ちます 上記のプロセス...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td> FlexStation 3 </td><td>分子デバイス</td><td> FLEX3 </td><td></td></tr><tr><td> 96ウェル、FlexStationのピペットチップ</td><td>分子デバイス</td><td> 9000-0912 </td><td></td></tr><tr><td> 96ウェルプレート</td><td>グライナーバイオワン</td><td> 655090 </td><td></td></tr><tr><td> 96ウェルクリアフラットボトムプレート</td><td>共演スター</td><td> 3595 </td><td></td></tr><tr><td> DMEM </td><td>インビトロジェン</td><td> 12430-104 </td><td></td></tr><tr><td>のOpti - MEM </td><td>インビトロジェン</td><td> 31985-070 </td><td></td></tr><tr><td>リポフェクタミン2000 </td><td>インビトロジェン</td><td> 11668-019 </td><td></td></tr><tr><td> AM蛍光- 4 </td><td>インビトロジェン</td><td> F14202 </td><td></td></tr><tr><td> PluronicF - 127 </td><td>インビトロジェン</td><td> P3000MP </td><td></td></tr><tr><td>ポリ- L -オルニチン</td><td>シグマ</td><td> P36551 </td><td></td></tr><tr><td>プロベネシド</td><td>シグマ</td><td> P8761 </td><td></td></tr><tr><td>ウシ血清アルブミン</td><td>シグマ</td><td> A9418 </td><td></td></tr></tbody></table>

cell-based calcium assay for medium to high throughput screening of trp channel functions using flexstation 3

分子デバイス&quot;FlexStation 3は、マルチウェルプレートの自動蛍光測定が可能な卓上型マルチモードマイクロプレートリーダーです。それは学術的設定の中〜高スループットスクリーン用に最適です。それは、マルチチャンネルピペッターやマシンを装備した統合型流体転送モジュールは、蛍光試薬の様々な蛍光の変化を監視するために一度に1列を読み取りますいます。例えば、FlexStation 3 +透過性のイオンチャンネルとGタンパク質共役型受容体細胞内遊離Ca 2 +濃度の変化を測定することにより、Ca 2 +の機能を研究するために使用されています。一過性受容体電位（TRP）チャネルは、多くの生理学的および病態生理学的機能に重要な役割を果たす非選択的カチオンチャネルの大きなファミリーです。 TRPチャネルのほとんどはカルシウム透過性であると活性化によりカルシウム流入を引き起こす。このビデオでは、我々はTRPA1チャネル、多数の有害な刺激のための分子センサーの薬理学的特性を研究するためにFlexStation 3のアプリケーションを示しています。 96ウェルプレートで培養一過性または安定的に人間のTRPA1チャネルを発現するHEK293細胞を、、のCa 2 +感受性蛍光色素、みたFluo - 4にロードされ、そしてこれらの細胞のリアルタイム蛍光の変化はのアプリケーションの実行前と実行中に測定されていますFlexStation 3のFLEXモードを使用して、TRPA1アゴニスト。推定されるTRPA1拮抗薬の効果に​​ついても検討した。データは、TRPA1活性化因子と阻害剤の濃度 - 反応関係を構築するためにSoftMax Proソフトウェアから転送されます。

Watch this Scientific Journal Video about Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3 at JoVE.com

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

このビデオでは、FlexStation 3を用いて、ヒトTRPA1チャネルの薬理学的特性を研究するための詳細なプロトコルを提供します。プロトコルは、細胞調製、染料ロードとマイクロプレートリーダー、FlexStation 3の動作の詳細について説明します。

FlexStation 3を用いてTRPチャネルの機能のハイスループットスクリーニングへの媒体のためのセルベースカルシウムアッセイ

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

cell-based-calcium-assay-for-medium-to-high-throughput-screening-trp

Research

JoVE Journal

Bioengineering

21.0K ビュー.  The University of Texas Health Science Center at Houston. このビデオでは、FlexStation 3を用いて、ヒトTRPA1チャネルの薬理学的特性を研究するための詳細なプロトコルを提供します。プロトコルは、細胞調製、染料ロードとマイクロプレートリーダー、FlexStation 3の動作の詳細について説明します。

ビデオ: Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

バイオマテリアルのハイスループット検出用高分子マイクロアレイ

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

生物工学

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

ハイスループット抗真菌薬スクリーニングのためのカンジダバイオチップ（ カルシウム BChip）

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

マイクロ流体プラットフォームを使用した高スループットのタンパク質発現ジェネレータ

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

生物学的液体培地中でスケーラブルな、メタリック高アスペクト比のナノコンポジットの生成

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Tomoautoの使用：ハイスループット自動クライオ電子断層撮影のためのプロトコル

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

医療機器のための細菌撥ポリマーのハイスループット同定

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

注射の再生療法と高スループットの薬剤のスクリーニングのための 3 D への放射能

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

中国ハムスター卵巣細胞培養におけるハイスループット自動マイクロバイオリアクターを用いたプロセス最適化

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

シュードモナスシリンゲpvに対する耐性のハイスループット同定苗フラッドアッセイを用いたトマトのトマト

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

ハイスループットマイクロ流体装置を用いた動的環境条件の生成

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...