この作品は、国立科学財団（助成金なし。DBI 0519984）と国立衛生研究所（助成金なし。5R01GM078279）によってサポートされていました..

1）植物材料の調製<ol><li>プロトプラストを酵素消化、細胞壁の適切な組み合わせが1を使用していることを提供する多くの異なった植物種や組織から得ることができます。本格的な実験が行われる前に、材料の小規模な消化は、組織のprotoplasting効率、酵素、などを評価し、細胞選別のためにパーセント陽性細胞を推定するためにお勧めです。ここでは、細胞のタイプは、特に緑色蛍光タンパク質（GFP）を発現していることをシロイヌナズナ実生の根から得られたプロトプラストを用いています。内皮と静止中心がP SCRでマークされています：：GFPとP WOX5で静止センター：：GFP 2,3（図1）。 </li><li>根が（0​​.22％w / vのムラシゲ及びスクーグ基礎培地[シグマ]、1％成長培地中にを通じて成長できるよう、ナイロンフィルター（NITEX250μmのメッシュ）で、苗が（図2aシグマ）phytatraysで水耕栽培されていますw / vのスクロース、0.05％（w / v）のMES [2 - （N -モルホリノ）エタンスルホン酸]、KOHでpH 5.7）。また、植物が垂直に配置さ1％寒天プレート（図2b）のナイロンフィルター（100μmのメッシュ）の上に成長させることができる。 </li><li>必要に応じて根の収穫のない唯一のエイズ上記フィルターの使用は、それはまた、苗木の容易な追加治療を容易にします。フィルタは、苗が関心の触媒を添加した新しいphytatraysまたは寒天プレートに一括して転送することができます。例えば、設定phytatrayは、細胞型特異的シロイヌナズナ実生4の窒素の治療への転写反応の解析に使用されています。 </li><li>あなたの蛍光マーカーが正しく（自分自身が治療によって影響される可能性が細胞型特異的マーカーとして、治療のオプションを使用する場合は特に）発現していることを顕微​​鏡で確認してください。このケースでは、苗は蛍光顕微鏡（;図1ニコン）の下に検査されます。細胞タイプ特異的蛍光マーカーのラインが正確にどの種類の細胞がマークされているかを判断し、発現の変動を決定するために共焦点顕微鏡下で最初に特徴付けされるべきであることに注意してください。 </li></ol> protoplasting溶液2）の調製<ol><li> 1.25パーセントw / vのセルラーゼ（ヤクルト）、0.3％w / vのMacerozyme（ヤクルト）、0.4 M、D -マンニトール、20mMのMESと脱イオン水に20mMのKClを（1Mストックから）に溶かし、5.7にpHを調整する1 Mトリス/ HCl pH7.5で。このソリューションは、僅かに濁ったようになります。 </li><li> 55に解決策を加熱℃で10分間（溶液は透明になります）と0.1％を追加する前に、それが室温に冷却させますw / vのBSA（ウシ血清アルブミン）、10mMのCaCl 2を 、および5mMβ-メルカプトエタノール。 </li></ol> 3）植物材料の収穫とprotoplasting <ol><li>ルーツは、メスとナイロンメッシュを離れてそれらをこすることによって収穫し、protoplasting溶液を含むフラスコに堆積される。一般的に、protoplasting溶液10mlを1500苗の根ごとに使用されます。 </li><li> 1時間室温で静かにフラスコ（75 rpm）を振る。より長いインキュベーション時間は、プロトプラストの収量を増加させることができるだけでなく、遺伝子発現に自分自身をprotoplastingの効果に追加されます。 </li><li> 40μmのセルストレーナー（BDファルコン）とのプロトプラスト溶液を濾過し、円錐形の15 mlチューブ（BDファルコン）を介してソリューションを分割する。 </li><li>この遠心分離の速度が使用されてプロトプラストの種類、それらの脆弱性および酵素処理中に生成細胞残渣の量に依存すること500 G.の注で10分間、スイングバケット遠心機でチューブをスピン。 </li><li>上清のほとんどを除去、残りの溶液中でプロトプラストを懸濁しますと（図3）顕微鏡でそれらを検査。 </li><li>プロトプラストの数と密度を推定するために血球計を利用してください。細胞密度は、試料注入速度、FACSで細胞を選別するために必要な秒あたりのイベント、したがって、合計時間を（また、4.3節を参照）が決定されます。 </li><li> 3.7）FACSに直接進むか、なしなどW5などのインキュベーション溶液、（154 mMのNaCl、125 mMの塩化カルシウム、5mMのKClを、5mMのMES、KOHで5.7にpHを調整）のプロトプラストまたはprotoplastingソリューションを洗浄し、再懸濁のどちらか酵素を追加しました。転写の変化を見ていたとき、それはそのようなバッファを変更するなど、遺伝子発現に影響を​​与える可能性の条件へのサンプルの曝露を最小限にするために重要であるプロトプラストは、それがゆえprotoplasting溶液中でプロトプラストを維持し、続行するために推奨されてインチに保たれているできるだけ早くFACSへ。 </li></ol>プロトプラストの4）蛍光活性化細胞選別<ol><li>電源をオンにして、セルソーターを準備する。ここで、FACSAria（BD）が使用されます。 </li><li> 100＆mでの流れのストリームを設定するU、m個のノズルと20 psiのシース圧。 </li><li>細胞密度および試料注入速度は、最高の歩留まりや、もっとも早く達成可能な速度が必要かどうかに基づいて、特定の実験に調整することができます。我々は、10,000,000個/ mlまでの濃度で正常にソートされている。 </li><li>プロトプラストの沈降を防ぐために、FACSのサンプル攪拌オプションを使用します。 1：FACSの目詰まりが問題になる場合は、3つの可能なトラブルシューティング手順があります。サンプルラインのフラッシュを実行します。 2。密度を減らすために、プロトプラスト懸濁液を希釈する。 3。遠心分離と再懸濁後ろ過工程（3.3）を繰り返すことにより、プロトプラスト溶液をクリーンアップします。 </li><li> 488 nmのレーザーで励起後の赤いスペクトル自己蛍光（RSA）のためのGFPと20分の610 nmの30分の530 nmでの前方散乱光（FSC）、側方散乱（SSC）とエミッションを測定する装置を準備します。これらは本質的にGFP陽性プロトプラストを単離するために使用される唯一のパラメータです。ここで、電圧の設定は以下の通りであった：FSC - 60V、SSC 250V、GFP 350VとRSA 335V。最適な電圧設定がすべてのFACSに異なるとなるとさらにセルソーターの存続期間全体を通じて調整が必要になることに注意してください。 </li><li>前方散乱対側方散乱のためのドットプロットを設定することで起動します。測定イベントはプロットの中心にされるように電圧設定を適用します。 </li><li>次に、緑と赤の蛍光シグナルのドットプロットを作成します。野生型（非GFP）をプロトプラスト懸濁液を見たときに測定されたイベントがプロットの中心角の人口が得られるように電圧設定を適用します。 GFPマーカーのラインから派生したプロトプラスト懸濁液は、野生型のサンプルでは見たことのない緑色蛍光イベントの明確な母集団を生成します。 </li><li> GFPとRSAとの間のスペクトルの重なりを調整するために補償の制約を設定します。適切な補償の制約の設定は、非GFPのプロトプラストと破片からGFP陽性プロトプラストの分離も可能になります。 RSA、マイナス17.91パーセントのGFP：ここで使用される制約は次のとおりです。 </li><li> GFP陽性のイベントを識別するためにゲートを設定し、非GFPのプロトプラストの負の制御は、ゲートの境界（図4）の定義を支援するために使用する必要があります。 </li><li>分析のうち、小さな破片を残すために前方散乱の遮断を実装します。 GFP陽性のカットの配置を決定するためのFSC対SSCプロット内のイベントを可視化する。ここでは、カットオフは5,000に設定した。 FACSは、残骸の高レベルのソートイベントとサンプルとして残骸をカウントすることに注意しては予想と異なるパーセントのGFPポジティブイベントがあるかもしれません。これは問題であるとは限りません。しかし、サンプルのより多くの破片は、長いソートがかかります。 </li><li>実験と分析される細胞の種類の豊富さに応じて、ソートされた細胞の最適な歩留まりや最適な純度のためにどちらかのFACSの精度モードを設定します。 </li><li> RNAの抽出には、RNAの抽出緩衝液の適切な量のコレクションチューブ（1.5ミリリットルマイクロチューブ）を用意。このセットアップでは、20,000ソートイベントは、抽出緩衝液350μlの（したRNeasyマイクロキット、QIAGEN）にソートされた約100μlの総容量が得られます。ソートの完了後に細胞懸濁液は、上部にプールできるようにサンプルを混ぜる。 </li><li>サンプルを保管または抽出をRNAに直接進みます。成功したマイクロアレイ解析は、わずか500ソートのイベントから抽出したRNAを用いて予備成形されています。ここで、我々はしたRNeasyマイクロ抽出キット（QIAGEN）、WT - OvationエキスパートのピコRNA増幅システムとFL -オベーションcDNAのビオチンモジュールV2（NuGEN）を使用。 </li></ol>代表的な結果 ：約1,500一週齢のP SCRの一つphytatray：GFPの苗木は60,000プロトプラスト（のような血球計で測定される）について得られた。 65000 FACS -処理されたイベントの2.6％がGFP陽性であると定義され、（図4b）ソートされた。 約1,500四日齢P WOX5の八プレート：：GFPの苗は、それぞれ（12,000合計）は3000万プロトプラスト（のような血球計で測定される）について得られた。 1600万FACS -処理されたイベントの0.063パーセントは、GFP陽性であると定義され、（図4c）ソートされた。 万ソートのイベントは通常、RNA抽出に使用され、20〜140 ngのトータルRNA（図5）にもたらすことができる。 <img src="/files/ftp_upload/1673/1673fig1.jpg" alt="図1" /> 図1。 シロイヌナズナの根における細胞型特異的GFPマーカーライン。蛍光顕微鏡画像は、微分干渉コントラスト（DIC）とMetamorphソフトウェア（Molecular Devices社）上で実行しているEclipse 90i顕微鏡（ニコン）でGFPフィルターで撮影された。 DICとGFPの画像は、可視化の目的のために重ねていた。この視覚的な実験で使用される2つのマーカーの行が表示されます。A）P SCR：：GFPとb）P WOX5：：GFP。 <img src="/files/ftp_upload/1673/1673fig2.jpg" alt="図2" /> 図2。植物の成長条件。苗木を水耕phytatrays（）または垂直に配置寒天プレート（B）上の環境のコントローラで増殖させた。 <img src="/files/ftp_upload/1673/1673fig3.jpg" alt="図3" /> 図3。 GFPの蛍光顕微鏡画像を発現する植物のプロトプラストは、微分干渉コントラスト（DIC）とMetamorphソフトウェア（Molecular Devices社）上で実行しているEclipse 90i顕微鏡（ニコン）でGFPのフィルターで撮影された。 DICとGFPの画像は、可視化の目的のために重ねていた。矢印は、バーストセル、細胞残渣およびGFP陽性プロトプラストを示している。つの白い線の間の距離は50μmである。 <img src="/files/ftp_upload/1673/1673fig4.jpg" alt="図4" /> 図4。 GFP陽性のプロトプラストの蛍光活性化細胞選別野生型（）P SCRから派生したプロトプラストは：。：GFP（B）またはP WOX5：：GFP（C）マーカーの線がFACSAria（BD）で分析し、ソートされた定義されたゲートを使用して対赤（20分の610 nmの、y軸）蛍光、緑（x軸30分の530 nm）のドットプロット上に。 100,000イベントは、各プロットに示されている。 GFPのソーティングゲート内のイベントは緑色で強調表示されています。 <img src="/files/ftp_upload/1673/1673fig5.jpg" alt="図5" /> 図5。 10.000ソートされた細胞からの代表的なRNA抽出。細胞がRNA抽出バッファー（QIAGEN）に直接ソートされたが、RNAを精製し、2100バイオアナライザ（アジレント）の濃度、純度および整合性がチェックされました。三つは、両方のマーカーの線が表示されているために複製します。

<ol>
	<li>Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+in+maize+and+Arabidopsis+mesophyll+protoplasts.">Signal transduction in maize and Arabidopsis mesophyll protoplasts.</a> Plant Physiol. 127, 1466-1475 (2001).</li><li>Wysocka-Diller, J. W., Helariutta, Y., Fukaki, H., Malamy, J. E., Benfey, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+SCARECROW+function+reveals+a+radial+patterning+mechanism+common+to+root+and+shoot.">Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot.</a> Development. 127, 595-603 (2000).</li><li>Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I., Friml, J., Heidstra, R., Aida, M., Palme, K., Scheres, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PIN+auxin+efflux+facilitator+network+controls+growth+and+patterning+in+Arabidopsis+roots.">The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots.</a> Nature. 433, 39-44 (2005).</li><li>Gifford, M. L., Dean, A., Gutierrez, R. A., Coruzzi, G. M., Birnbaum, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+nitrogen+responses+mediate+developmental+plasticity.">Cell-specific nitrogen responses mediate developmental plasticity.</a> Proc Natl Acad Sci U S A. 105, 803-808 (2008).</li><li>Bargmann, B. O. R., Birnbaum, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+fluorescent+selection+permits+precise,+rapid,+and+in-depth+overexpression+analysis+in+plant+protoplasts.">Positive fluorescent selection permits precise, rapid, and in-depth overexpression analysis in plant protoplasts.</a> Plant Physiol. 149, 1231-1239 (2009).</li><li>Petersson, S. V., Johansson, A. I., Kowalczyk, M., Makoveychuk, A., Wang, J. Y., Moritz, T., Grebe, M., Benfey, P. N., Sandberg, G., Ljung, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Auxin+Gradient+and+Maximum+in+the+Arabidopsis+Root+Apex+Shown+by+High-Resolution+Cell-Specific+Analysis+of+IAA+Distribution+and+Synthesis.">An Auxin Gradient and Maximum in the Arabidopsis Root Apex Shown by High-Resolution Cell-Specific Analysis of IAA Distribution and Synthesis.</a> Plant Cell. 21, 1659-1668 (2009).</li></ol>

プロトプラストは、原則として、植物組織の様々な由来することが、有利な条件の最適化は大幅にRNAの質と量を強化することができます。 protoplastingソリューションと使用される選択科目インキュベーションバッファーの両方が、この側面に影響を与えます。 多くの異なる蛍光タンパク質は、YFP、CFPまたはそれらの多くの変種および誘導体、 例えば、GFP、RFP、使?...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>250 &mu;m nylon mesh</td>
<td>&nbsp;</td>
<td>Sefar Filtration</td>
<td>NITEX 03-250/50</td>
<td>&nbsp;</td>
<tr>
<td>100 &mu;m nylon mesh</td>
<td>&nbsp;</td>
<td>Sefar Filtration</td>
<td>NITEX 03-100/47</td>
<td>&nbsp;</td>
<tr>
<td>Square petri dishes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-757-10k</td>
<td>&nbsp;</td>
<tr>
<td>Phytatrays</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1552</td>
<td>&nbsp;</td>
<tr>
<td>Murashige and Skoog Basal Medium (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5519</td>
<td>&nbsp;</td>
<tr>
<td>sucrose</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>S5-3</td>
<td>&nbsp;</td>
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M2933</td>
<td>&nbsp;</td>
<tr>
<td>KOH</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1767</td>
<td>10 M stock</td>
</tr>
<tr>
<td>Eclipse 90i microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cellulase R-10</td>
<td>&nbsp;</td>
<td>Yakult Pharmaceutical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Macerozyme R-10</td>
<td>&nbsp;</td>
<td>Yakult Pharmaceutical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-mannitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9546</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8041</td>
<td>1 M stock</td>
</tr>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3912</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>CALBIOCHEM</td>
<td>444203</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2536</td>
<td> 1 M stock</td>
</tr>
<tr>
<td>orbital shaker</td>
<td>&nbsp;</td>
<td>LAB-LINE</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>40 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>conical 15 ml tubes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352196</td>
<td>&nbsp;</td>
<tr>
<td>table centrifuge</td>
<td>&nbsp;</td>
<td>Sorvall</td>
<td>Legend RT</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3014</td>
<td>&nbsp;</td>
<tr>
<td>FACSAria</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5 ml microfuge tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>20170-38</td>
<td>&nbsp;</td>
<tr>
<td>RNeasy micro kit</td>
<td>&nbsp;</td>
<td>QIAGEN</td>
<td>74004</td>
<td>&nbsp;</td>
<tr>
<td>WT-Ovation Pico RNA Amplification System</td>
<td>&nbsp;</td>
<td>NuGEN</td>
<td>3300_12</td>
<td>&nbsp;</td>
<tr>
<td>FL-Ovation cDNA Biotin Module V2</td>
<td>&nbsp;</td>
<td>NuGEN</td>
<td>4200_12</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fluorescence activated cell sorting of plant protoplasts

遺伝子発現の高解像度、細胞型特異的な分析は非常に発達規制とあらゆる多細胞生物の環境刺激に対する反応の理解を深める。situハイブリダイゼーションおよびレポーター遺伝子の可視化でできる限られた範囲で、この目的のために使用されるが、高分解能の定量のためにすることにRT - PCRまたはハイスループットトランスクリプトーム全体の分析、特定の細胞型からのRNAの分離は必須です。特定の細胞型とそれに続く蛍光活性化細胞選別（FACS）で蛍光タンパク質マーカーを発現する組織の細胞の解離により、RNA抽出、cDNA合成/増幅とマイクロアレイ解析のために材料の十分な量を収集することができます。 細胞タイプ特異的蛍光レポーターラインの拡張セットは、植物の研究コミュニティに利用可能です。このケースでは、 シロイヌナズナの根の2つのマーカ線が使用されています：P SCR：：GFP（内皮および静止センター）とP WOX5：：GFP（静止センター）。苗の大量（数千）は水耕や寒天プレート上で成長し、さらなる分析のために十分なルートの材料を得るために収穫されます。植物素材の細胞の解離は、細胞壁の酵素消化によって実現されます。この手順では、高浸透圧誘導性の原形質分離と溶液中にプロトプラストを解放するために市販のセルラーゼ、ペクチナーゼ及びヘミセルラーゼを使用しています。 GFP陽性細胞のFACSは、488 nmレーザーで励起されたプロトプラストの赤色発光スペクトル対緑の可視化を利用しています。 GFP陽性のプロトプラストは、赤色発光の緑色のそれらの比の上昇によって区別す​​ることができます。プロトプラストは、通常のRNA抽出緩衝液に直接ソートされ、後での処理のために保存されています。 この手法は単純で実用的であることが明らかになりました。さらに、それはそれであっても非常に希少な種類の細胞（ 例えば、静止中心の細胞）のために、トランスクリプトーム解析のために細胞の十分な数字を分離するために困難なく使用できることが示されている。最後に、シロイヌナズナ実生の成長のセットアップは、セルソーティング（細胞の種類に固有の生物的または非生物的ストレス応答の解析のためなど ）する前に、植物の合併症の治療を可能にすることが示されている。植物プロトプラストのFACSのための潜在的な補足の使い方が説明されています。

Watch this Scientific Journal Video about Fluorescence Activated Cell Sorting of Plant Protoplasts at JoVE.com

Fluorescence Activated Cell Sorting of Plant Protoplasts

植物材料から、特定の種​​類の細胞を単離する方法が示されている。この手法は、特定の細胞型、細胞の解離と蛍光活性化細胞選別に蛍光タンパク質を発現するトランスジェニックマーカーラインを採用しています。さらに、成長のセットアップがの治療を容易にするここで確立されている<em&gt;シロイヌナズナ</em細胞選別の前に&gt;苗。

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 ...

fluorescence-activated-cell-sorting-of-plant-protoplasts

Research

JoVE Journal

Biology

24.6K ビュー.  New York University. 植物材料から、特定の種​​類の細胞を単離する方法が示されている。この手法は、特定の細胞型、細胞の解離と蛍光活性化細胞選別に蛍光タンパク質を発現するトランスジェニックマーカーラインを採用しています。さらに、成長のセットアップがの治療を容易にするここで確立されているシロイヌナズナ苗。

ビデオ: Fluorescence Activated Cell Sorting of Plant Protoplasts

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 ...

生物学

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

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. 
Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected 1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) 3. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.

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

Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

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 ...

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 ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

タバコプロトプラストと葉で二分子蛍光相補性によって可視化し、タンパク質間相互作用

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

浸透水透過係数（Pの測定<sub&gt; F</sub&gt;）球状細胞の：例として単離された植物プロトプラスト

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

発光時計レポーターとシロイヌナズナプロトプラストトランスフェにおける概日表現型の迅速分析

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

蛍光顕微鏡でストロミュール頻度を可視化

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

ゲルの表面上に成長する植物の根の光シート蛍光顕微鏡

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...