細胞型と発達段階特有のプロファイリングにブキャナン-ウォラストンラボでの作業は、欧州委員会の第 6次フレームワークProgammeにおけるアグロ-OMICSプロジェクト（グラント＃LSHG-CT-2006から037704）によってサポートされています。細胞型に特異的なプロファイリングのギフォード·ラボでの作業はBBSRC新治験助成（BB/H019502/1）によってサポートされています。 KC274とKC464 シロイヌナズナラインはAAR·ウェッブ（ケンブリッジ大学）から得た。

1。プロトプラストの生成<ol><li>かみそりの刃で1ミリメートル幅のストリップ（ 図1）への収穫葉材料とカット。のために最大15葉に単一のリーフ以上を含有するサンプルを簡単にカットする前に積み重ねることができます。 </li><li>直ちに20mlの溶液 （400mMのマンニトール、20mMのMES水和物は、20mMのKCl、10mMのCaCl 2、2mMのMgCl 2、0.1％BSAおよび2.5mMのβ-メルカプトエタノールを含む9センチメートルラウンドペトリ皿に葉片を転送protoplasting酵素を含むpHは5.7）（1.2％セルロース、R-10、0.6％セルロースRSと0.4パーセントmacerozyme R-10）とRNase及び転写酵素阻害薬（1×ProtectRNAおよび10μg/ mlのアクチノマイシンD）;優しく独立した葉片と真空度は2×5分間浸透させる。 </li><li> 3〜4時間室温およびプロトプラストを90 rpmに設定オービタルシェーカー上にシャーレを置きます。 <ol><li>インキュベーションの間葉片は、使用することによって、互いに分離されるべきである30分ごとの間隔で、フラットピンセットのセットです。 </li></ol></li><li> 50 mlチューブで70μmのセルストレーナー（フィッシャーサイエンティフィック）を置き、そっとを通じてprotoplastingソリューションをフィルタリングします。これは、プロトプラストが通過できるようにしながらふるいによって保持される葉片を、削除されます。 </li><li>ゆっくりとフィルター同じ篩4 mlの溶液を用いて葉片を洗浄します。 </li><li>丸底チューブにプロトプラストを転送し、ペレットプロトプラスト〜5分間300 xgでプロトプラスト溶液を遠心分離します。 </li><li>穏やかにプロトプラストを乱すことなく、できるだけ多くの上清を可能な限りを吸引。 </li><li> 、遠心分離機で5分間300 xgで5 mlの溶液を用いてプロトプラストを洗浄します。上清を除去</li><li>パスツールピペットやカットオフP1000チップを使用して、 ソリューションの適切な音量でプロトプラストを懸濁する。 </li></ol> 2。葉プロトプラストのFACS </p&gt; <ol><li>すぐにソートする前に、凝集プロトプラストを避けるために（ノンカット）P1000チップを用いてプロトプラストを再懸濁します。 </li><li> 50μmのfilconフィルター（BD Biosciences社）をソートチューブにプロトプラストを転送し、セルソーターのノズルの目詰まりを避けるために、必要に応じて希釈してください。プロトプラスト（これらの設定は、BDの流入セルソーターに最適な20 PSI（シース）と21から21.5 PSI（サンプル）、39.2 kHzの周波数と4000 &lt;事象発生率の圧力で100μmのノズルを使用してソートされます他のセルソーティング·マシン上（BD Biosciences社）。最適化）は、これらの設定を調整する必要があるかもしれません。 </li><li> GFP陽性プロトプラストを488nmのアルゴンレーザーを使用していて、30分の580バンドパスフィルタ（オレンジ）対40分の530バンドパスフィルター（緑）からの出力をプロットする識別されます。 GFP陽性プロトプラストは、高580 POPUの低530 /ロー580人口と瀕死/デッドプロトプラストと残骸の非GFPのプロトプラストと、高い530/low 580人口になりますlation（ 図2）。 <ol><li> RNAを抽出元となるプロトプラストをソートする場合、プロトプラストを還元剤（ 例えば 、β-メルカプトエタノール）を含む溶解緩衝液の少なくとも4つのボリュームに直接ソートする必要があります。また、単一試料のソート時間は、20〜30分であり、いずれかの時点で処理二から三のサンプルの最大値に制限されるべきである。 </li><li>ソートされたプロトプラストの可視化のために、プロトプラスト溶液の少なくとも8巻、その後プロトプラストを集中させる遠心分離によって収集に直接ソートする必要があります。 </li></ol></li></ol> 3。代表的な結果このプロトコルは、蛍光活性化細胞選別（ 図1）のために使用されるシロイヌナズナの葉からプロトプラストを得るための方法を説明します。 RNaseおよび転写阻害剤の存在下におけるプロトプラストの生成は細胞fを防ぎromは転写応答をマウントする。これは、自家蛍光の有意水準を持っている多くの死者と瀕死プロトプラストを生じさせるの結果を持っていません。この方法を用いて調製したプロトプラストをソートする場合には、ソートに使用される580nmの対530nmの蛍光のグラフにはいくつかの異なる集団を参照することが非常に一般的です。このプロトコルに使用されるソートゲートと一緒にこのような例は、 図3に示します。 プロトプラスト溶液の8巻にソートされ、濃縮を確認するために可視化のために収集することができます。 図2Gは、これは孔辺細胞にGFPを発現TP382ラインを使用しての例を示しています。 増幅前と後の両方で、この方法を用いて得られた収量の例は、 表1に示されている。算定された金額は、オベーションピコWTAのシステム（500 PG-50 ng）を、したがって、sを用いて増幅するために十分である定量PCR（ 図4）は、次世代シーケンシングやマイクロアレイのいずれかの方法でubsequent分析。 <img alt="図1" src="/files/ftp_upload/4214/4214fig1.jpg" /> 図1実験の全体的なワークフロー。 1）収穫はシロイヌナズナラインをGFP発現から関心のある葉。 2）迅速に約1mm幅のストリップに切断し、直ちにprotoplasting溶液及び真空浸透させるにこれらを転送します。 3）3〜4時間インキュベートする。 4）70μmのふるいを通してゆっくりと濾過プロトプラスト溶液を遠心分離により底チューブ、ペレットプロトプラストを丸める移す。 5）すぐに選別、再懸濁するプロトプラストおよびフィルタは50μmを通じて選別チューブに移す前に。 6）並べGFP陽性およびGFP陰性の実行可能なプロトプラスト。 7）プロトプラストは今どちらか視覚的に分析したり、そのような定量PCR、次世代シーケンシングやマイクロアレイなどの技術を使用して設定できます。 図2本研究で使用したKC464、KC274とTP382行。向軸表皮にGFPの発現を示すKC464葉のA）の横断面。 B）のプロトプラストはKC464葉由来。血管系にGFPの発現を示すKC274葉のC）の横断面。 D KC274葉由来）プロトプラスト。孔辺細胞におけるGFPの発現を示すE）TP382の葉。 F）のプロトプラストは、TP382の葉に由来する。 TP382葉由来プロトプラストの高い530/low 580集団からプロトプラストを含むGFPを示し、葉の細胞の複雑な背景に孔辺細胞をGFP発現のFACSによって得られた濃縮のG）の例。 AF：共焦点顕微鏡画像、G：蛍光顕微鏡画像。画像内のスケールバーは50μmである。 <img alt="図3" src="/files/ftp_upload/4214/4214fig3.jpg" /><html> 図3 30分の580 nmの（オレンジ）対40分の530 nm（緑）バンドパスフィルタ（BDの流入のセルソーター）からの出力の典型的なFACS買収ドットプロット。示さソートゲートは通常、ソート時に使用されたものである。単一の低530/low 580人口を示す阻害剤の非存在下でprotoplasted COL-4葉由来プロトプラスト、、のA）ドットプロット。阻害剤によるストレスや着色のために580蛍光のシフトを示す阻害剤の存在下でprotoplasted重量COL-4葉由来プロトプラスト、AからB）ドットプロット。高530/low 580集団で集団を含むGFPを示す阻害剤の存在下でprotoplastedそれぞれKC74、KC464とTP382ライン、、、から葉由来プロトプラストからCE）のドットプロット。 GFP（高530/low 580）集団内のイベントのパーセンテージが与えられている。 <a href="http://www.jove.com/files/ftp_upload/4214/4214fig3large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 e_content &quot;のfo：キープtogether.withinページ=&quot; always &quot;を&gt; <img alt="図4" src="/files/ftp_upload/4214/4214fig4.jpg" /> 図4：図3に示すように、代表的なゲーテッド人口画分におけるGFP発現。複製し2つの生物学は、その後行われたGFP特異的プライマー（ 表2）を用いてオベーションピコWTAのシステムと定量PCRを用いて増幅し、各分画の種類が得られた。示された値は、最も高い（赤）、最低（緑）と平均相対値である。高580人口はこのケースでは2、低530/low 580と単一の生物学的replicateは、単一の値のみが表示されているので、使用されていたため高530/low 580に分割されました。 ACT2（At3g18780）は、すべてのサンプルのための標準として用いた。葉：葉全体。未ソート：ソートされていないプロトプラスト。 GFP1：高530/low 580とRest1：低530/low 580、GFP2：高530/high 580とRest2： 図3とインサートで説明したように、低530/high 580、。結果は、検証Oアールソートされた画分（ 例えば、 図2G）におけるGFP細胞含量のf光学評価。 <a href="http://www.jove.com/files/ftp_upload/4214/4214fig4large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 <table border="1"><tbody><tr><td> サンプル </td><td> 葉数 </td><td> 細胞数 </td><td> RNAの収量 （NG） </td><td> RNAの収量 （増幅; NG） </td></tr><tr><td>全体の葉 全葉B </td><td> 1 1 </td><td> - - </td><td> 8001 10525 </td><td> 4623 *** 4572 *** </td></tr><tr><td>ソートされていないプロトプラスト ソートされていないプロトプラストのB </td><td> 5 5 </td><td> - - </td><td> 2433 2625 </td><td> 1152年*** 3321 *** </td></tr><tr><td> GFP1 Rest1 </td><td> 15 </td><td> 6039（0.39％*） 55389（6.40％*） </td><td> - ** - ** </td><td> 861 489 </td></tr><tr><td> GFP1 B Rest1 B </td><td> 15 </td><td> 10783（0.63％*） 57040（7.49％*） </td><td> - ** - ** </td><td> 537 420 </td></tr><tr><td> GFP2 Rest2 </td><td> 15 </td><td> 18337（1.49％*） 62405（73.49パーセント*） </td><td> - ** - ** </td><td> 360 411 </td></tr></tbody></table> 表1サンプルは、FACSを用いて収集し、ライブGFP発現プロトプラストを含有する画分を決定するために、定量PCRで分析した。またオベーションピコWTAシステム（NuGen）で増幅前後NGの総RNAの収量は、この表に含まれています。 GFP1、GFP2、Rest1とRest2画分を、 図4中の挿入で表示されます。総細胞ソート実行の細胞の*割合は分数のセルの数の横に括弧で与えられます。これらのパーセンテージはに相関していない収集レスト細胞のはるかに大きい数のためのGFP画分よりも早く停止したFACSの並べ替えなど、各サンプルのお互いが &#39;休息&#39;分数のために実行されます。製造元のプロトコールに従って増幅反応への入力として使用** 50 NG、。 <table border="1"><tbody><tr><td> プライマー </td><td> シーケンス </td></tr><tr><td> mGFP5-ER.fw </td><td> GCCTGAGGGATACGTGCAGGAGA </td></tr><tr><td> mGFP5-ER.rv </td><td> TGGCGGGTCTTGAAGTTGGCT </td></tr><tr><td> ACT2.fw </td><td> GCCATCCAAGCTGTTCTCTC </td></tr><tr><td> ACT2.rv </td><td> GCCATCCAAGCTGTTCTCTC </td></tr></tbody></table> 表2定量PCRプライマー。

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特別な利害関係は宣言されません。

我々は、プロトプラストの生成およびFACSを使用して後続の細胞選別のための葉にGFPを発現するシロイヌナズナ植物の使用を可能にする方法を説明してきました。この方法は、定量PCRやマイクロアレイのどちらかによって増幅、したがってその後の分析に使用するRNAの十分な量が得られます。ソートされた画分を、高度として定量PCR（ 図2）によって実証GFP含有細胞が濃縮されています。 葉片が阻害剤含有プロトプラスト溶液を真空に潜入されるまで生きてプロトプラストの高い収率を達成するには、手順の最初のステップの間に急速に働くことが重要です。また、1ミリメートルの葉片を生成するときに、これは過度の損傷、したがって低いプロトプラスト収量につながるとして、葉ではなく &#39;チョップ&#39;やマッシュにスライスまたはカットすることが非常に重要です。 ここで説明した方法と他publisheの決定的な違いdの方法はRNase阻害剤や転写阻害剤の使用です。ほとんどのメソッドは、プロトプラストをより簡単に生成することができ、そこからシロイヌナズナの根のために開発されています。しかし、肉を除いて、葉を扱うとき、それはプロトプラストの生成時間を増加させる必要がある。したがってプロトプラストの生成処理自体（データは示していない）の結果として、遺伝子発現の変化から守るために、これらの阻害剤を含めることが重要です。しかし、阻害剤の存在は、ストレスに対抗すると可塑性の喪失にこのリード線などのプロトプラストが受けやすくなる可能性のあるもの、オンまたはオフの遺伝子を切り替えることにより、どちらかの、転写応答をマウントすることができないことにつながる手順をprotoplasting。 我々はGFP発現シロイヌナズナライン数で正常ここで説明した方法を使用している。重要なのは、我々はどちらかに強くGFPを発現するGFPの行でこのメソッドを使用している核、またははるかに弱くnon-localized/diffuse細胞質方法では、ここで使用されてKC464とKC274線の場合と同様です。回線の両方の型は、メソッドと互換性があり、GFP発現プロトプラスト（ 図2および3）の高濃縮画分を得ることができます。フルオロフォアは、生きた細胞を選択する能力を維持するために死んだ/死細胞の自家蛍光とは著しく異なる蛍光を選択しなければなりませんが、方法は簡単、他のフルオロフォアに適合させることができる。

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>セルラーゼR-10 </td><td>メルフォード</td><td> C8001 </td></tr><tr><td>セルラーゼRS </td><td>メルフォード</td><td> C8003 </td></tr><tr><td> Macerozyme R-10 </td><td>メルフォード</td><td> M8002 </td></tr><tr><td> BSA </td><td>シグマアルドリッチ</td><td> A3912 </td></tr><tr><td> ProtectRNA </td><td>シグマアルドリッチ</td><td> R7397 </td></tr><tr><td>アクチノマイシンD </td><td>シグマアルドリッチ</td><td> A1410 </td></tr><tr><td> 70μmのセルストレーナー</td><td>フィッシャー</td><td> FB35181 </td></tr><tr><td> 50μmのfilconカップ型フィルター</td><td> BD Biosciences社</td><td> 340630 </td></tr></tbody></table>

cell specific analysis of arabidopsis leaves using fluorescence activated cell sorting

葉の原基の開始後、バイオマス蓄積は主に細胞増殖や葉1の拡大によって制御されます。しかし、 シロイヌナズナの葉は、多くの異なる細胞型といくつかの構造で構成された複雑な器官である。同時に、成長して葉が拡大停止と老化1を受けることが第一である葉柄から最も遠いセルと、開発のさまざまな段階で細胞が含まれています。リーフ内の異なる細胞が故に伸長または分化、分裂している、アクティブ、ストレスやデッド、および/または任意の一時点で、携帯型のサブ組での刺激に応答します。これはやりがいの葉のゲノム研究を行います。たとえば全体の葉から発現データを解析する際に、異なる細胞応答ゾーンまたは細胞型で動作遺伝子ネットワークからの信号が生成される不正確なプロファイルになり、混乱します。 この問題に対処するために、Sエベラル方法は細胞特異的遺伝子発現の研究​​を可能が記載されている。これらは、レーザーキャプチャーマイクロダイセクション（LCM）2またはプロトプラストの生成とそれに続く蛍光活性化細胞選別（FACS）3,4、原子力降水5とポリソーム6の免疫沈降のための最近記載そのままシステムに使用されるGFP発現植物が含まれる。 FACSを成功裏に根端からセルの識別情報との距離が遺伝子3,7の多数の発現プロファイルに有意な効果を持っていたことを示すなど、多くの研究が、使用されてきました。 GFPの行のFACSもroot窒素応答と側根の発達8、塩ストレスルート10の9オーキシン分布の間に細胞特異的転写調節を実証し、 シロイヌナズナの茎頂分裂組織11の遺伝子発現マップを作成するために使用されている。しかしFACSが以前自家蛍光12,13に基づくシロイヌナズナ葉由来プロトプラストをソートするために使用されてきたが、葉にGFPを発現シロイヌナズナライン上のFACSのこれまでの利用は4非常に限られてきました。以下のプロトコルでは、プロトプラストの生成レジームの影響を最小限に抑えながら、FACSと互換性のあるシロイヌナズナの葉のプロトプラストを得るための方法について説明します。我々は、向軸表皮14にKC274線、血管組織14とダブルGFPは内核局在化シグナルにリンクされている構築物を発現TP382 シロイヌナズナライン入力で GFPを発現するが、KC464 シロイヌナズナラインを使用する方法を実証しているGFPを発現する孔辺細胞（データは示されていない、 図2）。我々は現在、老化のさまざまな段階で、両方の細胞型の開発とストレス時の具体的な表現だけでなく、不均一な細胞集団を研究するためにこのメソッドを使用している。

Watch this Scientific Journal Video about Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting at JoVE.com

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

製造方法<em&gt;シロイヌナズナ</em&gt;特定の細胞集団の研究を可能にする、蛍光活性化細胞選別（FACS）と互換性のある葉プロトプラスト。このメソッドは、任意のと互換性があります<em&gt;シロイヌナズナ</em細胞のサブセットでGFPを発現&gt;行。

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 ...

cell-specific-analysis-arabidopsis-leaves-using-fluorescence

Universidad San Sebastian

Research

JoVE Journal

Biology

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

15.2K ビュー.  University of Warwick. 製造方法シロイヌナズナ特定の細胞集団の研究を可能にする、蛍光活性化細胞選別（FACS）と互換性のある葉プロトプラスト。このメソッドは、任意のと互換性がありますシロイヌナズナ行。

ビデオ: Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

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 ...

生物学

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

でFACSアシスト細胞型特異的発現プロファイリングを使用して組織と器官特異フィトクロム応答を調査シロイヌナズナ</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Heterokaryon Technique for Analysis of Cell Type-specific Localization

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling. These proteins display a diverse array of cellular functions including nuclear import/export of RNA and protein, transcriptional regulation, and apoptosis. Interestingly, major cellular reorganizations including cell division, differentiation and transformation, often involve such activities3,4,8,10. The detailed study of these proteins and their respective regulatory mechanisms can be challenging as the stimulation for these localization changes can be elusive, and the movements themselves can be quite dynamic and difficult to track. Studies involving cellular oncogenesis, for example, continue to benefit from understanding pathways and protein activities that differ between normal primary cells and transformed cells6,7,11,12. As many proteins show altered localization during transformation or as a result of transformation, methods to efficiently characterize these proteins and the pathways in which they participate stand to improve the understanding of oncogenesis and open new areas for drug targeting.
Here we present a method for the analysis of protein trafficking and shuttling activity between primary and transformed mammalian cells. This method combines the generation of heterokaryon fusions with fluorescence microscopy to provide a flexible protocol that can be used to detect steady-state or dynamic protein localizations. As shown in Figure 1, two separate cell types are transiently transfected with plasmid constructs bearing a fluoroprotein gene attached to the gene of interest. After expression, the cells are fused using polyethylene glycol, and protein localizations may then be imaged using a variety of methods. The protocol presented here is a fundamental approach to which specialized techniques may be added.

A flexible and efficient method for the characterization of cell type-specific protein localization and nucleocytoplasmic shuttling is described. This heterokaryon approach uses fluorescently-labeled fusion proteins to image protein localizations after cell fusion. The protocol is amenable to steady-state localizations or more dynamic determinations based on live cell imaging.

細胞種特異的局在の解析のためのヘテロカリオンのテクニック

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

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 ...

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 ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

イメージングのための簡単​​な方法シロイヌナズナ浸潤イメージング媒体としてPerfluorodecalinを使って葉

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

分裂酵母の定量的なライブセル蛍光-顕微鏡解析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

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 ...

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 ...