この作品は、米国国立科学財団（EAR-0920299およびEAR-0745808）からの補助金によって支えられて、米国国立科学財団（東アジア·太平洋担当）サマー研究所、アメリカ研究助成プログラムと大学院研究奨学金同窓会助成地質学会をオハイオ州立大学から。我々は彼らの洞察に満ちたコメントのためのエディタと二人の匿名査読者に感謝したいと思います。

1。 MTBのコレクション<ol><li>磁性細菌を収集する淡水サイト（MTB）を決定する際に、それが柔らかい泥質堆積物の層を有する池や動きの遅い流れから開始するのが最適であることが多い。このデモでは、コロンバスのオハイオ州立大学（OSU）、オハイオ州（アメリカ）のキャンパス内にOlentangy川の端でサンプルを収集した。これは私たちのデモのために便利な場所でしたが、ここで説明されたプロトコルは任意の水生場所に適用可能である。このプロトコルで使用される材料は、 表1に記載されています。水の深さは10〜100センチメートルである場所を探します。そのような場所では、土砂クリア、スクリュートップコンテナを使用しての最上部層を収集する必要があります。それは半分土砂と水で残量3分の1で満たされるまでコンテナに土砂と水をすくう。それが水で満たされるまで水没容器をそのままにして、しっかりと容器wを締めくくるi番目のそのねじぶた。これは、堆積物を混合する必要はありません。タオルで乾燥した容器の外側を拭いてから、研究室にサンプルを取る。それはあなたの研究室に戻ってサンプルを急いでする必要はありません。我々は、我々の研究室に戻し持ち込む前に数日間フィールドにプラスチック容器にMTBのサンプルを残してきた。あなたがフィールドに涼しい、日陰でサンプルを格納するようにMTBは限りヶ月に数週間のための実行可能でなければなりません。 </li><li>サンプルでは、​​あなたの研究室に入ったら、キャップを緩め、それが蒸発量を削減するための容器をカバーしておきます。暗い部屋で、引き出しの中に室温で容器を保管し、または完全にアルミホイルで容器をカバーしています。堆積物と微粒子が完全に数時間〜数日のために邪魔されずにサンプルを残すことによって、容器の底に沈殿することができます。それは堆積物を混合する必要はありませんが、MTBは邪​​魔されない環境を好む。プラスチック容器の透明な壁あなたは、粒子が底に沈殿していることを確認することができます。あなたのサンプルによって、MTBは何ヶ月も試料中に生き続けることができます。 </li></ol> 2。 MTBのアイソレーション<ol><li>あなたが約1cm堆積物-水界面（ 図1A）上記プラスチック容器の両側にMTB、場所の磁石を分離する準備ができたら。容器の底に沈殿を乱さないように注意してください。コンテナと反対側のもう1つの棒磁石の北側（ 図1A）の片側に棒磁石のS極を配置します。ほぼすべての磁石は、磁気攪拌棒や大型冷蔵庫のマグネットを使用することができる。何は、堆積物 - 水界面上に正しい高さに磁石をサポートするために使用することができます。我々は、段ボールやプラスチック製の箱の上に磁石を休んでいることが最善であることがわかりました、しかし、磁石は、プラスチック容器の外側にテープで固定されていることができます。いくつかに30分待って磁石に泳ぐ細菌のための時間。 </li><li>慎重に南極の棒磁石の位置の近くにコンテナ（ 図1A）、（北半球で収集されたサンプルのための）内側から水を集めるために滅菌ピペットを使用しています。この水は、南極の棒磁石に引き寄せされたMTBを含める必要があります。次に、キャピラリー競馬場は、さらにMTBを豊かにするために使用されるべきである。 </li></ol> 3。 MTBの競馬場<ol><li>走磁性細菌とサンプルを豊かにするために、毛細管競馬場が必要である（ 図1B及び1C）。これらはクリアプラスチック容器から細胞を単離する前に行う必要があります。 </li><li>競馬場を作るために5.75インチ（146ミリメートル）のガラスパスツールピペットを使用しています。ピペットの上部をカットするダイヤモンドペンまたはファイルを使用して、ピペットの長さは重要ではありませんが、それは水の約1〜2 mlを含んでいることができるはずです。次に、先端を溶かすためにブンゼンバーナーを使用するようにそれは（ 図1C）密封されたとなります。結果のピペットは、開放端と密封端を持っている必要があります。 </li><li>これらの競馬場のいくつかの後、オートクレーブを行います。また、綿、いくつかの長い金属針をオートクレーブする必要があります。 </li><li>ろ過シリンジに取り付け長い金属針を用いてオートクレーブした競馬場に、 図1Aに示すように土砂と水の界面付近から採取し、ろ過した試料水を追加します。フィルターの孔径は、水から破片や汚染物質を除去するために0.22ミリメートルである必要があります。ガラスキャピラリー内に気泡がないことを必ず確認することが重要です。 </li><li>滅菌綿棒（ 図1B）と競馬場の底に差し込みます。封印先端（ 図1C）から1cm -それが0.5であるので、競馬場の密封された端の方に綿をプッシュするために金属針を使用します。 </li><li>滅菌ピペットを用いて、試料にMTB含有水（2.2節）を追加R準備されたMTBのレーストラック（ 図1B）のeservoir（開放端）。 </li></ol> 4。 MTBの濃縮<ol><li>競馬場は、サンプル流体で満たされたら、水平な面の上に、その側にそれを置く（ 例えば 、卓上型）と競馬場の密封された先端部（ 図1Bの隣に（北半球の）棒磁石のS極を配置及び図1C）。 </li><li>綿を通って移動し、MTBの場合は5〜30分待ちます。その後、競馬場の先端付近の流体を収集する必要があります。長すぎる待って、キャピラリの先端にそのような他の運動性細菌などの汚染物質を、導入することができます。必要に応じて、競馬場の先端を見るとMTBが競馬場の先端に集めて見て光学顕微鏡を使用することができます。これは、あなたがそれを綿栓を通って移動し、MTBのにかかる時間を決定することができます。 </li><li>優しく綿栓の近くの小さな傷を作り、競馬場の端を折り、ダイヤモンドナイフを使用しています。 </li> &lt;LI&gt;競馬の先端から流体を除去するために細い針（25または27ゲージ）を1mlシリンジを使用してください。この液体試料は現在、濃縮MTBを含める必要があります。 </li></ol> 5。光学顕微鏡で観察MTB <ol><li>カバースリップ上に濃縮されたMTBのサンプルの液滴（10〜20μl）を置きます。 </li><li>迅速ので、ドロップが現在ダウンして直面しているとカバースリップの上からぶら下がってカバースリップを反転させます。 </li><li>スライドガラス上に載っているOリングの上にカバースリップを置きます。 Oリングは、その後わずかに小さい直径カバースリップ（; 図2約1cm）を持っている必要があります。 </li><li>光顕微鏡ステージ上にこのハンギングドロップを置き、ドロップのいずれかの端に焦点を当てる。しかし、ハンギングドロップ法（ 図2）のために使用することは困難である石油を必要としないが、ほとんどは、高開口数（ 例えば 、0.93 NA）を持っているので、60X乾燥対物レンズは非常によく動作します。 </li><li>絞首刑dに近い棒磁石の南の端を置きますROPとMTBは磁石（ 図3）に最も近いドロップの端に向かって移動を開始します。数分以内に多くのMTBはドロップの端（ 図3）であるべきである。細菌は磁石の極を反転させることにより、磁気であることを自分で証明してから、細菌が反対方向に泳ぐ観察する。 </li></ol> 6。透過型電子顕微鏡（TEM）によるMTB観測<ol><li>銅グリッド上に濃縮されたMTBの滴（約20μL）を配置し、細菌が10分のために解決することができます。 </li><li>クリーンフィルタ一枚の紙で余分な水分をオフウィック。 </li><li>必要に応じて、グリッドに悪影響を2％酢酸ウラニル、2％リンタングステン酸のpH 7.2、または2.5％のモリブデン酸ナトリウム、7,8,9で染色することができます。これは、濃縮されたMTBでグリッドをインキュベートした後、直ちに染色一滴の上に銅グリッドを配置することによって行われます。負の汚れでグリッドをインキュベートし、倍はstに応じて異なりますアインは、使用した後、きれいなろ紙の切れ端で流体を切る芯。 </li><li>透過型電子顕微鏡（TEM、 図4）を使用して、MTBを守ってください。ここでMTBを説明された仕事のためにFormvar安定化と炭素コーティングされた200メッシュの銅グリッド（テッド·ペラ＃01800）に吸着させた。グリッドは、最大10分までのために細胞懸濁液の一滴にカーボン側を下に置かれ、その後すぐに30秒間水の一滴にグリッドを配置することによって、1時間洗浄した。染色のために、グリッドは30秒〜5分間、2％酢酸ウラニル（テッド·ペラ＃19481）のドロップに置かれ、その後完全に濾紙を用いて乾燥した。グリッドは、TEMは200kVで高角度環状暗視野STEMを用いて80kVまたはFEI Tecnai F20のいずれかで、FEI Tecnaiスピリットを用いて分析した。 </li></ol>

<ol>
	<li>Blakemore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+bacteria.">Magnetotactic bacteria.</a> Science. 190, 377-379 (1975).</li><li>Blakemore, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+bacteria.">Magnetotactic bacteria.</a> Annual Reviews in Microbiology. 36, 217-238 (1982).</li><li>Bazylinski, D. A., Frankel, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Biomineralization+of+Magnetite+(Fe3O4)+and+Greigite+(Fe3S4)+in+a+Magnetotactic+Bacterium.">Controlled Biomineralization of Magnetite (Fe3O4) and Greigite (Fe3S4) in a Magnetotactic Bacterium.</a> Applied and Environmental Microbiology. 61, 3232-3239 (1995).</li><li>Lefevre, C. T., Menguy, 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+Cultured+Greigite-Producing+Magnetotactic+Bacterium+in+a+Novel+Group+of+Sulfate-Reducing+Bacteria.">A Cultured Greigite-Producing Magnetotactic Bacterium in a Novel Group of Sulfate-Reducing Bacteria.</a> Science. 334, 1720-1723 (2011).</li><li>Simmons, S. L., Bazylinski, D. 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=South-seeking+magnetotactic+bacteria+in+the+Northern+Hemisphere.">South-seeking magnetotactic bacteria in the Northern Hemisphere.</a> Science. 311, 371-374 (2006).</li><li>Wolfe, R., Thauer, 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=A+'capillary+racetrack'+method+for+isolation+of+magnetotactic+bacteria.">A 'capillary racetrack' method for isolation of magnetotactic bacteria.</a> FEMS Microbiology Letters. 45, 31-35 (1987).</li><li>Rodgers, F. G., Blakemore, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+structure+in+a+many-celled+magnetotactic+prokaryote.">Intercellular structure in a many-celled magnetotactic prokaryote.</a> Archives of Microbiology. 154, 18-22 (1990).</li><li>Moench, T. T., Konetzka, W., 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+novel+method+for+the+isolation+and+study+of+a+magnetotactic+bacterium.">A novel method for the isolation and study of a magnetotactic bacterium.</a> Archives of Microbiology. 119, 203-212 (1978).</li><li>Balkwill, D., Maratea, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+a+magnetotactic+spirillum.">Ultrastructure of a magnetotactic spirillum.</a> Journal of Bacteriology. 141, 1399-1408 (1980).</li><li>Lins, U., Freitas, F., 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=Simple+homemade+apparatus+for+harvesting+uncultured+magnetotactic+microorganisms.">Simple homemade apparatus for harvesting uncultured magnetotactic microorganisms.</a> Brazilian Journal of Microbiology. 34, 111-116 (2003).</li><li>Jogler, C., Lin, W., 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=Toward+Cloning+of+the+Magnetotactic+Metagenome:+Identification+of+Magnetosome+Island+Gene+Clusters+in+Uncultivated+Magnetotactic+Bacteria+from+Different+Aquatic+Sediments.">Toward Cloning of the Magnetotactic Metagenome: Identification of Magnetosome Island Gene Clusters in Uncultivated Magnetotactic Bacteria from Different Aquatic Sediments.</a> Applied and Environmental Microbiology. 75, 3972-3979 (2009).</li><li>Lin, W., Li, J., 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=Newly+Isolated+but+Uncultivated+Magnetotactic+Bacterium+of+the+Phylum+Nitrospirae+from+Beijing,+China.">Newly Isolated but Uncultivated Magnetotactic Bacterium of the Phylum Nitrospirae from Beijing, China.</a> Applied and Environmental Microbiology. 78, 668-675 (2012).</li><li>Li, J., Pan, 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=Biomineralization,+crystallography+and+magnetic+properties+of+bullet-shaped+magnetite+magnetosomes+in+giant+rod+magnetotactic+bacteria.">Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria.</a> Earth and Planetary Science Letters. 293, 368-376 (2010).</li><li>Oestreicher, Z., Valerde-Tercedor, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetosomes+and+magnetite+crystals+produced+by+magnetotactic+bacteria+as+resolved+by+atomic+force+microscopy+and+transmission+electron+microscopy.">Magnetosomes and magnetite crystals produced by magnetotactic bacteria as resolved by atomic force microscopy and transmission electron microscopy.</a> Micron. 43, 1331-1335 (2012).</li></ol>

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走磁性細菌は、必ずしもすべての水生環境8で発見されていないが、それらが発生した場合、それらは100のオーダーで見つけることができます- 2ミリリットル当たり1000個の細胞。光学顕微鏡を用いて、MTBを観察するためには、あなた自身のサ ​​ンプル8で約50細菌/ mlが必要になります。あなたのサンプル中にないか、または少数のMTBがない場合は、どちらかあなたの...

<table border="1"><tbody><tr><td> アイテム名 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>スライドガラス</td><td>フィッシャー·サイエンティフィック</td><td> S95933 </td><td></td></tr><tr><td>ガラスパスツールピペット</td><td>フィッシャー·サイエンティフィック</td><td> 13から678-6A </td><td></td></tr><tr><td> Oリング</td><td>金物店</td><td></td><td></td></tr><tr><td>カバースリップ</td><td>フィッシャー·サイエンティフィック</td><td> 12-542B </td><td></td></tr><tr><td>棒磁石</td><td>フィッシャー·サイエンティフィック</td><td> S95957 </td><td></td></tr><tr><td>コンテナ</td><td>任意の</td><td></td><td>すべてプラストICまたは少なくとも0.5リットルを保持することができ、密閉できるガラス容器</td></tr><tr><td>コットン</td><td>任意の</td><td></td><td></td></tr><tr><td> 60X乾式レンズ付顕微鏡</td><td>ツァイス</td><td></td><td> 60X乾いたレンズは絶対に必要というわけではないが、これは油を使用せずに高NAを与える</td></tr><tr><td>ダイヤモンドペン</td><td>フィッシャー·サイエンティフィック</td><td> 08から675 </td><td></td></tr><tr><td> 0.22ミリメートルフィルター</td><td>フィッシャー·サイエンティフィック</td><td> 09-719C </td><td></td></tr><tr><td> 1mlシリンジ</td><td>フィッシャー·サイエンティフィック</td><td> NC9788564 </td><td></td></tr><tr><td>微量遠心チューブ</td><td>フィッシャー·サイエンティフィック</td><td> 02-681-320 </td><td></td></tr><tr><td> Formvar /カーボン200メッシュ、銅グリッド</td><td>テッド·ペラ社</td><td> 01800 </td><td></td></tr><tr><td>酢酸ウラニル</td><td>テッド·ペラ社</td><td> 19481 </td><td></td></tr><tr><td> TecnaiスピリットTEM </td><td> FEI </td><td></td><td></td></tr><tr><td> Tecnai F20 S / TEM </td><td> FEI </td><td></td><td></td></tr></tbody></table>

collection, isolation and enrichment of naturally occurring magnetotactic bacteria from the environment

走磁性細菌（MTB）は、最初、特にマサチューセッツ州（米国）の塩性湿地に収集する堆積物試料から1975年1に記載された水生微生物である。それ以来、MTBは世界2世界中から層状水と土砂の列が発見されました。すべてのマウンテンバイクに共通する一つの特徴は、マグネタイト（Fe 3 O 4）および/ ​​またはグレイジャイト鉄（Fe 3 S 4）、またはその両方3、4の細胞内、膜結合型磁気ナノ結晶であるmagnetosomesを含むことである。南半球では、それらは通常、磁石3,5の北の端に惹かれながら、北半球では、MTBは、典型的には、棒磁石の南の端に魅了されています。環境試料からMTBを隔離しようとするときに、このプロパティを利用することができる。 MTBを豊かにするための最も一般的な方法の1つは、天然源から堆積物と水を収集するために透明なプラスチック容器を使用することです淡水池など。北半球では、棒磁石の南の端は、堆積物 - 水界面でちょうど堆積物の上に容器の外側に接して配置されています。しばらくすると、細菌がピペットを用いて磁石の近くに容器内から削除することができ、その後キャピラリー競馬6と磁石を使用してさらに充実。かつて豊かに、細菌はハンギングドロップ法を用いた顕微鏡スライド上に配置し、光学顕微鏡で観察または銅グリッド上に堆積され、透過型電子顕微鏡（TEM）を用いて観察することができます。 この方法を使用して、孤立したMTBは、行動、鞭毛の種類と数、細胞の細胞形態、磁性結晶の形状、magnetosomes数、各セル内magnetosome鎖の数の組成を水泳などの特性を決定するために顕微鏡的に研究することができるnanomineral結晶、細胞内の液胞が存在する。

磁石は、環境試料（ 図1A）に含まれる磁性細菌（MTB）を単離するために使用することができる効果的なツールです。キャピラリー競馬場（ 図1B）は 、彼らはまた、環境試料中に含まれ、非走磁性微生物から分離することができる綿栓を介してそれらを誘致するためにMTBの磁気特性を使用しています。 <img alt="図1" src="/...

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Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

我々は、天然水に適用できる磁性細菌（MTB）を収集する方法を示しています。 MTBを単離して、細菌の自然な磁気を利用しています比較的簡単なセットアップを使用して堆積物試料から濃縮することができます。孤立したMTBは、次に光学および電子顕微鏡の両方を用いて詳細に調べることができます。

当然のことながら環境から磁性細菌の発生の収集、分離、濃縮

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

collection-isolation-enrichment-naturally-occurring-magnetotactic

Research

JoVE Journal

Biology

23.0K ビュー.  The Ohio State University. 我々は、天然水に適用できる磁性細菌（MTB）を収集する方法を示しています。 MTBを単離して、細菌の自然な磁気を利用しています比較的簡単なセットアップを使用して堆積物試料から濃縮することができます。孤立したMTBは、次に光学および電子顕微鏡の両方を用いて詳細に調べることができます。

ビデオ: Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

StemCell Technologies社からRosetteSepキットと人間の血液からNK細胞の濃縮

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

生物学

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

アメリカカブトガニ、カブトガニポリフェムスから採血

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

ヒト血清から小非コードRNAの分離

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. Birds are widely used subjects in biological research and are ideal for studies involving repeated sperm samples. However, few resources are currently available for those wishing to learn how to collect and extract DNA from avian sperm. Here we describe cloacal massage, a gentle, non-invasive manual technique for collecting avian sperm. Although this technique is established in the literature, it can be difficult to learn from the available descriptions. We also provide information for extracting DNA from avian semen using a commercial extraction kit with modifications. Cloacal massage can be easily used on any small- to medium-sized male bird in reproductive condition. Following collection, the semen can be used immediately for motility assays, or frozen for DNA extraction following the protocol described herein. This extraction protocol was refined for avian sperm and has been successfully used on samples collected from several passerine species (Passer domesticus, Spizella passerina, Haemorhous mexicanus, and Turdus migratorius) and one columbid (Columba livia).

This protocol describes cloacal massage, a non-invasive manual technique for collecting semen from birds, and DNA extraction from avian sperm using a commercial extraction kit with modifications.

排せつマッサージと精子から DNA の隔離による鳥の精液採取

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ability to biomineralize magnetosomes, which are magnetic nanometer-sized crystals of magnetite (Fe3O4) or greigite (Fe3S4) surrounded by a lipid bilayer membrane, within their cytoplasm. For most known magnetotactic bacteria, magnetosomes are assembled in chains inside the cytoplasm, thereby conferring a permanent magnetic dipole moment to the cells and causing them to align passively with external magnetic fields. Because of these specific features, magnetotactic bacteria have a great potential for commercial and medical applications. However, most species are microaerophilic and have specific O2 concentration requirements, making them more difficult to grow routinely than many other bacteria such as Escherichia coli. Here we present detailed protocols for growing three of the most widely studied strains of magnetotactic bacteria, all belonging to the genus Magnetospirillum. These methods allow for precise control of the O2 concentration made available to the bacteria, in order to ensure that they grow normally and synthesize magnetosomes. Growing magnetotactic bacteria for further studies using these procedures does not require the experimentalist to be an expert in microbiology. The general methods presented in this article may also be used to isolate and culture other magnetotactic bacteria, although it is likely that growth media chemical composition will need to be modified.

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

We present a procedure for growing several strains of Magnetospirillum in two different types of growth media. Magnetospirillum gryphiswaldense strain MSR-1 is grown in both liquid and O2 concentration gradient semi-solid media while M. magneticum strain AMB-1 and M. magnetotacticum strain MS-1 are grown in liquid medium.

属磁性の走磁性細菌の成長: MSR 1、AMB 1 MS 1 株

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

成人ネズミ涙腺および顎下腺からの筋上皮細胞の単離

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

オルガノイド培養のためのヒト肺上皮前駆細胞の単離と濃縮

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

都市環境のスワッピング - 環境貯留層からのSARS-CoV-2のサンプリングと検出のためのパイプライン

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

真核細胞からのトランスレーショナル複合体の迅速な インビボ 固定と分離

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

ヒト変形性膝関節からの組織採取とRNA抽出

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...