この研究はオーストラリアの国立保健医療研究評議会（プロジェクトの助成金がない。6288480）とスコビーとクレアマッキノントラストによってサポートされていました。

1。 RT反応をマイクロミキシング<ol><li>マイクロミキシングを持つRT反応を行う前に、RT反応の所望の温度にマイクロミキサーを平衡化します。 <ol><li> 37内部のマイクロミキサーを置く° C（またはRTの供給業者が推奨する温度）RT反応の開始前に少なくとも1時間のためのインキュベーター。 </li></ol></li><li>入力のトータルRNA（例えば0.1-1pg/μl）、代わりにNG -μgの単一セルの金額を使用することを除いて、逆転写酵素の供給業者の（Promega社から例えばMMTV - RT、キアゲンからOmniscript）の指示にしたがってRTミックスを、設定するサプライヤーが推奨する量。 <ol><li>我々は標準的な滅菌ヌクレアーゼフリー200μL薄壁PCRチューブを使用してください。 </li></ol></li><li>彼らはミキシングの準備ができているとすぐに安全にマイクロミキサーに位置RTチューブ。マイクロミキサーは、RT反応の間、37℃のインキュベーターの内部に残るはず。 </li><li>マイクロミキシングの適切な周波数と振幅を選択します。この例では150Hzの使用。 </li><li>のマイクロミキサーを切り替えて、RT反応（60分）期間中、常にそれを残す。 </li><li>マイクロミキサーのスイッチをオフにし、RT反応が完了すると、RTのチューブを取り出します。 </li><li>氷上でRTのチューブをセットし、そのような定量的ポリメラーゼ連鎖反応（定量PCRまたはリアルタイムPCR）として、さらにアプリケーションで通常どおり進みます。 </li></ol> 2。代表的な結果： RT反応のコンテキスト内でマイクロミキシングの利点は、他の混合方法（例えば、揺れボルテックスまたは粉砕）が使用されている場合に相対的cDNAの収量の増加です。これは、cDNAを用いて定量的ポリメラーゼ連鎖反応（定量PCRまたはリアルタイムPCR）のその後の分析により、例えば、見ることができます。図2Aは、直前のRTのインキュベーション（黒、&quot;粉砕&quot;にRT反応の粉砕に比べて最初の5分（青、&quot;micromixed5&quot;）または全体の60分間マイクロミキシングの効果（赤、&quot;micromixed60&quot;）を示しています）。この場合、前述のRT反応液量は25μlのであり、それはトータルRNAの2.5pg含まれている。 cDNAを表すNurr - 1 mRNAを検出するように設計されたプライマーを用いて定量PCRのトレースは、上記の表示され、平均値±この同じ実験の3反復で50％の最大蛍光に達するまでのサイクル数のSEMのは、以下にプロットされます。アスタリスクは、統計学的有意差（Tukeyの多重比較で、一方向ANOVA）を表します。粉砕に比べて大きな改善をもたらした前述のRT反応の最初の5分間マイクロミキシングは、前述のRT反応の全体の60分間マイクロミキシングのに対し、粉砕を介して非有意な改善をもたらしたことに注意すること、の大きさは約15定量PCRと同等であったサイクル、平均で。 マイクロミキシングのメリットも得、その後の定量PCR産物の融解曲線分析で見ることができます。図2Bに示す実験では、前述のRT反応の全体の60分間マイクロミキシングにのみ生産された最初の5分または粉砕のために一貫性のない定量PCRをマイクロミキシングのに対し、重複して試料中の一貫性のある定量PCRのシグナル（図2BA、2赤のトレース）となりましたトレース（図2BA、2青色のトレースと2本の黒い痕跡、それぞれ）。グレーのトレースは、定量PCRの反応から除外されたcDNAをしたネガティブコントロール（&quot;NEG。&quot;）です。この実験から、定量PCRの製品が（すなわち融解曲線分析）、その温度をランプによって変性されたそれは、適切なPCR産物（すなわち、mRNAのはるかに高い濃度が逆転写と定量PCRによって増幅された際に得られたものと同一であったアンプリコン、ことは明らかでしたデータは示されていないが）のみ60分（図2BB、2赤のトレース）のためにmicromixedされていた以下のRT反応存在していた。他のすべて（すなわちmicromixed5、粉砕およびネガティブコントロール）（例えばプライマーダイマー）の代替アンプリコンの製品を生成した。 <img src="/files/ftp_upload/3144/3144fig1.jpg" alt="図1"/> 図1標準的な実験RT反応へのマイクロミキシングを適用するためのプロトコルのフロー図。物理的原理に関与、マイクロミキサーとどのように社内のマイクロミキサーを構築するためのより詳しい説明を参照13,14に記載されています<img src="/files/ftp_upload/3144/3144fig2.jpg" alt="図2"/> 図2。そうでなければ標準的な実験RT反応でmRNAの単一セルの量を構成するRT反応の前に適用されるマイクロミキシングのメリットを示す代表的な定量PCRデータ。Nurr - 1遺伝子をコードするcDNA表すmRNAを検出する定量PCRのトレースの例。前述のRT反応は、25μlの体積のトータルRNAの2.5pg含有し、℃で60分間、37℃インキュベーター中で行った。 RT反応は、Rの粉砕によって混合し、Tは、インキュベーション（黒痕跡、&quot;粉砕&quot;）する前に混ぜてRT反応の最初の5分間の間にmicromixed（青トレース、&quot;micromixed5&quot;）またはRT反応（赤いトレース、&quot;micromixed60&quot;の全体の60分間micromixed ）。平均±最大蛍光50％に達するために必要な定量PCRのサイクル数のSEMのは、この実験のn = 3の繰り返しのために以下にプロットされます。アスタリスクは有意差（Tukeyの多重比較を一元配置ANOVA）を示します。Bは Nurr - 1のcDNA産物、25μlの体積で、 ​​トータルRNAの25pgを含むRT反応は、次のこの時間を調べるもう一つの定量PCR実験。定量PCRのトレース（）と同様に得られる定量PCR産物の融解曲線解析は（b）の異なる混合条件として使用されています。ないcDNAは前述のRT反応に含まれていないれたネガティブコントロールも（灰色の痕跡、&quot;NEG。&quot;）が含まれています。 mRNAのはるかに高い濃度でRT反応も行うと（b）micromixed60用（赤のトレース）の試料のものと同一の融解曲線のピークを（データは示さず）を製造した。

<ol>
	<li>Livesey, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+Funct+Genomic+Proteomic.">Brief Funct Genomic Proteomic.</a> 2 (1), 31-31 (2003).</li><li>Aumann, T. D., Gantois, I., Egan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Exp Neurol. 213 (2), 419-419 (2008).</li><li>Ottino, J. M., Wiggins, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Philos Transact A Math Phys Eng Sci. 362 (1818), 923-923 (2004).</li><li>Losey, M. W., Jackman, R. J., Firebaugh, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Microelectromech. Syst. 11, 709-709 (2002).</li><li>Bontoux, N., Dauphinot, L., Vitalis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lab Chip. 8 (3), 443-443 (2008).</li><li>Marcus, J. S., Anderson, W. F., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anal Chem. 78 (9), 3084-3084 (2006).</li><li>Marcus, J. S., Anderson, W. F., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anal Chem. 78 (3), 956-956 (2006).</li><li>Warren, L., Bryder, D., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proc Natl Acad Sci U S A. 103 (47), 17807-17807 (2006).</li><li>Jiao, Z. J., Huang, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microfluidics Nanofluidics. 6, 847-847 (2009).</li><li>Ahmed, D., Xiaole, M., Juluri, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microfluidics Nanofluidics. 7, 727-727 (2009).</li><li>Paxton, W. F., O'Hara, M. J., Peper, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anal Chem. 80, 4070-4070 (2008).</li><li>Autom, L. a. b. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 11, 399-399 (2006).</li><li>Petkovic-Duran, K., Manasseh, R., Zhu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 47 (4), 827-827 (2009).</li><li>Boon, W. C., Petkovic-Duran, K., White, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 50 (2), 116-116 (2011).</li><li>Liu, R. H., Lenigk, R., Druyor-Sanchez, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anal Chem. 75 (8), 1911-1911 (1911).</li><li>Riley, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ann+Rev+Fluid+Mech.">Ann Rev Fluid Mech.</a> 33, 43-43 (2001).</li><li>Whitehill, J., Neild, A., Ng, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Applied Physics Letters. 96 (5), 053501-053501 (2010).</li></ol>

著者らは、開示することは何もない。

ここで説明する標準的な実験RT反応にマイクロミキシングのアプリケーションの方法は、もちろん、どのような方法（例えば、細胞溶解、レーザーキャプチャーマイクロダイセクション）を経由して収穫されたmRNAを含むことができる。また、任意のブランドまたはRT試薬、任意の温度（マイクロミキサーの材料の許容範囲内）の種類、および任意の期間を含むことができる。例えば、我々は?...

<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>トータルRNAは、スナップ凍結急性準備成体マウスの脳スライスから単離された</td><td></td><td></td><td></td></tr><tr><td> PicoPure RNAアイソレーションキット</td><td>アークトゥルス、CA、アメリカ合衆国</td><td> KIT0204 </td><td>キットは現在、アプライドバイオシステムズから入手できます。 </td></tr><tr><td> DNA -フリーDNase処理と除去試薬</td><td>アンビオン</td><td> AM1906M </td><td></td></tr><tr><td>ランダムヘキサマープライマー</td><td>プロメガ</td><td> C1181 </td><td></td></tr><tr><td> AMV - RT </td><td>プロメガ</td><td> M5101 </td><td></td></tr><tr><td> dNTPのセット</td><td>プロメガ</td><td> U1240 </td><td></td></tr><tr><td> RNasin リボヌクレアーゼ阻害剤</td><td>プロメガ</td><td> N2111 </td><td></td></tr><tr><td> Nuclease - Free Waterを</td><td>プロメガ</td><td> P1193 </td><td></td></tr><tr><td> SYBR グリーンPCRマスターミックス</td><td>アプライドバイオシステム</td><td> 4309155 </td><td></td></tr><tr><td> HPRT前進（20mer）：CTT TGC TGA CCT GCT GGA TT </td><td></td><td></td><td></td></tr><tr><td> HPRTリバース（20mer）：TAT GTC CCC CGT TGA CTG AT </td><td></td><td></td><td></td></tr><tr><td> Nurr1フォワード（23mer）：CAG CTC CGA TTT CTT AAC TCC AG </td><td></td><td></td><td></td></tr><tr><td> Nurr1リバース（23mer）：GGT GAG GTC CAT GCT AAA CTT GA </td><td></td><td></td><td></td></tr><tr><td>光度計1000分光光度計。 </td><td>サーモサイエンティフィック</td><td></td><td></td></tr><tr><td>コルベットローター遺伝子のRG - 6000 </td><td>コルベットライフサイエンス</td><td></td><td>コルベットライフサイエンスは、2008年7月にQIAGENに買収された</td></tr></tbody></table>

increasing cdna yields from single-cell quantities of mrna in standard laboratory reverse transcriptase reactions using acoustic microstreaming

細胞の挙動との相関遺伝子発現は、理想的には単一細胞レベルで行われます。それが転写を逆にすることができます前に、単セル（1-5 1 - 50pgのトータルRNAの％、または0.01 - 2.5pg mRNAは、セル1あたり）に存在する不安定なmRNAの少量がほとんど劣化するため、これは容易に達成されていません安定したcDNAコピーに。例えば、標準的な実験用試薬とハードウェアを使用して、遺伝子のわずかな数は、定性的にセル2ごとに評価することができます。 mRNAの単セルの量を構成する標準的な実験の逆転写酵素（RT）反応（マイクロリットルの量で、すなわち標準試薬）の効率を向上させる一つの方法は、それが低下する前にmRNAをcDNAに変換できるので、より迅速に試薬を混合することです。しかし、マイクロリットルスケールでの液体の流れが層流であるため、これは自明ではない、すなわち混合の現在利用​​可能なメソッド（、揺れボルテックスと粉砕つまりは）効果的に試薬を混合するために十分なカオス運動を生成するために失敗する。この問題を解決するために、マイクロスケールの混合技術は、3,4を使用する必要があります。マイクロ流体ベースのミキシング技術の数が正常にRT反応の収量5-8を増加させるが開発されている。しかし、マイクロ流体技術は、比較的高価であり、まだ広く利用できない特殊なハードウェアを必要とする。安く、より便利なソリューションが望まれる。本研究の主な目的は、mRNAの単セルの量を構成する標準的な実験RT反応の小説&quot;マイクロミキシング&quot;技術の応用が著しく、そのcDNA収量を増加させる方法を説明することです。遺伝子発現の解析（2）より多くの定量;（1）セルごとに分析される遺伝子の大きい数と発現量の低い遺伝子の（3）良好な検出：我々は、cDNAの収量が可能な約10 100倍、増加見つける単一細胞インチマイクロミキシングは音響は、9-12小障害物の周りに伝播する音波が障害物の近くに平均流を作る現象をmicrostreamingに基づいています。我々は、主要な簡素化と音響microstreamingベースのデバイスを（&quot;マイクロミキサー&quot;）を開発しました。音響microstreamingは、システムが湾曲13の小さな半径を持つ液体-空気界面を持って確保することによってオーディオ周波数で達成することができます。チューブ内の溶液のマイクロリットルの容積のメニスカスの曲率の適切な小さい半径を提供します。オーディオ周波数の使用は、ハードウェアが安価と13の多彩なことができる、と彼らは標準的な実験室sonicatorsとすることができるように、核酸と他の生化学試薬が破損していないことを意味します。

Watch this Scientific Journal Video about Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming at JoVE.com

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

我々は、そうでなければ標準的な実験の逆転写反応でmRNAの単一セルの量からcDNA収量を増加させるための新しい方法を説明します。目新しさは、ボルテ​​ックスまたは粉砕、揺れよりも効果的にマイクロリットルスケールでの流体を混合するために、音響microstreamingの現象を利​​用したマイクロミキサーの使用に存在する。

アコースティックMicrostreamingを使用して標準的な実験系逆転写反応でmRNAの単セルの物理量からcDNA収量を増加させる

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

increasing-cdna-yields-from-single-cell-quantities-mrna-standard

Research

JoVE Journal

Bioengineering

10.1K ビュー.  University of Melbourne. 我々は、そうでなければ標準的な実験の逆転写反応でmRNAの単一セルの量からcDNA収量を増加させるための新しい方法を説明します。目新しさは、ボルテ​​ックスまたは粉砕、揺れよりも効果的にマイクロリットルスケールでの流体を混合するために、音響microstreamingの現象を利​​用したマイクロミキサーの使用に存在する。

ビデオ: Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

標準的なインクジェットプリンタを使用して一時的な細胞膜孔を作成する

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

生物工学

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

単一細胞分泌物の時空間イメージング用ラベルフリー技術

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

単一細胞のゲノムの配列のための超高スループット マイクロ流体プラットフォーム

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

A Converging Strategy for the Generation of a Virtually Sequenced cDNA Library from Unreferenced Pacific Oysters

The access to biological material of reference species, which were used previously in key experiments such as in the development of novel cell lines or genome sequencing projects, are often difficult to provide for further studies or third parties due to the consumptive nature of the samples. Although now widely distributed over the Pacific coasts of Asia, Australia and North America, individual Pacific oyster specimens are genetically quite diverse and are therefore not directly suitable as the starting material for gene libraries. In this article, we demonstrate the use of unreferenced Pacific oyster specimens obtained from regional seafood markets to generate cDNA libraries. These libraries were then compared to the publicly available oyster genome, and the closest related library was selected using the mitochondrial reference genes Cytochrome C Oxidase subunit I (COX1) and NADH Dehydrogenase (ND). The suitability of the generated cDNA library is also demonstrated by cloning and expression of two genes encoding the enzymes UDP-glucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS), which are responsible for the biosynthesis of UDP-xylose from UDP-glucose.

We describe a strategy for how to use RNA samples from unreferenced Pacific oyster specimens, and evaluate the genetic material by comparison with publicly available genome data to generate a virtually sequenced cDNA library.

参照されていない太平洋カキからの仮想配列cDNAライブラリの生成のための収束戦略

The access to biological material of reference species, which were used previously in key experiments such as in the development of novel cell lines or ...

Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high cell isolation and a controlled environment. However, the survival of hippocampal cells in vitro is compromised: most cells die during the first week of culture. It is therefore of great importance to identify ways to increase the durability of neural cells in culture.
Calcium carbonate in the form of crystalline aragonite derived from the skeleton of corals can be used as a superior, active matrix for neural cultures. By nurturing, protecting, and activating glial cells, the coral skeleton enhances the survival and growth of these cells in vitro better than other matrices.
This protocol describes a method for cultivating hippocampal cells on a coralline matrix. This matrix is generated by attaching grains of coral skeletons to culture dishes, flasks, and glass coverslips. The grains assist in improving the environment of the cells by introducing them to a fine three-dimensional (3D) environment to grow on and to form tissue-like structures. The 3D environment introduced by the coral skeleton can be optimized for the cells by grinding, which enables control over the size and density of the grains (i.e., the matrix roughness), a property that has been found to influence glial cells activity. Moreover, the use of grains makes the observation and analysis of the cultures easier, especially when using light microscopy. Hence, the protocol includes procedures for generation and optimization of the coralline matrix as a tool to improve the maintenance and functionality of neural cells in vitro.

Dissociated hippocampal cell culture is a pivotal experimental tool in neuroscience. Neural cell survival and function in culture is enhanced when coralline skeletons are used as matrices, due to their neuroprotective and neuromodulative roles. Hence, neural cells grown on coralline matrix show higher durability, and thereby are more adequate for culturing.

生物学的に活性なサンゴリンマトリックスを用いた解離神経細胞培養の耐久性向上

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

音響セパレータを用いたCHO採取細胞培養液の一次解明

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

Standard Test Method ASTM D 7998-19 for the Cohesive Strength Development of Wood Adhesives

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the adhesive cure, and the effect of adhesive penetration on the wood interphase; thus, normal testing of a neat adhesive film is generally not useful. Most tests of wood adhesive bond strength are slow, laborious, can be strongly influenced by the wood and do not provide information on the kinetics of cure. Test method ASTM D 7998-19, however, can be used for fast evaluation of the strength of wood bonds. The use of a smooth, uniform, and strong wood surface, like maple face-veneer, and sufficient bonding pressure reduces the adhesion and wood strength effects on bond strength. This method has three main applications. The first is to provide consistent data on bond strength development. The second is to measure the dry and wet strengths of bonded lap shear samples. The third is to better understand the adhesive heat resistance by quickly evaluating thermal sensitivity and distinguishing between thermal softening and thermal degradation.

We present a procedure, ASTM D7998-19, for a rapid and more consistent evaluation of both dry and wet strength of adhesive bonds on wood. The method can also be used to provide information on strength development as a function of temperature and time or strength retention up to 250 &#176;C.

木材接着剤の凝集力開発のための標準試験方法ASTM D 7998-19

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

磁気温熱療法とポト温熱併用がん治療のための磁気、音響、光三重応答性マイクロバブル

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

ヒト人工多能性幹細胞由来心筋細胞における単細胞光学作用電位測定

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, function at both the cellular and tissue levels,and regulate the mechanical systems in the body. Numerous biological processes are force-dependent, including motility, adhesion, and division of single-cells, as well as contraction and relaxation of organs such as the heart, bladder, lungs, intestines, and uterus. Given its importance in maintaining proper physiological function, cellular contractility can also drive disease processes when exaggerated or disrupted. Asthma, hypertension, preterm labor, fibrotic scarring, and underactive bladder are all examples of mechanically driven disease processes that could potentially be alleviated with proper control of cellular contractile force. Here, we present a comprehensive protocol for utilizing a novel microplate-based contractility assay technology known as fluorescently labeled elastomeric contractible surfaces (FLECS), that provides simplified and intuitive analysis of single-cell contractility in a massively scaled manner. Herein, we provide a step-wise protocol for obtaining two six-point dose-response curves describing the effects of two contractile inhibitors on the contraction of primary human bladder smooth muscle cells in a simple procedure utilizing just a single FLECS assay microplate, to demonstrate proper technique to users of the method. Using FLECS Technology, all researchers with basic biological laboratories and fluorescent microscopy systems gain access to studying this fundamental but difficult-to-quantify functional cell phenotype, effectively lowering the entry barrier into the field of force biology and phenotypic screening of contractile cell force.

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

マイクロパターンエラストマーを使用したシングルセル収縮性の簡素化されたハイスループット分析

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, ...