建設的な議論、Jozsef コウノトリ、ミッチェル リッチモンド、Jarrad Gollihue とアンドレア ・ サンチェスに感謝します。表現型の写真のため博士 Sharyn ペリー。この材料は、組合契約第 1355438 の下での国立科学財団によってサポートされる作業に基づいています。

1. 希釈ライブラリを作成します。
<ol><li>化合物ライブラリーからの対応するプレートに一致することを確保する 625 希釈ライブラリ プレートを手でラベル付けします。さらに、5 ガロン タンク コンソール ドライブを介してそれらを渡すことによってフローとフロー ホースに、マルチ チャネルのヒント洗浄自動実験器具ポジショナ (ALP) アウトを接続 (材料の表を参照してください)。</li>
	<li>コンピューターにアクセスして、水を循環するためにマルチ チップ洗浄 ALP にデバイス コント ローラーの接続を介して洗浄ポンプをオンにします。これは、プロトコルの最後で自動的にオフになります。</li>
	<li>負荷、手、スタッカー 10 によってホテル A ~ D (図 4スタッカー) に以下の順番で、スタッカー カルーセルに接続第 1 室、4 つの 96 ウェル V で AP96 P20 ピペット チップ 1 箱-注文の図書館と 2 つの低いプレートからストック濃度を含む 2 つの上部プレート 2-5 の部屋で底板を空に (図 5スタッカー)。また、部屋 6 で 4 つの 96 ウェル V AP96 P20 ピペット チップの 1 つのボックスをロード-7-9 の部屋で順序付けられたライブラリの 2 つの低いプレート ストック濃度を含む 2 つの上部プレートの底板 (図 5スタッカー) を空にします。</li>
	<li>設定まで、手で P3, P7, 上 300 mL 70 %etoh バスに 300 mL の水貯蔵所をデッキ先端ローダー ALP (TL1) とマルチ チップ洗浄 ALP (TW1) (図 4図 5、デッキ デッキ)。</li>
	<li>オペレーティング ソフトウェアを使用して、スタッカー 10 から AP96 P20 ピペット チップを提示し、ヒント ローダー ALP に移動します。 注意: 1.5 1.12 すべてからロボットの動作ソフトウェアを処理液に行っています。材料表を参照してください。</li>
	<li>ホテルから部屋 2 を示し、別のすべての 4 つの 96 ウェル V-下部プレート、デッキに下部を配置する 2 つの P4、P8、P5、P9 のトップ 2 (図 4)。</li>
	<li>ヒント ローダー 96 チャンネル 200 μ L の頭の上に ALP AP96 P20 ピペット チップを読み込みます。300 mL の水貯蔵所から 90 μ L を吸引し、96 ウェル V に分配-P4 の底希釈板。P8 の板には、この手順を繰り返します。</li>
	<li>P5 の化合物ライブラリー プレートをミックスするには、繰り返し吸引・ 3 回 15 μ L を吐出します。さらに、P5 の化合物ライブラリー プレートから 10 μ L を吸引し、P4 の希釈プレートに 10 μ L を分注します。</li>
	<li>計 3 回繰り返し吸引・ 50 μ L を吐出で P4 のプレートのソリューションをミックスします。一度混ぜ、きれいな吸引・吐出 70% の 70 μ L で AP96 P20 ピペット チップ吸引・吐出水 4 の 110% のボリュームによってマルチ チャンネル チップ洗浄 ALP でそれらを洗浄、P7 から江藤回します。</li>
	<li>P8 ・ P9 にプレートの 2 番目のペアについて手順 1.8 1.9 繰り返します。作成される 2 番目の 96 ウェル V-下希釈プレート、プレートから順に下の先頭スタック: P4、P5 P8、P9。その後、1 つの空の静的 ALP; にスタックを配置します。か P1、P2、P6、P10、P11、P12、P13。</li>
	<li>ホテルの部屋 5 が空になるまで、手順 1.6 1.10.部屋 6 に到達、ヒント ローダー ALP に新しい AP96 P20 ピペット チップを移動および空の静的 ALP に使用される AP96 P20 ピペット チップを配置する時に 1.5 を手順を繰り返します。</li>
	<li>部屋 9 のホテル A が空になるまで、手順 1.6 1.10.ただし、ホテル B に進むためにプレートとデッキのヒントする必要があります再読み込みするホテル A に</li>
	<li>手で 300 mL 水タンク再入力します。このステップが重要であり、コンピューターのプログラムが一時停止 '続行'、次のステップを実施する前にヒットするユーザーを必要とする、このメッセージの詳細を組み込むことができます。</li>
	<li>残りのホテルは、隣のホテルに進む前に各時間フル 300 mL 水タンクを確保する 1.5 1.13 の手順を繰り返します。</li>
</ol>2. スクリーニング プレートにメディア シード混合物を追加します。
<ol><li>½ 培地と培 (MS) メディアを作る 0.1 %4.3 g MS 塩、MES の 0.50 g 1.0 g 寒天培地 1 L ・ ディ ・ H2o. 添加による寒天培地調整 5.7 を pH が 5 M 水酸化カリウム添加 pH プローブを監視しながら。</li>
	<li>15 と 30 分間 1% の漂白剤と SDS でそれらの動揺によって種子を滅菌し、遠心分離によって水の等しい容積と 4 回をすすぎ。種が生殖不能にされたら、vernilization の 7 日間を 24 時間から 4 ° C でそれらを配置します。シロイヌナズナの生物学的リソース センターでは、殺菌、vernilization、および成長の22の追加のメソッドについて説明します。</li>
	<li>密度 0.1 g/100 mL で手でメディアに種子を追加します。この密度は、96 ウェル プレートのウェルあたり 3-10 種の平均の結果します。</li>
	<li>手で 4 つの 96 ウェル フラットの場所-(図 6、ホテル)、ホテル A の部屋 1 と 2 で底板。ヒント ローダー ALP 上 AP96 P250 ピペット チップのボックスを配置、300 mL 貯水池配合手順で作成したメディア シード混合物 2.1 2.3 の P3、および 300 mL 貯水池 70% EtOH P7 に (図 4、デッキおよび図 6、デッキ)。 注: を 2.8 2.5 オペレーティング ソフトウェアで行われます。</li>
	<li>ホテル A の部屋 1 と 2 を提示し、別の 4 つのプレートのスタック。空の静的アルプス (P4、P5、P6、P8、P9、P10、P11、および P12) のそれぞれに 1 つのプレートを配置します。AP96 P250 ピペット チップ 96 チャンネル 200 μ L の頭の上をロードします。</li>
	<li>P3 で 300 mL メディア種子貯蔵所から 90 μ L を吸引し、最初の 96 ウェル フラットに分配-底板。メディア シード混合物を含むすべての 8 つのプレートまで、このプロセスを繰り返します。</li>
	<li>吸引・吐出 70% でいっぱい 300 mL 貯水池から 70 μ L で AP96 P250 ピペット チップをきれい P7 の江藤。吸引・吐出 110% の水量 4 倍でマルチ チャンネルのヒント洗浄の ALP でヒントを洗う、TL1 でヒントをアンロードし、プレートを手で収集します。</li>
</ol>3. スクリーニング プレートに小さな分子を追加します。
<ol><li>手による負荷、ルーム ホテル 1、2 の 96 ウェル V に AP96 P250 ピペット チップのボックス-2、4、6、および 8、客室に 2 つ 96 平底スクリーニング プレート 3、5、7、および 9 (図 4の部屋に底希釈図書館板、スタッカーおよび図 7、ホテル)。さらに、マルチ チップ洗浄 ALP の 5 ガロン タンクからホースを接続します。 注: 3.10 を通じて 3.2 オペレーティング ソフトウェアで行われます。</li>
	<li>P7; で 300 mL 70 %etoh 洗浄タンクを格納するデッキを構成します。メディア シード貯水池は P3 で、デッキ上左ことができます (図 4、デッキおよび図 7デッキ)。さらに、マルチ チャンネル チップ洗浄アルプを通して水を循環させるデバイス コント ローラーの接続を介してコンソール ドライブ オンにします。これは、プロトコルの最後で自動的にオフになります。</li>
	<li>ホテルから AP96 P250 ピペット チップ ボックスが表示され、ヒント ローダー ALP に移動します。</li>
	<li>96 ウェル V を提示-デッキと静的 ALP P4 と P8 の上の 1 つの場所の 1 つにホテルの部屋の 2 A から底希釈ライブラリ板。96 ウェル フラットを提示-下スクリーニング プレートから部屋のデッキにホテル A の 3 と静的 ALP P5、P9 の 1 つを配置します。</li>
	<li>ヒント ローダー 96 チャンネル 200 μ L の頭の上に ALP AP96 P250 ピペット チップを読み込みます。</li>
	<li>96 ウェル V をミックス-底希釈板の吸引・吐出 50 μ L 3 回で P4。次に、このプレートから 10 μ L を吸引し、96 ウェル フラットに分配-P5 の下スクリーニング プレート。</li>
	<li>吸引・吐出 50 μ L 3 回で P5 の打席でソリューションをミックスします。吸引・吐出 70% の 70 μ L でエタノール AP96 P250 ピペット チップをきれい P7、吸引・吐出量 110% でマルチ チップ洗浄 ALP のヒント水 4 回洗いで貯水池から江藤。</li>
	<li>について 2 番目の 96 ウェル V、3.5、3.6 手順-希釈ライブラリの底板 (P8) と 96 ウェル フラット-スクリーニングの底板 (P9)。</li>
	<li>スタック 2 96 ウェル V-底希釈ライブラリ板一緒と一緒に 2 つの 96 ウェル フラット底スクリーニング プレート。静的アルプス P1 や P2、P6、P10、P11、P12、P13 にプレートを移動します。</li>
	<li>手順 3.4 3.9 3 回、合計 8 回のプレートのスクリーニングに希釈した化学物質を追加します。最後に、視覚構造をスクリーニング プレートの各ウェルに種子の数をチェックし、滅菌と種子春化シードが 3 つより少ない種とそれらの井戸を補います。</li>
</ol>4. インキュベーションとプレートをスクリーニングの可視化
<ol><li>インキュベート 96 ウェル平底スクリーニング プレート 4 日 22 ° c 環境室で 16/8/明暗サイクルの乾燥証拠コンテナー内。96 ウェル平底スクリーニング プレート解剖顕微鏡の下で視覚化します。さらに調査のため異常な表現型すべてを記録します。</li>
</ol>

<ol>
	<li>Blackwell, H. E., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+genetic+approaches+to+plant+biology.">Chemical genetic approaches to plant biology.</a> Plant Physiol. 133 (2), 448-455 (2003).</li><li>Dejonghe, W., Russinova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+chemical+genetics:+From+phenotype-based+screens+to+synthetic+biology.">Plant chemical genetics: From phenotype-based screens to synthetic biology.</a> Plant Physiol. 174 (1), 5-20 (2017).</li><li>McCourt, P., Desveaux, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+chemical+genetics.">Plant chemical genetics.</a> New Phytol. 185 (1), 15-26 (2010).</li><li>Lumba, S., Cutler, S., McCourt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+nuclear+hormone+receptors:+A+role+for+small+molecules+in+protein-protein+interactions.">Plant nuclear hormone receptors: A role for small molecules in protein-protein interactions.</a> Annu Rev Cell Dev Biol. 26, 445-469 (2010).</li><li>Hicks, G. R., Raikhel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opportunities+and+challenges+in+plant+chemical+biology.">Opportunities and challenges in plant chemical biology.</a> Nat Chem Biol. 5 (5), 268-272 (2009).</li><li>De Rybel, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+the+root+cap+in+root+branching+revealed+by+the+non-auxin+probe+naxillin.">A role for the root cap in root branching revealed by the non-auxin probe naxillin.</a> Nat Chem Biol. 8 (9), 798-805 (2012).</li><li>Koornneef, M., Meinke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+Arabidopsis+as+a+model+plant.">The development of Arabidopsis as a model plant.</a> Plant J. 61 (6), 909-921 (2010).</li><li>Serrano, M., Kombrink, E., Meesters, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+designing+chemical+screening+strategies+in+plant+biology.">Considerations for designing chemical screening strategies in plant biology.</a> Front Plant Sci. 6, 131 (2015).</li><li>Yoshitani, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+structure-based+strategy+for+discovery+of+small+ligands+binding+to+functionally+unknown+proteins:+Combination+of+in+silico+screening+and+surface+plasmon+resonance+measurements.">A structure-based strategy for discovery of small ligands binding to functionally unknown proteins: Combination of in silico screening and surface plasmon resonance measurements.</a> Proteomics. 5 (6), 1472-1480 (2005).</li><li>Macarron, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+high-throughput+screening+in+biomedical+research.">Impact of high-throughput screening in biomedical research.</a> Nat Rev Drug Discov. 10 (3), 188-195 (2011).</li><li>DeBolt, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morlin,+an+inhibitor+of+cortical+microtubule+dynamics+and+cellulose+synthase+movement.">Morlin, an inhibitor of cortical microtubule dynamics and cellulose synthase movement.</a> Proc Natl Acad Sci U S A. 104 (14), 5854-5859 (2007).</li><li>Christian, M., Hannah, W. B., Luthen, H., Jones, A. M. <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+auxins+by+a+chemical+genomics+approach.">Identification of auxins by a chemical genomics approach.</a> J Exp Bot. 59 (10), 2757-2767 (2008).</li><li>Drakakaki, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clusters+of+bioactive+compounds+target+dynamic+endomembrane+networks+in+vivo.">Clusters of bioactive compounds target dynamic endomembrane networks in vivo.</a> PNAS. 108 (43), 17850-17855 (2011).</li><li>Armstrong, J. I., Yuan, S., Dale, J. M., Tanner, V. N., Theologis, A. <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+inhibitors+of+auxin+transcriptional+activation+by+means+of+chemical+genetics+in+Arabidopsis.">Identification of inhibitors of auxin transcriptional activation by means of chemical genetics in Arabidopsis.</a> Proc Natl Acad Sci U S A. 101 (41), 14978-14983 (2004).</li><li>Brown, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+molecule+with+differential+effects+on+the+PTS1+and+PTS2+peroxisome+matrix+import+pathways.">A small molecule with differential effects on the PTS1 and PTS2 peroxisome matrix import pathways.</a> Plant J. 65 (6), 980-990 (2011).</li><li>De Rybel, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+inhibition+of+a+subset+of+Arabidopsis+thaliana+GSK3-like+kinases+activates+brassinosteroid+signaling.">Chemical inhibition of a subset of Arabidopsis thaliana GSK3-like kinases activates brassinosteroid signaling.</a> Chem Biol. 16 (6), 594-604 (2009).</li><li>Arkin, M. R., Tang, Y., Wells, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+inhibitors+of+protein-protein+interactions:+progressing+toward+the+reality.">Small-molecule inhibitors of protein-protein interactions: progressing toward the reality.</a> Chem Biol. 21 (9), 1102-1114 (2014).</li><li>St Onge, R., Schlecht, U., Scharfe, C., Evangelista, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+chemical+genetics+in+yeast+for+discovery+of+chemical+probes+targeting+metabolism.">Forward chemical genetics in yeast for discovery of chemical probes targeting metabolism.</a> Molecules. 17 (11), 13098-13115 (2012).</li><li>Vassilev, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+activation+of+the+p53+pathway+by+small-molecule+antagonists+of+MDM2.">In vivo activation of the p53 pathway by small-molecule antagonists of MDM2.</a> Science. 303 (5659), 844-848 (2004).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+genetic+interrogation+of+natural+variation+uncovers+a+molecule+that+is+glycoactivated.">Chemical genetic interrogation of natural variation uncovers a molecule that is glycoactivated.</a> Nat Chem Biol. 3 (11), 716-721 (2007).</li><li>Walsh, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+field+of+chemical+genetics:+Potential+applications+for+pesticide+discovery.">The emerging field of chemical genetics: Potential applications for pesticide discovery.</a> Pest Manag Sci. 63 (12), 1165-1171 (2007).</li><li>. Seed Handling Available from: <a target="_blank" href="https://abrc.osu.edu/seed-handling">https://abrc.osu.edu/seed-handling</a> (2013)</li><li>Knoth, C., Salus, M. S., Girke, T., Eulgem, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synthetic+elicitor+3,5-dichloroanthranilic+acid+induces+NPR1-dependent+and+NPR1-independent+mechanisms+of+disease+resistance+in+Arabidopsis.">The synthetic elicitor 3,5-dichloroanthranilic acid induces NPR1-dependent and NPR1-independent mechanisms of disease resistance in Arabidopsis.</a> Plant Physiol. 150 (1), 333-347 (2009).</li><li>Conway, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scalable+96-well+Plate+based+iPSC+culture+and+production+using+a+robotic+liquid+handling+system.">Scalable 96-well Plate based iPSC culture and production using a robotic liquid handling system.</a> J Vis Exp. , (2015).</li><li>Daniszewski, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+cell+culture+systems+and+their+applications+to+human+pluripotent+stem+cell+studies.">Automated cell culture systems and their applications to human pluripotent stem cell studies.</a> SLAS Technol. , (2017).</li><li>Popa-Burke, I., Russell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compound+precipitation+in+high-concentration+DMSO+solutions.">Compound precipitation in high-concentration DMSO solutions.</a> J Biomol Screen. 19 (9), 1302-1308 (2014).</li><li>Partridge, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+high-throughput+system+for+phenotypic+screening+of+chemical+libraries+on+C.+elegans+and+parasitic+nematodes.">An automated high-throughput system for phenotypic screening of chemical libraries on C. elegans and parasitic nematodes.</a> Cold Spring Harb Protoc. , (2017).</li></ol>

著者は、彼らは競合する金銭的な利益があることを宣言します。

このプロトコルは、シロイヌナズナの前方の化学遺伝学画面を達成するために研究者を支援するために設計されています。我々 は、代表的な結果を提供するまでに9,13,23シロイヌナズナの実行最大の前方の化学遺伝学スクリーンの 1 つ 50,000 化合物 (図 2および図 3) の画面から。...

過去 20 年間で植物学の分野の研究者長足化学遺伝学アプローチ、フォワードおよびリバースを使用して細胞壁生合成、細胞骨格、ホルモンの生合成の私達の理解を改善し、シグナル伝達、重力屈性、病態、プリン生合成、内1,2,3,4,5を人身売買します。前方の化学遺伝学手法を用いた関心の表現型の識別し特定のプロセスの遺伝的基盤を理解する研究者ができます。逆に、逆の化学遺伝学は、事前に決められた蛋白質ターゲット6と対話する化学物質を求めています。シロイヌナズナは、マップ、および注釈、そのゲノムは小さいためプラント生物学のこれらの発見の最前線にされています。短い世代時間があり、異常な細胞内機械7の識別を容易に利用可能な複数の変異/記者行があります。
前方の化学遺伝学的画面の進行状況、初期審査と金利8化合物のターゲットを決定する 2 つの主要なボトルネックがあります。小分子選択の速度の増加の主要な援助は、自動化と自動機器9の使用です。液体ハンドリング ロボットは小分子の大規模なライブラリを処理するための優れたツールを生物科学10で進行中の運転に尽力しています。ここで提示されたプロトコルは.の急速なペースで生理活性小分子の同定を有効化審査に関連するボトルネックを軽減するために設計されていますこの手法は、労力とも原則として探偵に経済的なコストを減らしている間オペレーターに代わって時間の負担を減少します。
これまでほとんどの化学ライブラリ分析は 10,000 のそして 20,000 化合物として 150,000 と 709,11,12,13,14,ようないくつかのいくつかの間抱いてください。15,16. ここに紹介されたプロトコル (材料表参照) 50,000 化合物の小分子ライブラリで実装されていました、これまでに実施したシロイヌナズナの画面より大きい前方化学遺伝学の一つ。このプロトコルは除草剤検出、発見殺虫剤、殺菌剤に関連した特に効率性の向上と前方の化学遺伝学に関する速度の現在の傾向とフィットを発見、創薬、・がん生物学17 ,18,19,20,21。実装されているシロイヌナズナと、このプロトコルでは、簡単にできる培養細胞、胞子、および可能性のある昆虫に適応 96-、384 - または 1536 ウェル プレート内液体培地で。サイズが小さいため、シロイヌナズナ、96 ウェル プレートでのスクリーニングを受けやすい。ただし、井戸の間で均等に種子を配布するは難しい問題です。手播種は労働集約的、正確、96 ウェルのプレートに種子を分配する機器がありますが、購入する高価。ここでは、精度で小さな損失だけでこのステップを回避できる方法を示す.
このメソッドの全体的な目標は、経由での使用液体ハンドリング ロボットの精度を損なうことがなくより管理しやすいシロイヌナズナに対する大規模な化合物ライブラリーをスクリーニングすることでした。このメソッドを使用して初期希釈シリーズ管理と解剖顕微鏡の下で迅速なサンプルの迅速な視覚化を許可以降の表現型画面を完了するのにかかる時間を減少させることによって研究者の効率を改善します。新規生理活性小分子の同定。図 1は、4 つのステップでこのプロトコルの主要な成果を示しています。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57393/57393fig1.jpg"> 図 1: 前方の化学遺伝学画面の全体的なワークフロー 。4 主な手順のそれぞれのいくつかの詳細を説明するプロトコルの概要です。1: 化学ライブラリ、2 を受信: 3 希釈ライブラリを作る: スクリーニング プレートと 4: インキュベートとスクリーニング プレートを可視化します。<a href="/files/ftp_upload/57393/57393fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

<table>
	<tbody>
		<tr>
			<td>Keyboard</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Used for protocol design and operating the Biomek FX</td>
		</tr>
		<tr>
			<td>Mouse</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Used for protocol design and operating the Biomek FX</td>
		</tr>
		<tr>
			<td>Computer Screen</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Used for protocol design and operating the Biomek FX</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Used for protocol design and operating the Biomek FX</td>
		</tr>
		<tr>
			<td>DIVERSet Diverse Screening Library</td>
			<td>ChemBridge</td>
			<td>N/A</td>
			<td>Chemical library</td>
		</tr>
		<tr>
			<td>Biomek Software</td>
			<td>Beckman Coulter</td>
			<td>N/A</td>
			<td>Runs and designs the Biomek FX</td>
		</tr>
		<tr>
			<td>Device Controller</td>
			<td>Beckman Coulter</td>
			<td>719366</td>
			<td>Operates the water pump/tip washing station</td>
		</tr>
		<tr>
			<td>Stacker Carousel Pendent</td>
			<td>Beckman Coulter</td>
			<td>148240</td>
			<td>Manual operation of Biomek Stacker Carousel</td>
		</tr>
		<tr>
			<td>Biomek Stacker Carousel</td>
			<td>Beckman Coulter</td>
			<td>148520</td>
			<td>Rotary unit that houses all FX Stacker 10&#39;s</td>
		</tr>
		<tr>
			<td>FX Stacker 10</td>
			<td>Beckman Coulter</td>
			<td>148522</td>
			<td>Elevator unit that houses components for screen</td>
		</tr>
		<tr>
			<td>FX Stacker 10</td>
			<td>Beckman Coulter</td>
			<td>148522</td>
			<td>Elevator unit that houses components for screen</td>
		</tr>
		<tr>
			<td>FX Stacker 10</td>
			<td>Beckman Coulter</td>
			<td>148522</td>
			<td>Elevator unit that houses components for screen</td>
		</tr>
		<tr>
			<td>FX Stacker 10</td>
			<td>Beckman Coulter</td>
			<td>148522</td>
			<td>Elevator unit that houses components for screen</td>
		</tr>
		<tr>
			<td>Biomek FX</td>
			<td>Beckman Coulter</td>
			<td>https://www.beckman.com/liquid-handlers</td>
			<td>Robot that performs the desired operations</td>
		</tr>
		<tr>
			<td>Accuframe</td>
			<td>Artisan Technology Group</td>
			<td>76853-4</td>
			<td>Frames arm to place components corretly</td>
		</tr>
		<tr>
			<td>Framing Fixture</td>
			<td>Beckman Coulter</td>
			<td>719415</td>
			<td>Centers arm in the Accuframe</td>
		</tr>
		<tr>
			<td>Multichannel Tip Wash ALP</td>
			<td>Beckman Coulter</td>
			<td>719662</td>
			<td>Washes the tips after the ethanol bath</td>
		</tr>
		<tr>
			<td>Tip Loader ALP</td>
			<td>Beckman Coulter</td>
			<td>719356</td>
			<td>Pneumatically loads tips onto the arm</td>
		</tr>
		<tr>
			<td>Air Compressor</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Provides air for pneumatic tip loading</td>
		</tr>
		<tr>
			<td>MasterFlex Console Drive</td>
			<td>Cole-Parmer</td>
			<td>77200-65</td>
			<td>Pump used to circulate water through the Multichannel Tip Washer</td>
		</tr>
		<tr>
			<td>Air Hose</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Provides air from air compressor to Tip Loader</td>
		</tr>
		<tr>
			<td>Water Hose</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Provides water from 5 Gallon Reserviour to Tip Washer</td>
		</tr>
		<tr>
			<td>Static ALP&#39;s</td>
			<td>Beckman Coulter</td>
			<td>Comes with Biomek FX</td>
			<td>Supports equipment for the Screen</td>
		</tr>
		<tr>
			<td>5 Gallon Reserviour</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Recirculates the dirty water from cleaning the tips</td>
		</tr>
		<tr>
			<td>Grippers</td>
			<td>Beckman Coulter</td>
			<td>Comes with Biomek FX</td>
			<td>Grabs and moves the equipment to the correct places</td>
		</tr>
		<tr>
			<td>96-Channel 200 &#181;L Head</td>
			<td>Beckman Coulter</td>
			<td>Comes with Biomek FX</td>
			<td>Holds the 96 tips used within the screen</td>
		</tr>
		<tr>
			<td>AP96 P200 Pipette Tips</td>
			<td>Beckman Coulter</td>
			<td>717251</td>
			<td>Used to make the screening library</td>
		</tr>
		<tr>
			<td>96 Well Flat Bottom Plate</td>
			<td>Costar</td>
			<td>9018</td>
			<td>Aids in visulization of screen</td>
		</tr>
		<tr>
			<td>96 Well V-Bottom Plate</td>
			<td>Costar</td>
			<td>3897</td>
			<td>Aids in storing of dilution library</td>
		</tr>
		<tr>
			<td>AlumaSeal 96 Sealing Film</td>
			<td>MedSci</td>
			<td>F-96-100</td>
			<td>Seals for storage both the chemicle library and dilution library</td>
		</tr>
		<tr>
			<td>Plastic ziplock sandwich bags</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Used to ensure a humid environment for screen</td>
		</tr>
		<tr>
			<td>AP96 P20 Pipette Tips</td>
			<td>Beckman Coulter</td>
			<td>717254</td>
			<td>Used in the dilution library creation</td>
		</tr>
		<tr>
			<td>Growth Chamber</td>
			<td>Percival</td>
			<td>AR36L3</td>
			<td>Germinates seeds for phenotypic visulization</td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Holds seeds to add into wells where liquid seeding failed seed adequatly</td>
		</tr>
		<tr>
			<td>Toothpick</td>
			<td>Local Provider</td>
			<td>N/A</td>
			<td>Pushes seeds from spatula to wells</td>
		</tr>
		<tr>
			<td>Murashige and Skoog Basal Salt Mixture</td>
			<td>PhytoTechnology Laboratories</td>
			<td>M524</td>
			<td>Add to MS media mixture</td>
		</tr>
		<tr>
			<td>MES Free Acid Monohydrate</td>
			<td>Fisher Scientific</td>
			<td>ICN19483580</td>
			<td>Added to MS media to decrease pH</td>
		</tr>
		<tr>
			<td>Agar Powder</td>
			<td>Alfa Aesar</td>
			<td>9002-18-0</td>
			<td>Increases thickness of media to support seed suspension</td>
		</tr>
		<tr>
			<td>5M KOH</td>
			<td>Sigma-Aldrich</td>
			<td>484016</td>
			<td>Increases pH to adequate levels</td>
		</tr>
		<tr>
			<td>1L Media Storage Bottle</td>
			<td>Corning</td>
			<td>1395-1L</td>
			<td>Holds enough media for a screen</td>
		</tr>
		<tr>
			<td>Polypropylene Centrifuge Tubes</td>
			<td>Corning</td>
			<td>431470</td>
			<td>Sterilizes seeds prior to vernilization</td>
		</tr>
		<tr>
			<td>pH Probe</td>
			<td>Davis Instruments</td>
			<td>YX-58825-26</td>
			<td>Used for making media</td>
		</tr>
		<tr>
			<td>ALPs (Automated Labware Positioners) Users Manual</td>
			<td>Beckman Coulter</td>
			<td>PN 987836</td>
			<td>Aids in setting up the accompaning equipment for the Biomek FX</td>
		</tr>
		<tr>
			<td>Biomek 2000 Stacker Carousel Users Guide</td>
			<td>Beckman Coulter</td>
			<td>609862-AA</td>
			<td>Aids in setting up the Stacker Carousel</td>
		</tr>
		<tr>
			<td>Biomek FX and FXP Laboratory Automation Workstations Users Manual</td>
			<td>Beckman Coulter</td>
			<td>PN 987834</td>
			<td>Used to frame the Multichannel Pod</td>
		</tr>
		<tr>
			<td>Biomek FXP Laboratory Automation Workstation Customer Startup Guide</td>
			<td>Beckman Coulter</td>
			<td>PN B32335AB</td>
			<td>Used to aid in setting up the Biomek FX</td>
		</tr>
		<tr>
			<td>Biomek Software User&#39;s Manual</td>
			<td>Beckman Coulter</td>
			<td>PN 987835</td>
			<td>Used to set up and understand the Software</td>
		</tr>
	</tbody>
</table>

optimizing the use of a liquid handling robot to conduct a high throughput forward chemical genetics screen of arabidopsis thaliana

化学遺伝学はますます伝統的な遺伝学遺伝子重複や致死性のために手に負えないことができる植物の形質をデコードに採用されています。ただし、生理活性されている合成小分子の確率が低いです。したがって、分子の数千人は、関心のあるものを見つけるためにテストする必要があります。リキッドハンド ロボット システム、エラーの最小化/標準化に加えて化合物ライブラリーをスクリーニングする速度を増加させるサンプルの大規模な数を処理する設計されています。ベンチ上のマルチ チャンネル液体を用いたプロトコルシロイヌナズナ(シロイヌナズナ) に 50,000 の小分子のライブラリの高スループット転送化学遺伝学画面を達成するために最小限必要とする開発されたハンドリング ロボット技術者の関与です。これらのプロトコルで目に見える表現型の変化を引き起こした 3,271 小分子を発見されました。1,563 化合物による短い根、変更 1,148 化合物着色、383 化合根毛と他、非分類変更と発芽抑制 177 化合物。

正確かつ効率的に解剖顕微鏡の下で濃度のスクリーニングで小分子添加に基づく表現型を特徴付ける能力はシロイヌナズナ.の化学遺伝学前方のこのメソッドの最終的な目標50,000 すべての化合物を上映したときに観察された表現型多様ないくつかの異なるクラス (図 2) に分割できます。図 3A-Fは、解剖顕?...

Watch this Scientific Journal Video about Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana at JoVE.com

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

高スループット転送化学遺伝学の画面シロイヌナズナを行う液体処理ロボットの使用を最適化します。

合成小分子の高スループット画面は、モデル植物シロイヌナズナの行われました。液体ハンドリング ロボットの開発、このプロトコルは、前方の化学遺伝学スクリーン、植物生理に影響を与える小さな分子の発見を加速の速度を上げます。

Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or ...

optimizing-use-liquid-handling-robot-to-conduct-high-throughput

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

6.6K ビュー.  University of Kentucky. 合成小分子の高スループット画面は、モデル植物シロイヌナズナの行われました。液体ハンドリング ロボットの開発、このプロトコルは、前方の化学遺伝学スクリーン、植物生理に影響を与える小さな分子の発見を加速の速度を上げます。

ビデオ: Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of Arabidopsis thaliana

Adaptation of a Haptic Robot in a 3T fMRI

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing radiation, millimeter spatial accuracy of anatomical and functional data 2, and nearly real-time analyses 3. Haptic robots provide precise measurement and control of position and force of a cursor in a reasonably confined space. Here we combine these two technologies to allow precision experiments involving motor control with haptic/tactile environment interaction such as reaching or grasping. The basic idea is to attach an 8 foot end effecter supported in the center to the robot 4 allowing the subject to use the robot, but shielding it and keeping it out of the most extreme part of the magnetic field from the fMRI machine (Figure 1).
The Phantom Premium 3.0, 6DoF, high-force robot (SensAble Technologies, Inc.) is an excellent choice for providing force-feedback in virtual reality experiments 5, 6, but it is inherently non-MR safe, introduces significant noise to the sensitive fMRI equipment, and its electric motors may be affected by the fMRI's strongly varying magnetic field. We have constructed a table and shielding system that allows the robot to be safely introduced into the fMRI environment and limits both the degradation of the fMRI signal by the electrically noisy motors and the degradation of the electric motor performance by the strongly varying magnetic field of the fMRI. With the shield, the signal to noise ratio (SNR: mean signal/noise standard deviation) of the fMRI goes from a baseline of &tilde;380 to &tilde;330, and &tilde;250 without the shielding. The remaining noise appears to be uncorrelated and does not add artifacts to the fMRI of a test sphere (Figure 2). The long, stiff handle allows placement of the robot out of range of the most strongly varying parts of the magnetic field so there is no significant effect of the fMRI on the robot. The effect of the handle on the robot's kinematics is minimal since it is lightweight (&tilde;2.6 lbs) but extremely stiff 3/4" graphite and well balanced on the 3DoF joint in the middle. The end result is an fMRI compatible, haptic system with about 1 cubic foot of working space, and, when combined with virtual reality, it allows for a new set of experiments to be performed in the fMRI environment including naturalistic reaching, passive displacement of the limb and haptic perception, adaptation learning in varying force fields, or texture identification 5, 6.

The adaptation and use of a haptic robot in a 3T fMRI is described.

3TのfMRIの中の触覚ロボットの適応

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing ...

生物工学

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 ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

無水物ナノ粒子のハイスループット糖鎖の合成と機能化

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Designing a Bio-responsive Robot from DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in parallel computing2,3 , recognize and bind molecular or cellular targets4,5 , catalyze chemical reactions6,7 , and generate calculated responses in a biological system8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13, and several variations on this theme have since been realized using various techniques11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16. In this technique, the folding of a long (&gt;7,000 bases) single-stranded DNA 'scaffold' is directed to a desired shape by hundreds of short complementary strands termed 'staples'. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18 .
 
The current paper will focus on the caDNAno 2.0 software19 developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21 to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here, we describe how DNA origami can be utilized to design a robotic robot capable of sensing biological cues and responding by shape shifting, subsequently relayed to a desired effect.

DNA折り紙からバイオ応答ロボットの設計

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in ...

High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. The geometry of the monolith can be adapted for specific pipette tips ranging in volume from 1.0 to 5.0 ml. The large porosity of the monolith enables viscous or complex samples to readily pass through it with minimal fluidic backpressure. Bi-directional flow maximizes residence time between the monolith and sample, and enables large sample volumes to be processed within a single TruTip. The fundamental steps, irrespective of sample volume or TruTip geometry, include cell lysis, nucleic acid binding to the inner pores of the TruTip monolith, washing away unbound sample components and lysis buffers, and eluting purified and concentrated nucleic acids into an appropriate buffer. The attributes and adaptability of TruTip are demonstrated in three automated clinical sample processing protocols using an Eppendorf epMotion 5070, Hamilton STAR and STARplus liquid handling robots, including RNA isolation from nasopharyngeal aspirate, genomic DNA isolation from whole blood, and fetal DNA extraction and enrichment from large volumes of maternal plasma (respectively).

TruTip is a simple nucleic acid extraction technology whereby a porous, monolithic binding matrix is inserted into a pipette tip. Consequently, the sample preparation format is compatible with most liquid handling instruments, and can be used for many medium to high-throughput clinical applications and sample types.

共通液ハンドリングロボットでTruTipテクノロジーを使用して臨床検体からのDNAやRNAのハイスループット、自動抽出

TruTip is a simple nucleic acid extraction  technology whereby a porous, monolithic binding matrix is inserted into a  pipette tip. The geometry of the ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

ポリ（εカプロラクトン）エレクトロ糸にヒト間葉系幹細胞の添付ファイルの最適化

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

ハイスループットの使用<em&gt;インビトロ</emオーラル多種バイオフィルムを開発するために&gt;マイクロ流体システム

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

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 ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

モジュラー DNA デバイスの液体ハンドリング アセンブリを自動化

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

3D空間の細胞培養のための細胞骨格などの生体適合性液晶エラストマー発泡体の合成

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

高スループット転送化学遺伝学の画面シロイヌナズナを行う液体処理ロボットの使用を最適化します。

要約

さらに動画を探す

この動画の章

3TのfMRIの中の触覚ロボットの適応

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

無水物ナノ粒子のハイスループット糖鎖の合成と機能化

DNA折り紙からバイオ応答ロボットの設計

共通液ハンドリングロボットでTruTipテクノロジーを使用して臨床検体からのDNAやRNAのハイスループット、自動抽出

ポリ（εカプロラクトン）エレクトロ糸にヒト間葉系幹細胞の添付ファイルの最適化

ハイスループットの使用

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

モジュラー DNA デバイスの液体ハンドリングアセンブリを自動化

3D空間の細胞培養のための細胞骨格などの生体適合性液晶エラストマー発泡体の合成

高スループット転送化学遺伝学の画面シロイヌナズナを行う液体処理ロボットの使用を最適化します。

要約

さらに動画を探す

この動画の章

3TのfMRIの中の触覚ロボットの適応

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

無水物ナノ粒子のハイスループット糖鎖の合成と機能化

DNA折り紙からバイオ応答ロボットの設計

共通液ハンドリングロボットでTruTipテクノロジーを使用して臨床検体からのDNAやRNAのハイスループット、自動抽出

ポリ（εカプロラクトン）エレクトロ糸にヒト間葉系幹細胞の添付ファイルの最適化

ハイスループットの使用

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

モジュラー DNA デバイスの液体ハンドリング アセンブリを自動化

3D空間の細胞培養のための細胞骨格などの生体適合性液晶エラストマー発泡体の合成

モジュラー DNA デバイスの液体ハンドリングアセンブリを自動化