著者らは、AVのマーカーでセルブロックの切断セクションを実証するためにクリスチャートランド、HT（ASCP）を感謝。

はじめに： これはHistoGelTM（サーモサイエンティフィック）（HG）を使用して液体ベースの細胞診（LBC）標本からのセルブロックの準備のためのShidhamの方法を説明するビデオです。他のランダムなアプローチと比較して、以下は単独で散在バラセル（1-5）と比較的hypocellular標本から細胞のブロックを製造するためのこのプロトコルの2つの重要な機能です。 <ol><li>このプロトコルは、セルのブロックの切断面に平行な平面に沿って細胞を濃縮するための手順が含まれます。 </li><li>また、以下の目的のうちの2つを提供するAV -マーカー（図1b）、のビーコンのようなダークインクルージョンが含まれています。 <ol style="list-style-type:lower-alpha;"><li>細胞が濃縮されるレベルを可視化へ。暗い色のビーコンが切削中にさらされているときに目的の細胞と細胞のブロックの面積は現在histotechnologistによって可視化することができます。監視するには、この能力は、細胞の大部分でレベルを切断、またはサンプルの細胞の最も高い濃度でのレベルにあまりにも表面的なカットではないのを妨げる。 </li><li>別のスライド上でシリアルセルブロックのセクションのロケータの基準点として機能する。この基準点は、SCIPのアプローチ（6,7）と座標の免疫反応性のパターンの評価のための細胞の特定の細胞またはグループを検索するためのビーコンとして機能します。 </li></ol></li></ol> PROTOCOL（図2） サンプルの調製。 <ol><li>平底ガラス管（直径15mmさx 45mm）（2.4〜図2.1）に残留LBC子宮頸部細胞診標本を転送する。大きなプラスチック製のキャリアのチューブ（28 × 85ミリメートル）と遠心分離機にガラス管を置きます。キャリアのチューブからガラス底のチューブを外し、上清を取り除き。 </li><li>ガラス管は、蓋を（次のステップで加熱水の流出を防止するため）と大きな平底のキャリアのプラスチックチューブの内側に戻される</li><li>ガラス管を含むキャリアのプラスチック製のチューブは、その後、ふたを遠心分離機に入れ（ 細胞がガラス管の平らな底に垂直に落ちるように旋回カップではなく、固定アングルカップ付き ）、および1805でG（3000 rpmをスピンしているローター半径- 17センチメートル）5分（図2.5）のため。 </li><li>試験管を遠心分離器から垂直に削除され、小さなガラス管は、細胞を沈殿ペレットを乱すことなく、より大きなキャリアのプラスチックチューブから鉗子で除去する。 </li><li>試験片とガラス管が非キャップされ、上清は下部の堆積物の細胞の平らな層（図2.6）を乱さないように注意しながらオフ注がれています。 </li></ol>基準座標AV -マーカーの包含およびゲルの追加<ol><li>暗いビーコンAV -マーカー （2 × 2mmの大きさについて、フラットは、暗い色、sectionable材料の断片を表面化）（図3）ガラス管（図2.7）への道標として追加されます。 </li><li>ミディアムパワーで10秒間電子レンジで溶かしてHGのアリコートを液化する。 </li><li> 、チューブに溶融HGの0.5 mlを加え素早く堆積物と混合し、（図2.8）、それを要約（HGが凝固を開始できるようにすることなく、迅速に次のステップに進みます）。 </li><li>キャリアのプラスチックチューブに温かい（45℃）水（図2.9）の約2.5 mlを追加します。 </li><li>小さなキャップガラス管は、暖かい水とプラスチック製のチューブ内に配置されます。 （この手順は、次のステップの間に固化するからHGを維持する必要がある）（図2.9）。 </li><li>キャリアのプラスチック製のチューブは、（ 細胞がガラス管の平らな底に垂直に落ちるように旋回カップと固定されていない角のカップ付き ）遠心分離機に入れ、と5のための1805 G（3000 rpmの、ローター半径- 17センチメートル）で回転さ分。この遠心分離のステップの目的は、AV -マーカーをプッシュすると近い最後のパラフィン包埋細胞のブロック（図1d）の切断面の層に細胞を集中することです。 </li><li>チューブを下部に、サンプルの細胞と沈殿した薄い層を乱さないように注意しながら遠心機から静かにして垂直に削除されます。 </li><li>大きなプラスチック製のチューブは非キャップされており、小さいガラス管は、試料の細胞の堆積層を乱すことなく、ピンセットで垂直に削除されます。 </li><li>小さなガラス管は、HG（図2.11）を冷却固化するために15分間垂直位置で冷凍です。 </li></ol>最終処理のための試料を用いてゲルのボタンなど、セルブロックの除去<ol><li>下部に集中/堆積物試料の層を持つ固化HGディスクは、注射器（図2.12）と23ゲージの針を通して10％ホルマリンを潮吹きで平坦な底部のガラス管から外れている。 </li><li>針は、試料（図2.12）と固化HGディスクの周辺部での管の側面に沿って挿入されます。 </li><li> needlホルマリンがゆっくりとシリンジを介してプッシュされる一方、電子は管の側面に沿って回転される。暗い色のビーコンAV -マーカーとガラス管の平底（図2.12）からそれに集中標本とともにHGボタンの分離でこの結果。 </li><li>セルブロック（試料の細胞とゲルのボタン）は、標識カセット内に配置し、パラフィン包埋セルブロック（図2.13）を調製するために組織の処理のために送信されます。 </li></ol>埋め込みと試料の切断<ol><li>ディスクは切断面のようなダウン暗いビーコンマーカー側（図1）とパラフィンに埋め込まれています。 </li><li>ビーコンなどの暗い色のAV -マーカーが露出し、はっきりと見えるようになるまでブロックは切片です。 </li><li> 3〜4ミクロンの切片を試料から単独で散在する細胞のほとんどが含まれている必要がありますこのレベルからカットされています。 </li><li>セクションは、さらに染色、免疫組織化学染色、または示されるように他のテスト用のガラススライド上に収集されます。セクションをマウントするために使用するスライドの種類を含めこれらのテストのためのプロトコルが異なる場合があります。一般的に免疫染色のために、コーティングされたスライドは、免疫染色のステップ中に浮遊し、スライドからセクションの損失を防ぐために使用されます。 </li></ol> （アルファベット順）使用される略語：CISH、in situハイブリダイゼーション試験発色、FISH、蛍光in situハイブリダイゼーション試験、FFPE、ホルマリン固定パラフィン包埋、FNA、穿刺吸引、HG、HistoGel™（サーモサイエンティフィック）; LBC、液体ベースの細胞診、PCRのポリメラーゼ連鎖反応; <img src="/files/ftp_upload/1316/1382fig1.jpg" alt="図1" /> 図1。Shidhamのプロトコルによって調製された細胞のブロックの構造。 <img src="/files/ftp_upload/1316/1382fig2.jpg" alt="図2" /> 図2。ShidhamのプロトコルによってLBC検体からのセルブロックを調製するためのさまざまな手順の概要。 <img src="/files/ftp_upload/1316/1382fig3.jpg" alt="図3" /> 図3。バナナの皮からAV -マーカーの調製。 <img src="/files/ftp_upload/1316/1382fig4.jpg" alt="図4" /> 図4とし、AV -マーカーなしのセルブロックとセクションの比較。 <img src="/files/ftp_upload/1316/1382fig5.jpg" alt="図5" /> 図5。AV -マーカーがある場合とない場合のセルのブロックのセクションの細胞数の比較。

<ol>
	<li>Shidham, V. B., Epple, J., Shidham, V. B., Atkinson, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+14,+Appendix+I:+Collection+and+processing+of+effusion+fluids+for+cytopathologic+evaluation.">Chapter 14, Appendix I: Collection and processing of effusion fluids for cytopathologic evaluation.</a> Cytopathologic Diagnosis of Serous Fluids. , .</li><li>Varsegi, G., D'Amore, K., Shidham, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p16INK4a+Immunocytochemistry+as+an+Adjunct+to+Cervical+Cytology+-+Potential+Reflex+Testing+on+Specially+Prepared+Cellblocks+from+Residual+Liquid+Based+Cytology+(LBC)+Specimens.">p16INK4a Immunocytochemistry as an Adjunct to Cervical Cytology - Potential Reflex Testing on Specially Prepared Cellblocks from Residual Liquid Based Cytology (LBC) Specimens.</a> Modern Pathology 22: 98th Annual Meeting of United States and Canadian Academy of Pathology. , 97a-97a (2009).</li><li>Nigro, K., Tynski, Z., Wasman, J., Abdul-Karim, F., Wang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+cell+block+preparation+methods+for+nongynecologic+ThinPrep+specimens.">Comparison of cell block preparation methods for nongynecologic ThinPrep specimens.</a> Diagn Cytopathol. 35, 640-643 (2007).</li><li>Saleh, H. A., Hammoud, J., Zakaria, R., Khan, A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Thin-Prep+and+cell+block+preparation+for+the+evaluation+of+Thyroid+epithelial+lesions+on+fine+needle+aspiration+biopsy.">Comparison of Thin-Prep and cell block preparation for the evaluation of Thyroid epithelial lesions on fine needle aspiration biopsy.</a> CytoJournal. 5, 3-3 (2008).</li><li>Kyroudi, A., Paefthimiou, M., Symiakaki, H., Mentzelopoulou, P., Voulgaris, Z., Karakitsos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+diagnostic+accuracy+with+a+cell+block+preparation+from+thin-layer+endometrial+cytology:+a+feasibility+study.">Increasing diagnostic accuracy with a cell block preparation from thin-layer endometrial cytology: a feasibility study.</a> Acta Cytol. 50, 63-69 (2006).</li><li>Atkinson, B. F., Shidham, V. B., Atkinson, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+5:+Immunocytochemistry+of+effusion+fluids:+introduction+to+SCIP+approach+(Chapter+5.">Chapter 5: Immunocytochemistry of effusion fluids: introduction to SCIP approach (Chapter 5.</a> Cytopathologic Diagnosis of Serous Fluids. , (2007).</li><li>Shidham, V. B., Shidham, V. B., Atkinson, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+15,+Appendix+II:+Immunocytochemistry+of+effusions+processing+and+commonly+used+immunomarkers.">Chapter 15, Appendix II: Immunocytochemistry of effusions processing and commonly used immunomarkers.</a> Cytopathologic Diagnosis of Serous Fluids. , (2007).</li></ol>

セルブロックは、様々な細胞診標本（1）の評価のための貴重なツールです。最も重要なことは、試料のアーキテクチャの詳細に加えて、セルのブロックは、免疫細胞化学などの補助的な研究は、発色/蛍光in - situハイブリダイゼーション試験（FISH / CISH）とのin - situ PCRの評価を可能にする。セルブロックの調製のための様々な方法が記載されている。しかし、これらのほとんどは、比較的多くの細胞を含む、組織のFNA吸引のようなmicrofragmentsや滲出液（1,3,4,5）などのいくつかの漿液性の液体で、非婦人科細胞診標本に適しています。 セルブロックは、主に免疫細胞化学的評価のための機会を提供するので、それらの処理は、好ましくは、ホルマリン固定パラフィン包埋（FFPE）組織のそれのようになります。最終的にセルブロックのセクションの免疫細胞化学的評価後に得られた結果は、FFPE組織切片で主に行わ発表された文献とのそれらと比較されます。プロトコル内の任意の変化は、潜在的にルーチンFFPEのために使用される以外のさまざまな固定液と試薬のシーケンスを通じて処理セルブロックで得られた結果の妥当性が損なわれ、無効にする。いくつかの商用技術は他の固定液、試薬の露出への曝露のプロトコルを介して処理の欠点を持つことができます。 セルブロックの準備の様々な方法は、堆積物および組織断片のかなりの量の試料のために用意されています。主に、濃縮された堆積物は、いくつかのゲルまたは凝集の原理によってサポートされています。操作しやすいボタンは、外科病理標本/生検のように処理した後、パラフィンに埋め込まれています。 使用するゲルは、ゼラチン、寒天、フィブリノーゲン/プラズマトロンビン、およびそのようなHGのような他の市販のゲルを含む。濃度のメソッドは、遠心分離によって沈殿物の単純なペレットからの膜の様々なタイプに沿って細胞の濃度に変化する。例としては：ミリポア、collodin（Celloidin）バッグまたはガラススライド（1）細胞診塗抹標本から細胞をこすりを。我々はまた、寒天とゼラチンの様々な組み合わせを試すことによってゲルの多様性を評価した。組み合わせのどれもワンピースの試料から埋め込まれた細胞と凝固ゲルの簡単に操作しやすいディスクを入手するために会社に十分な一貫性を達成していない。 HGは、適切な一貫性を示し、我々の経験でHGのセルブロックのセクションでの免疫染色の結果が優れている。 前述のようにcervicovaginal細胞診のための液体ベースの細胞診（LBC）標本は、一般的に非婦人科標本よりも携帯電話です。さらに、婦人科LBC標本は主にcervicovaginal粘膜から個々の散在剥離した表層の細胞が含まれています。このため、セルブロックのセクション内の適切な細胞充実性は、特別なアプローチなしでは達成されないことがあります。ブロックはセクションカット時のhistotechnologistが見ることができないで、これらの単独で散在する細胞成分として、細胞がセクションに表示されて開始されるレベルは高く評価することはできず、どちらかほとんどの細胞でレベルを越えてカットしてかどうか、見逃されることがあるサンプルセルの最高濃度（図4）でレベルに十分に深くカット。 Shidhamのプロトコルは、培地と従来のラボ機器（2）（図2）を埋め込むようにHGを使用して、これらの問題の両方に対応しています。 このプロトコルは、2つの主な機能（図1）以下が含まれます。 <ol><li>隣接およびセルブロック（図5）の切断面に平行な狭い面で単独で散在して細胞の濃度と整列。 </li><li>道標としてビーコンのような暗いAV -マーカーの包含。これは、次の機能を得るために重要です。 <ol style="list-style-type:lower-alpha;"><li>識別し、細胞をセルブロックに集中してレベルを監視すること。 濃い色の道標の暴露は、セルブロックで単独で散在して細胞のほとんどが （図4）が配置されると予想されるレベルを強調しています。これによセルブロックへの伐採 （ほとんどの細胞でレベルを越えて切断）やアンダーカット （サンプルセルの最高濃度でレベルに十分深くカットされていない）と最高濃度に対応するセルブロックのレベルからのセクションの選択を可能に細胞の。 </li><li>セクションで暗い色の道標にも別のスライド上のセルのブロック（図4）の別の連続切片で全く同じ細胞を調査し、識別するための基準点として機能します。別のセクション（6,7）で同じセルに従うようにSCIPのアプローチにより、特定のセルの座標プロパティなどimmunoprofileを解釈し、評価しながら、これは非常に重要です。 </li></ol></li></ol> OUもののRプロトコルは、液体ベースの子宮頸部細胞診標本のための標準化と報告されている、それはまた、さらにAVマーカーを改善促進するような滲出液、FNA、ブラッシング、嚢胞の内容等、多くの他の非婦人科標本の診断率を高めるために使用することができますこれらの標本のセルブロックの切片の免疫組織化学評価中にSCIPのアプローチの応用。埋媒体は、関連するステップで適切な変更を加えて他の試薬に置き換えてもよい。 HGは、室温（1）においてトロンビンによってゲル化するプラズマ（フィブリノーゲン）に置き換えてもよい。

cell block preparation from cytology specimen with predominance of individually scattered cells

このビデオでは、個々に散在する細胞と小細胞群を含む液体ベースのcervicovaginal細胞診標本からのセルブロックの調製のためのShidhamの方法を示しています。この手法は、従来の実験装置でHistoGel（サーモサイエンティフィック）を使用しています。 セルブロックのセクションの使用は非婦人科細胞診の評価のための貴重な補助的なツールです。彼らは、細胞病理学者が病変のアーキテクチャを含む、追加の形態学的試料の詳細を調べることができます。最も重要なことは、彼らは、in situハイブリダイゼーション試験（FISH / CISH）とのin - situポリメラーゼ連鎖反応（PCR）は、このような免疫細胞化学などの補助的な研究の評価が可能になります。従来のセルブロックの調製技術は、主に体液の滲出液および穿刺吸引生検のために一般的に、非婦人科細胞診標本に適用されている。 液体ベースのcervicovaginal標本は多くの個々の散在のセルとの非婦人科比べ相対的に小さい携帯電話です。このため、セルブロックのセクション内に十分な細胞数は達成することは困難である。さらに、ブロックの切削histotechnologistは、細胞が最も高い濃度にさらされているレベルを可視化することはできません。したがって、セクションは、テスト用スライドガラスに転送するように選択されることができる適切なレベルを監視するのは困難です。その結果、目的の細胞と細胞のブロックの面積はどちら過去切断または十分深くカットしないことで、見逃されることがある。 Shidhamのメソッドの現在のプロトコルは、これらの問題に対処します。このプロトコルは、婦人科、液体ベースの細胞診標本のための標準化と報告ですが、それはまた、滲出液、FNA、ブラッシング、セルブロックのセクションの診断材料の品質向上のための嚢胞の内容などのような非婦人科標本に適用することができます。

Watch this Scientific Journal Video about Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells at JoVE.com

Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells

個々に散在する細胞と小細胞群を含む細胞診標本からAV -マーカー付きセルブロックの調製のためのShidhamの方法。

個別散在セルの優位を持つ細胞診標本からのセルブロックの準備

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually ...

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73.2K ビュー.  University of Wisconsin - Milwaukee. 個々に散在する細胞と小細胞群を含む細胞診標本からAV -マーカー付きセルブロックの調製のためのShidhamの方法。

ビデオ: Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

冷凍細胞から出発培養：ヒトES細胞

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

マウス胚性幹細胞から造血幹細胞の導出

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Midgastrulaステージショウジョウバエの胚からの神経培養の準備

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual dissociations the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions.  The gentleMACS Dissociator can optimally dissociate mouse spleen, combining timesaving and standardization with user-safety.

This video describes how to dissociate mouse spleens using the gentleMACS Dissociator, an automated bench-top device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation, spleens are filtered, centrifuged, and resuspended for further applications.

This video describes a simple, time-saving technique for automated tissue dissociation using the gentleMACS Dissociator to prepare single-cell suspensions of mouse splenocytes.

gentleMACS Dissociatorとマウス脾臓からの単細胞懸濁液の調製

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.
S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24&deg;C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.
Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Preparation of Drosophila S2 cells for Light Microscopy

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

の準備ショウジョウバエ</em光学顕微鏡用> S2細胞

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

Preparation of Quality Inositol Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

品質イノシトールピロリン酸塩の調製

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

培養細胞からの染色体の準備

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

基礎脊椎動物の系統からの一次筋原前駆細胞/筋芽細胞培養液の調製

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

仙台ウイルスのヒト体細胞からの効率的な次世代ヒューマン誘導多能性幹細胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

集束イオンビームを用いて低温電子顕微鏡標本の準備

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...