このプロジェクトの資金は、国立科学財団助成金OCE1029607（XMまで）とMCB0702125（MAMへ）とゴードンとベティムーア財団（MAMへ）によって提供されていました。

1。水のサンプルの処理<ol><li>大きな粒子とbacteriovoresを削除するには、1μmの孔径のメンブレンフィルターを通してフィルター10L環境水。カーボイの水ろ液を集める。 </li><li> 4ミリリットル、新たに調製したパラホルムアルデヒド溶液（、10％PFA）を含む3滅菌エッペンドルフチュー​​ブ（50ml）に移転36ミリリットルろ液それぞれ。細胞を維持するために、室温で2時間インキュベートする。真空濾過によって0.22μmの孔サイズの白いメンブレンフィルター上に細胞を集めます。真空濾過によってフィルターを介して生理食塩水（PBS）をリン酸緩衝10 mlを渡すことでフィルタを洗浄してください。ネガティブコントロールとしてフィルタにラベルを付けて、-20℃で保管します</li></ol> ステップ1.3から1.4には、DOC -限られた条件を確立するためのオプションです。 <ol start="3"><li>カーボイに無機態窒素とリン（5μMのNH 4 Clを、5μMのNaNO 3を 、及び1μMのNaH 2 PO 4、最終濃度）の混合物を追加。 </li><li>時折扇動で48時間その場の温度で、暗所でインキュベートする。 </li></ol> 2。縮図の設立とインキュベーション<ol><li>小宇宙を確立するために、ステップ1.4のカーボイから800 mlの水のサンプルで6個の1リットルのガラスフラスコの各々を埋める。各縮図にBrdUを（10μM、最終濃度）を追加します。よく混ぜる。 </li><li>小宇宙の三つに1 mlのモデルDOCの化合物溶液を追加し、これらは、DOCの改正として機能します。残りの3つの小宇宙に1 mlの滅菌PBSを追加、これらは無添加のコントロールとして機能します。 </li><li>インキュベーターシェーカーにすべての小宇宙をインキュベートし、100rpmで振とうしながらその場の温度で、暗所でインキュベートする。 </li><li> 0の時点で50 mlの滅菌エッペンドルフチュー​​ブ、8、16、および24時間にそれぞれの縮図と転送からの水のサンプルの36 mlを収集する。すぐにコレクションチューブに新鮮なPFAを4 ml（10％）を追加し、細胞を維持するために、室温で2時間インキュベートする。 </li><li>フィルタは、0.22μmのポアサイズのポリカーボネートメンブランフィルターを介して細胞を維持。 10ミリリットルのPBSでフィルターを洗う。次のステップに進んで、または-20℃でフィルターを保存する</li></ol> 3 BrdUの取り込みのためのその場免疫検出で <ol><li>室温でフィルタ（DOC修正サンプル、無添加のコントロールとネガティブコントロール）を解凍。 </li><li>リゾチーム溶液1mlを適用する[10 100 mMトリス、50 nMのEDTAのmg / mlのリゾチーム卵白液（pH = 8）]フィルタで細菌細胞をカバーする4。室温で30分間インキュベートする。吸引下でそれまでの10 mlのPBSを渡すことでフィルタを洗浄してください。 </li><li> 4フィルタで細菌細胞をカバーするために[100 mMトリス、50 nMのEDTA（pHは= 8）で2 mg / mlのプロテイナーゼK]プロテイナーゼK溶液1mlを追加。室温で30分間インキュベートする。吸引下でそれまでの10 mlのPBSを渡すことでフィルタを洗浄してください。 </li><li>フィルター上の細菌細胞をカバーするために1ミリリットルエキソヌクレアーゼソリューション[5 mMのMgCl 2のエキソヌクレアーゼIII（50 U / ml）及び50mMのTris - HCl] 5を追加。 37℃で30分間。吸引下でそれまでの10 mlのPBSを渡すことでフィルタを洗浄してください。 </li><li>メーカーの指示に従って、フレームシールのインキュベーション室（Bio - Rad）を組み立てる。 </li><li>アルコール洗浄乾燥した表面に滅菌ブレードを使用してeighthsにフィルタをスライス。 </li><li>鉗子を使用して、1組み立てたフレームシールインキュベーションチャンバー（8台のフレームシール室が、各フィルタのサンプルのために必要とされる）に、フィルタの第八節を置きます。細胞を含むフィルタセクションの側では上向きに直面しているはず。フィルターの奥の水分は、スライドにフィルタスティックをできるようになります。フィルタが乾燥した場合、スライドにフィルタセクションを配置する前に、チャンバーの中央にDIH 2 Oの小さなドロップ（2μl）を適用する。 </li></ol> ステップ3.8から3.20には、メーカーの指示から変更手順に従って、In Situ細胞増殖キットのロシュから試薬をFLUOS使用。 PBSを除き、すべての試薬 ​​はキット内で提供されています。 <ol start="8"><li>均等にシールが適用されると、オーバーフローを引き起こすことなく、インキュベーション室内の全体のフィルタセクションをカバーするのに十分なインキュベーション緩衝液（0.5％ウシ血清アルブミン、PBS中の0.1％Tween20を、）を適用します。 </li><li>インキュベーションチャンバーのフレーム上にポリエステルのフレームカバーを置きます。しっかりとインキュベーションチャンバーを密封するために下に押します。フィルター上に気泡を避けてください。暗所で室温にて10分間密閉チャンバーをインキュベートする。 </li><li>ポリエステルシールを取り外して、インキュベーション室を開きます。 （注：時々チャンバーを開くには、スライドからのフレームシールをプルアップされますその場合は、次の手順のための新しいチャンバーを準備する。。） </li><li>室の一角からインキュベーションバッファー出力ピペット。フィルターとの接触を避ける。 </li><li>優しくに100μlのPBSをピペッティングすることにより、フィルタを洗うとチャンバーからの3倍。 </li><li>使用直前にメーカーが推奨する手順に従って、抗BrdU - FLUOSワーキング溶液を調製。 </li><li>ピペットで120μlの抗BrdU - FLUOSは、フィルター上に溶液を取り組んで均一に表面全体を覆​​うように注意してください。 </li><li>新しいポリエステルカバーとチャンバーを再シール。フィルターの上に気泡を避けてください。 37℃暗所でチャンバーをインキュベート℃で3時間。このステップでは、フルオレセインイソチオシアネート（FITC） を用いたin situ でのBrdU法人のDNAにラベルを付けます。 </li><li>ポリエステルのカバーを取り外し、インキュベーションチャンバーを開きます。抗BrdU - FLUOSワーキングソリューションからピペット。 PBSでフィルタ3回洗浄する。 </li><li>インキュベーションチャンバーから滅菌表面にフィルターを転送する。滅菌ブレードを使用して小片にフィルタセクションをスライス。 </li><li> 2 mlのマイクロチューブにフィルター部分、それぞれを含む1mlのPBSを転送します。しっかりとパラフィルムとチューブとシールをキャップ。 37℃、10分間200rpmで。 </li><li> 5分間の最大速度でボルテックスし、渦の上にチューブを固定します。滅菌15ミリリットルのエッペンドルフチュー​​ブにピペットで上清を。さらに5回のためのインキュベーションとボルテックスの手順を繰り返します。各サンプルに対して同じ15エッペンドルフチュー​​ブに上清を組み合わせる。通常は、80％の細胞が懸濁液中に回収することができる。 </li><li> 4で再懸濁した細胞と上清℃にて保存してください。 2日以内にソート。 </li></ol> 4。 FACS解析 BD FACSAriaフローサイトメーターと対応するソフトウェアのための手順を説明します。 <ol><li>メーカーが推奨する手順に従って、フローサイトメーターの設定を最適化する。これが伴います：必要なブレークオフパラメータとスイートスポットを設定するには、増幅コントロールを調整し、実験のシース圧のためにレーザーの遅延時間と面積のスケーリング係数を最適化し、FSCとSSCの電圧、FSC閾値、FSC蛍光スケーリング、蛍光PMT電圧の設定を最適化、など</li><li> FITCと側方散乱（SSC）の蛍光強度に基づいて、フローサイトメーター（FCM）のネガティブコントロールのサンプルを実行します。陰性対照には細胞はFITC - SSCの取得の表示により視覚化することができるようになるまで、FITCしきい値を増やします。 </li><li>無添加のコントロールサンプルを実行し、FITCとSSCの買収に基づいて、10,000個の細胞の分布パターンを調べる。すべてのセルを囲むと、&quot;低強度細胞&quot;（LIS）としてそれを指定するためにゲートを定義します。 </li><li> DOC -修正サンプルを実行し、FITCとSSCに基づいて、10,000個の細胞の分布パターンを調べる。いくつかの細胞は、プリセットLIのゲートに表示されます。 LIはより高い蛍光強度を（HIS）を持つ細胞を囲むように別のゲートを定義します。ゲートセルの相対量を表示するには、統計情報を取得する。 </li><li> &quot;1滴を浄化する&quot;モードで500μlのPBSを含むコレクションチューブにソートHI細胞。 HISの数が500,000カウントに達したときにソートを終了する。 </li></ol> 5。 16S rRNA遺伝子のフィルタPCR増幅 フィルタPCR手順はKirchmanらから変更されています。6 <ol><li>白、0.22μmの孔サイズ、25ミリ直径、ポリカーボネートメンブレンフィルター上にフィルターソートされた細胞。滅菌ブレードを使用していない細菌の細胞が含まれていないフィルターの端を切り落とす。 8等しい大きさにフィルターをスライス。 </li><li>細胞がチューブの内側を向くように、PCR反応チューブに単一のフィルタピースを置きます。 PCRの反応チューブに45μlのPCRグレードの水を加えます。水に完全にフィルター水没。 </li><li> 2 ROCHE illustra PuReTaq読み取り- to - Goの、PCR反応チューブにPCRビーズを簡単にボルテックスを追加。 PCRの反応チューブのそれぞれに、2μlのよう27Fおよび1492R 7のような順方向と逆の16S rRNA遺伝子のプライマー（0.4μMの各プライマーの最終濃度）、のそれぞれを追加します。 </li><li> （オプション）吸着増幅阻害剤を助けるためにPCR反応液に1μlのウシ血清アルブミン溶液を（BSA、終濃度が30μg/100μL）を追加します。一BSAを追加しないことを選択した場合、水の代わりに、1μlを加える。 </li><li>サーマルサイクラーでのPCR増幅を行います。 PCRプログラムダウンタッチが推奨されて、これは順番に62から52に減少アニーリング温度を有し° Cを1 ° C 52のアニーリング温度で15サイクルが続く11サイクル、℃でのサイクルごとにすべてのサイクルは、（95℃）変性（62℃〜52℃）アニーリング、および拡張（72℃）50の持続時間のステップが含まれています。最初の3分の変性と最後の10分延長のステップはまた、PCRのプログラムに含まれています。 </li><li>エチジウムブロマイド染色した1％アガロースゲル電気泳動でPCR増幅を確認してください。 QIAGEN QIAquickゲル抽出キットでゲルとクリーンから消費税のPCRアンプリコンを。 </li><li>各サンプルに2つの追加のPCR増幅を行う、するたびに新しいフィルタセクションを使用してください。 PCRゲルpurificati後で、一緒に同じサンプルのアンプリコンをプール。精製された16S rDNAのアンプリコンは、現在そのようなクローンのライブラリー構築と塩基配列決定などの分類学的同定を可能にする分子解析、多数のための準備が整いました。 </li></ol> 6。代表的な結果： 代表的な結果は例としてプトレシン分解菌の研究を使用して記述されます。水試料は、グルジアの沿岸のサイトから収集し、上記の手順に従って処理した。 FACS分析は、プトレシン加えて、高BrdUの取り込み率（図1）を示す、高FITC蛍光強度を持つ細菌のグループの発展を誘導することを明らかにした。これらの細胞は、高のBrdU取り込み細胞（HIS）として指定され、主にプトレシン分解菌を含むことが期待された。彼は唯一のBrdUの取り込み（LIS）の低レベルを持つ細胞が含まれている無添加のコントロールに欠落していた。 LISは、主に追加さプトレシンを使用することができませんでしたbacterioplanktonを含むことが期待された。彼は、分離されたチューブに分類し、メンブレンフィルター上に採取された。 16S rRNA遺伝子のアンプリコンは、フィルタPCRを用いて、高HI細胞が得られた。 <img src="/files/ftp_upload/2855/2855fig1.jpg" alt="図1"/> 図1無添加制御とインキュベーションの24時間後に採取した（ここで例としてプトレシン）モデル化合物-改正されたサンプルのフローサイトメトリー分析。細胞の分布の分析は、積極的にBrdUの取り込みのレベルに関連してFITC標識（x軸）、の（1）蛍光強度に基づいていた、と（2）側方散乱（SSC、y軸）は、積極的に細胞に関連しているサイズ。ゲートの表記は、BrdUの取り込み、（; LI、低のBrdU取り込みHI、高のBrdU取り込み）のレベルに基づいています。 HIとLI細胞の相対的な割合は、対応するゲートに示されています。

<ol>
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利害の衝突は宣言されません。

水生環境での個々のDOC成分を代謝するbacterioplanktonの種レベルの識別を可能にするために我々のアプローチのカップルBrdUの取り込み、FA​​CSおよび16S rDNAの解析。のみ活性細菌の解析を可能にするため、休眠細胞が含まれていない代謝活動に基づいて、BrdU取り込みアッセイラベル細菌細胞、。我々のアプローチでは、BrdUの取り込みは、その後に可視化するBrdUの取り込みのレベルが異なる免?...

<table border="1"><tbody><tr><td> の名前 </td><td> タイプ </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> BrdUを</td><td>試薬</td><td>シグマ</td><td> B5002 - 5G </td><td></td></tr><tr><td>リゾチーム</td><td>試薬</td><td>シグマ</td><td> L6876 - 5G </td><td></td></tr><tr><td>プロテイナーゼK </td><td>試薬</td><td>シグマ</td><td> P2308 - 25mgを</td><td></td></tr><tr><td> In Situ細胞増殖キット、FLUOSで</td><td>キット</td><td>ロッシュ</td><td> 11810740001 </td><td>抗BrdU - FLUOSと製造元が推奨する速度よりも反応あたりのインキュベーションバッファーをより多く消費する。 </td></tr><tr><td>フレームシールのインキュベーションチャンバー</td><td>材料</td><td> Bio - Rad社</td><td> SLF - 1201 </td><td></td></tr><tr><td>ポリカーボネート膜フィルター（142 - MM -直径、1.0μmの孔径） </td><td>材料</td><td>ミリポア</td><td> FALP14250 </td><td></td></tr><tr><td>ポリカーボネート製メンブレンフィルター（25mmの直径、0.2μmの孔径） </td><td>材料</td><td>ミリポア</td><td> FGLP02500 </td><td></td></tr><tr><td> illustra PuReTaqのReady - To - GoのPCRビーズ</td><td>キット</td><td> GEヘルスケア</td><td> 27-9559-01 </td><td></td></tr><tr><td> QIAquickゲル抽出キット</td><td>キット</td><td> QIAGEN </td><td> 28704 </td><td></td></tr><tr><td> FailSafeはPCRシステム</td><td>キット</td><td> EPICENTRE </td><td> FS99060 </td><td></td></tr></tbody></table>

bromodeoxyuridine (brdu) labeling and subsequent fluorescence activated cell sorting for culture-independent identification of dissolved organic carbon-degrading bacterioplankton

微生物は、水生環境における多数の溶存有機炭素（DOC）基質の分解を仲介する主要なエージェントです。しかし、自然の中でDOCの特定のプールに変換細菌の分類群の同定には、技術的な課題となっています。 ここでは、アプローチを説明しているカップルのブロモデオキシウリジン（BrdU）の取り込み、蛍光活性化細胞選別（FACS）、および16S水生環境において分解する特定のDOCの化合物の能力bacterioplanktonのカルチャに依存しない識別を可能にするrRNA遺伝子に基づく分子解析。三連のbacterioplanktonの小宇宙を、BrdUとモデルのDOCの化合物（DOC修正）、または唯一のBrdU（無添加制御）の両方を受け取るよう設定されております。 BrdUの代替は、新たに合成された細菌DNAおよびBrdU標識DNAのチミジンの位置は容易に免疫検出1,2できます。 24時間のインキュベーションを通じて、付加価値DOCの化合物を使用することができるbacterioplanktonが選択的に活性化し、それゆえにDOCの改正と細胞に非応答性細胞よりBrdU取り込み（HI細胞）のレベルが高いことが期待されているは、さらにコントロール（低BrdU取り込み細胞、LI細胞）。蛍光免疫検出した後、HIの細胞が区別され、物理的に蛍光活性化細胞選別（FACS）3でLI細胞から分離。ソートされたDOC -応答性細胞は（HI細胞）のDNAを抽出し、分類学的PCR、クローンのライブラリー構築と塩基配列決定を含め、その後の16S rRNA遺伝子ベースの解析によって識別されます。

Watch this Scientific Journal Video about Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degradin

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

環境bacterioplanktonは、有機炭素（DOC）化合物とDNA標識試薬、ブロモデオキシウリジン（BrdU）を溶解モデルとインキュベートされています。その後、DOC分解細胞は、蛍光活性化細胞選別（FACS）を使用して自身の昇格BrdUの取り込みに基づいてバルクコミュニティから分離されています。これらの細胞は、その後の分子生物学的解析によって識別されます。

ブロモデオキシウリジン（BrdU）標識と溶存有機の文化に依存しない識別に対する後続の蛍光活性化細胞選別カーボン分解Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

bromodeoxyuridine-brdu-labeling-subsequent-fluorescence-activated

Research

JoVE Journal

Biology

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

20.8K ビュー.  Kent State University. 環境bacterioplanktonは、有機炭素（DOC）化合物とDNA標識試薬、ブロモデオキシウリジン（BrdU）を溶解モデルとインキュベートされています。その後、DOC分解細胞は、蛍光活性化細胞選別（FACS）を使用して自身の昇格BrdUの取り込みに基づいてバルクコミュニティから分離されています。これらの細胞は、その後の分子生物学的解析によって識別されます。

ビデオ: Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

ヒトCD40活性化B細胞の生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

生物学

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

植物プロトプラストの蛍光活性化細胞選別

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

磁気活性化細胞選別（LIMACS）を使用して、脂質小胞を介したアフィニティクロマトグラフィー：タンパク質脂質相互作用を解析する新手法

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

の細胞特異的な分析<em&gt;シロイヌナズナ</em&gt;蛍光活性化細胞選別を使用した葉

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

細胞トラッキング染料を使用して細胞分裂監視のための最適化された染色と増殖モデリング方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

蛍光活性化細胞選別（FACS）によって新鮮な組織からの正常および癌関連線維芽細胞の分離

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies.

超微細構造解析のための酵母エンドソーム系のナノゴールドラベル付け

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

メチルトランスフェラーゼと補因子類似体核酸およびタンパク質の配列特異的標識化

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

磁気活性化細胞を分離し、滑膜液由来間葉系幹細胞が家兎モデルからの浄化戦略を並べ替え

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

巨大ウイルス混合物分離のための蛍光活性化細胞選別の代替戦略としての単一細胞マイクロ吸引

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...