我々は、FACSソーティングと彼らの助けのために博士Yitzchak Oschry博士と点の他鷺-アシフに感謝します。この研究は、助成合意nの下欧州連合第7次フレームワーク計画（FP7/2007-2013）から°[276890]、イスラエル癌学会（＃20110078）から、イスラエルがん研究基金（ 研究からNEへの補助金によって支えられてキャリア開発賞 ）。

1。マウスから乳腺や皮膚組織を解剖する<ol><li>マウスから所望の組織を切開する前に、次の消耗品と試薬を準備します。 備品： <ul><li>手術ツール（洗剤で洗浄した後、70％エタノールに浸漬）。 </li><li>発泡スチロールの表面には、解剖時にマウスを休ませる。 </li><li>解剖時にマウスを保持するためにピンや針。 </li><li>磁気攪拌棒（オートクレーブ）を含む消化のために蓋付きのガラスの瓶、。 </li><li> 37℃水浴℃、撹拌プレート上で休んでいる間、潜水マグネチックスターラーと水没消化jarをするのに十分な水を含む。 </li></ul>試薬： <ul><li> FACS緩衝液I：（4℃）：ダルベッコのリン酸緩衝生理食塩水、CMF（カルシウムマグネシウム無料）+0.5％BSAをバッファ</li><li>コラゲナーゼ溶液（使用直前に作られる）： 0.05グラムコラゲナーゼII 0.05グラムコラゲナーゼIV 0.01グラムのデオキシリボヌクレアーゼ 20ミリリットルFACS緩衝液Iを使用するまで氷上でコラゲナーゼ溶液を保管してください。 </li><li> PharmLyse（塩化アンモニウムの溶解試薬）。 </li><li> FACS緩衝液II：ダルベッコのリン酸緩衝生理食塩水、CMF + 1％FCSをバッファ。 </li></ul></li><li> 乳腺の解剖 （また、17を参照）。 <ul><li>二酸化炭素（CO 2）の吸入を使ってメスのマウスを安楽死させる。 </li><li>発泡スチロールの表面に、その背中の上にマウスを置き、しっかりと25ゲージ針（ 図1A）を使用して、すべての四肢をピン。 70％エタノールで寛大にマウスを吹き付けます。 </li><li>ピンセットを用いて、正中線で腹部の皮膚を引き上げて、鋭いハサミで小さな切開を加える。 </li><li>腹部または胸部の空洞に穴をあけることを回避しながら切開から始まり、動物の首に皮膚をカット。 </li><li> &quot;Y字&quot;で、その結果、足に向かって正中切開から後脚の皮膚を切り取ります。 </li><li>マウス本体から離れて皮を引き、突き止める、露光乳腺は皮膚の下側（ 図1B）に接続されている。 </li><li>マウスの背に向かって乳腺を区切るには小さな鋭いハサミで結合組織を切断するときに優しく、皮膚から乳腺を区切るには綿棒を使用しています。 </li><li>腹部腺（＃4、5）はこのプロトコルのために最適です。また、第2 回乳腺を使用していますが、これらの腺は、解剖学的に混合した組織由来の細胞で、その結果、分離するのは難しいかもしれません胸筋に近接して配置されていることを念頭に置くことができます。 </li><li>注：腫瘍組織を切開する際には、壊死領域を削除することを推奨します。 </li></ul></li><li> 皮膚組織の解剖：脱毛に対処することなく、皮膚組織を取得する最も簡単な方法は、マウスの耳を使用することです。組織の大規模な量が必要な場合は、マウスの胴体から毛を剃ると肌を分析することができます。 <ul><li> CO 2吸入を使用してマウスを安楽死させる。 </li><li>鋭いはさみを使用して、氷の上でのPBS少量（〜0.5ミリリットル）を含む消化瓶に直ちに耳と場所の両方を遮断する。 </li></ul></li></ol> 2。乳腺/肌単一細胞懸濁液の調製<ul><li>氷の上でPBSの小さなボリュームを含む消化瓶に乳腺/肌を置きます。組織はその後、PBSで洗浄血まみれX2れている場合、そして過剰のPBSを捨ててください。 </li><li>湾曲したハサミで徹底的にミンチ。 </li><li> 20mlのコラゲナーゼ溶液（注​​：コラゲナーゼ溶液のこの量は、効率的に15匹までから乳腺または腫瘍組織と耳の約5グラムを解離する）を追加します。 </li><li> 37にプレートをかき混ぜる時にすぐ℃の水浴を置き、中間速度で攪拌しながら15分間インキュベートする。 </li></ul> 注：他の組織を消化し ​​た場合、最適なインキュベーション時間は変わる場合があります。 <ul><li>反応を停止し、30 mlの冷DMEM + 10％FCSを追加します。 </li><li>組織/メディア混合througを痛める50 mlコニカルチューブの上に置かれヘクタール70μmのセルストレーナー。 </li><li> 4℃で450×gで5分間コニカルチューブを遠心</li></ul> 3。赤血球の溶解 注：それらを屠殺する前にマウスをPBSで心臓潅流した場合は、この手順は必要ありません。 <ul><li>細胞ペレットを乱すことなく、真空に取り付けられたガラスピペットを用いて、上清を吸引します。 </li><li>室温で5分間、10mlのPharmLyse液とインキュベートでRBC、再懸濁した細胞ペレットを溶解する。 </li><li> FACS緩衝液Iの約40ミリリットルを添加して溶液を中和PharmLyse </li><li> 4℃、450×gで5分間細胞でコニカルチューブをスピン</li><li>上清を吸引します。 </li></ul> 4。内因FCのブロッキング<ul><li>オプション：次のステップのために適切なボリュームを確認するには、Count細胞。 </li><li> 1〜3ミリリットルFACS緩衝液Iに細胞を再懸濁します（o応じn個の細胞数、および使用する抗体の量）。 </li><li> 1:50希釈液（10μg/ ml）で到達し、10月20日 &#39;、氷上でインキュベートしFcBlock（抗マウスCD16/CD32）を追加します。 いいえFcBlockを洗い流しする必要はありません。 </li></ul> 5。染色<ul><li>非ステンド制御と単一色コントロール（使用する蛍光抗体の数に応じて）として使用するエッペンドルフチュー​​ブに100μlのアリコートを取り出します。 </li><li>ラベルの線維芽細胞に：1:50（10μg/ ml）を希釈に（フルオロフォアに直接結合）抗PDGFRα追加します。 </li><li>他の細胞集団（ 例えば、免疫細胞、マクロファージ、内皮細胞など）を標識するために：1:50希釈し、表面マーカーに適切な蛍光標識抗体を加える。 </li></ul> 注：PDGFRαは、線維芽細胞のための堅牢なマーカーであるが、それは任意の二重陽性を排除するために免疫細胞や上皮細胞の抗体を含むことが推奨され集団およびこうして孤立した線維芽細胞の純度を高める。 <ul><li>単色コントロールエッペンドルフチュー​​ブのそれぞれに各抗体の2μlを添加する。 </li><li>暗闇の中で氷上で1時間抗体で細胞をインキュベートします。 </li><li>洗浄するには、次のようにFACS緩衝液Iでチューブを記入し、4℃で450×gで5分間スピン</li><li>約2×10 6細胞/ ml、またはいずれかの利用可能なFACS選別機で選別するための最も適切な濃度の濃度になるように私をバッファリングし、FACSで上清を、再懸濁した細胞を吸引します。 </li></ul>オプション：細胞をソーティングの持続時間のために、FACS緩衝液IIに再懸濁させてもよい。これは、細胞の生存率を向上させることができる。しかしながら、血清中の因子への曝露は、テストされ、考慮されるべき効果の遺伝子発現を有することができる。 <ul><li>細胞塊を防止するためのチューブにフィルター細胞：フィルタートップとFACSチューブに標識された細胞を転送します。 </li><li>死細胞を除外するには、7AAD（0.5μg/ ml）を追加したり、プロップサンプルにidiumヨウ化物（PI）（非ステンドコントロールを除く）。 </li></ul>注：セルを無駄にしないように、あなたは、FACSで録音した後、未染色試料に7AAD/PIを追加し、7AAD専用コントロールとして再び使用することができます。 <ul><li>分析しながら、サンプルを氷上で保管してください。 </li></ul> 6。 FACSによる細胞の分離 （注：プロトコルのこの部分は、以前のFACSソーターを動作させるの知識、 例えば BD FACS AriaII、または熟練した技術者の支援が必要です）。 <ul><li> FACSソーターソフトウェア（BD FACSDiva）を使用して、前方散乱（FSC）、側方散乱光（SSC）、7AAD、FITC、PEおよびラベル細胞（ 図2）に使用される任意の他の蛍光色素分子を分析するために、適切な解析プロットを準備します。 </li><li>セルソーターに各サンプルをロードする前に、渦は簡単に細胞を再懸濁する。 </li><li> CELからゲートに、全細胞集団のためのゲーティングを設定するために、無染​​色試料を分析することから始めリットルの破片、および自家蛍光やバックグラウンド染色を決定する。 </li><li>全細胞集団からの生細胞の集団を決定し、ゲートに染色されていないサンプル+ 7AADを分析します。 </li><li>このような補償などのパラメータを決定して、キャリブレーションする各単一カラーコントロールを分析します。 </li><li>並べ替えのためにゲートの位置を定義するために染色されたサンプルを分析します。 </li><li>いったん所望の細胞集団のパラメータとゲートが設定されている、エッペンドルフチュー​​ブまたは培養培地またはRNA溶解緩衝液（例えば、RLTバッファーまたはトリゾールなど）を含む15mlのコニカルチューブにラベルされた細胞集団を選別開始。 </li></ul>注：多数のセルをソートする場合、それは細胞が溶解緩衝液を希釈し、収量を減少させるでしょうに伴うシース液の大きなボリュームとしては、RNA溶解用緩衝液に直接ソートすることは推奨されません。 <ul><li>デ、新鮮な培地またはRNA溶解用緩衝液にソートが終了した直後に、転送ダウン細胞をスピンあなたの実験の目的で保留中。 </li><li>文化単離された細胞への順序でソートする場合は、無菌のソートを実行します。細胞のよりよい回復のために、代わりにFACS緩衝液IIのフェノールレッド不含DMEM +5％FCSを使用し、20％FCSを含む培地に細胞を回収します。 </li><li>培養は、線維芽細胞をソートしたときは、それらの遺伝子発現の変化につながる線維芽細胞の活性化では、この結果として、プラスチックの上にコラーゲンコートしたプレート上alwaysplate、決して直接。 </li></ul>

<ol>
	<li>Kalluri, R., Zeisberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+in+cancer.">Fibroblasts in cancer.</a> Nat. Rev. Cancer. 6, 392-401 (2006).</li><li>Shimoda, M., Mellody, K. T., Orimo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carcinoma-associated+fibroblasts+are+a+rate-limiting+determinant+for+tumour+progression.">Carcinoma-associated fibroblasts are a rate-limiting determinant for tumour progression.</a> Seminars in cell &amp; developmental biology. 21, 19-25 (2010).</li><li>Orimo, A., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+stromal+fibroblasts+in+tumors.">Heterogeneity of stromal fibroblasts in tumors.</a> Cancer Biol. Ther. 6, 618-619 (2007).</li><li>Ostman, A., Augsten, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-associated+fibroblasts+and+tumor+growth--bystanders+turning+into+key+players.">Cancer-associated fibroblasts and tumor growth--bystanders turning into key players.</a> Current opinion in genetics &amp; development. 19, 67-73 (2009).</li><li>Allinen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+the+tumor+microenvironment+in+breast+cancer.">Molecular characterization of the tumor microenvironment in breast cancer.</a> Cancer Cell. 6, 17-32 (2004).</li><li>Bhowmick, N. A., Neilson, E. G., Moses, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblasts+in+cancer+initiation+and+progression.">Stromal fibroblasts in cancer initiation and progression.</a> Nature. 432, 332-337 (2004).</li><li>Orimo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblasts+present+in+invasive+human+breast+carcinomas+promote+tumor+growth+and+angiogenesis+through+elevated+SDF-1/CXCL12+secretion.">Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion.</a> Cell. 121, 335-348 (2005).</li><li>Pietras, K., Ostman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+interactions+with+the+tumor+stroma.">Hallmarks of cancer: interactions with the tumor stroma.</a> Experimental cell research. 316, 1324-1331 (2010).</li><li>Erez, N., Truitt, M., Olson, P., Arron, S. T., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-Associated+Fibroblasts+Are+Activated+in+Incipient+Neoplasia+to+Orchestrate+Tumor-Promoting+Inflammation+in+an+NF-kappaB-Dependent+Manner.">Cancer-Associated Fibroblasts Are Activated in Incipient Neoplasia to Orchestrate Tumor-Promoting Inflammation in an NF-kappaB-Dependent Manner.</a> Cancer Cell. 17, 135-147 (2010).</li><li>Sugimoto, H., Mundel, T. M., Kieran, M. W., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+fibroblast+heterogeneity+in+the+tumor+microenvironment.">Identification of fibroblast heterogeneity in the tumor microenvironment.</a> Cancer Biol. Ther. 5, 1640-1646 (2006).</li><li>Neumann, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+and+passaging+alters+gene+expression+pattern+and+proliferation+rate+in+rheumatoid+arthritis+synovial+fibroblasts.">Cell culture and passaging alters gene expression pattern and proliferation rate in rheumatoid arthritis synovial fibroblasts.</a> Arthritis research &amp; therapy. 12, R83 .</li><li>Sherr, C. J., DePinho, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence:+mitotic+clock+or+culture+shock.">Cellular senescence: mitotic clock or culture shock.</a> Cell. 102, 407-410 (2000).</li><li>DeNardo, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+complexity+in+breast+cancer+predicts+overall+survival+and+functionally+regulates+response+to+chemotherapy.">Leukocyte complexity in breast cancer predicts overall survival and functionally regulates response to chemotherapy.</a> Cancer Discovery. 1, 54-67 (2011).</li><li>Pahler, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+in+tumor-promoting+inflammation:+impairment+of+macrophage+recruitment+evokes+a+compensatory+neutrophil+response.">Plasticity in tumor-promoting inflammation: impairment of macrophage recruitment evokes a compensatory neutrophil response.</a> Neoplasia. 10, 329-340 (2008).</li><li>Pietras, K., Pahler, J., Bergers, G., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+paracrine+PDGF+signaling+in+the+proangiogenic+tumor+stroma+revealed+by+pharmacological+targeting.">Functions of paracrine PDGF signaling in the proangiogenic tumor stroma revealed by pharmacological targeting.</a> PLoS Med. 5, e19 (2008).</li><li>Quante, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow-derived+myofibroblasts+contribute+to+the+mesenchymal+stem+cell+niche+and+promote+tumor+growth.">Bone marrow-derived myofibroblasts contribute to the mesenchymal stem cell niche and promote tumor growth.</a> Cancer Cell. 19, 257-272 (2011).</li><li>Plante, I., Stewart, M. K., Laird, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Mammary+Gland+Development+and+Function+in+Mouse+Models.">Evaluation of Mammary Gland Development and Function in Mouse Models.</a> J. Vis. Exp. (53), e2828 (2011).</li><li>Xouri, G., Christian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+function+of+tumor+stroma+fibroblasts.">Origin and function of tumor stroma fibroblasts.</a> Seminars in cell &amp; developmental biology. 21, 40-46 (2010).</li></ol>

特別な利害関係は宣言されません。

組織培養で行われた実験は、有益であるとin vivoで検証することができる機能的な原則を提案することができますが、それは文化11,12中の細胞の遺伝子発現に大きな変化が起こることが知られている。線維芽細胞における遺伝子発現プロファイルの作成時手順組織培養を避けるために、私たちは新鮮なマウスやヒト組織から通常の分離、ならびに癌関連線維芽細胞を可能にする?...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong試薬の&gt;名前</strong</td&gt;<td<strong&gt;会社概要</strong</td&gt;<td<strong&gt;カタログ番号</strong</td&gt;<td<strong&gt;コメント（オプション）</strong</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM</td&gt;<td&gt;ギブコ</td&gt;<td&gt; 41965</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PBS</td&gt;<td&gt;生物産業</td&gt;<td&gt; 02-023-1A</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;コラゲナーゼII</td&gt;<td&gt;ワージントン</td&gt;<td&gt; LS4176</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;コラゲナーゼIV</td&gt;<td&gt;ワージントン</td&gt;<td&gt; LS4188</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;デオキシリボヌクレアーゼ</td&gt;<td&gt;ワージントン</td&gt;<td&gt; LS2007</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PharmLyse</td&gt;<td&gt; BD</td&gt;<td&gt; 555899</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;セルストレーナー70μmの</td&gt;<td&gt; SPL</td&gt;<td&gt; 93070</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;精製された抗マウスCD16/CD32</td&gt;<td&gt; BDファーミンジェン</td&gt;<td&gt; 553142</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;を介してプローブ（7AAD）</td&gt;<td&gt;電子バイオサイエンス</td&gt;<td&gt; 00-6993-50</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;アンチマウスCD140a-PE（PDGFRA）</td&gt;<td&gt;電子バイオサイエンス</td&gt;<td&gt; 12-1401-81</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;アンチマウスF4/80- FITC</td&gt;<td&gt; Cederlane</td&gt;<td&gt; CL8940F</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM W / Oフェノールレッド</td&gt;<td&gt;ギブコ</td&gt;<td&gt; 31053</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; I型コラーゲン</td&gt;<td&gt; BD Biosciences社</td&gt;<td&gt; 354236</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table

isolation of normal and cancer-associated fibroblasts from fresh tissues by fluorescence activated cell sorting (facs)

癌関連線維芽細胞（CAFS）は、特定の乳癌では、多くの癌の腫瘍間質内で最も顕著な細胞型であり、その卓越した存在は、しばしば予後不良の1,2に関連付けられています。 CAFSは、間質の線維芽細胞の活性化亜集団であり、それらの多くは筋線維芽細胞マーカーのα-SMA 3を発現している 。 CAFSは、地元組織の線維芽細胞からだけでなく、骨髄由来細胞から由来する腫瘍の開発に採用され、腫瘍微小環境4の影響下にCAFの表現型を採用しています。 CAFSは、腫瘍細胞増殖、血管新生、および浸潤5-8を促進し、そのシグナル伝達を介して腫瘍の開始、成長と進行を促進することが示された。我々は、CAFSは早く前腫瘍段階9時から、腫瘍促進炎症を介在することによって腫瘍の成長を高めることが実証された。腫瘍の成長を促進する上で重要な役割をCAFSプレイの増加する証拠にもかかわらず、勉強CAFSは、CAF-特異的なマーカーと腫瘍微小環境10に共存する多くのサブタイプを持つこれらの細胞の広大な異質性の欠如のために継続的な課題となっている。また、in vitroで線維芽細胞を研究し、それらの遺伝子発現プロファイルはしばしば組織培養11,12で変更されているという事実によって妨げられる。この問題に対処するために、新鮮なマウスおよびヒト組織からの線維芽細胞の公平な遺伝子発現プロファイリングを可能にするために、我々は、蛍光活性化細胞ソーティング（FACS）13,14は、前のプロトコルに基づいた方法を開発した。我々のアプローチは新鮮なマウスおよびヒト組織から線維芽細胞を単離するための表面マーカーとしてPDGFRαを利用に依存しています。 PDGFRαは豊富正常線維芽細胞およびCAFS 9,15両方で表される。このメソッドは、正常線維芽細胞およびCAFSの純粋な集団の単離を可能に含むが、α-SMA +活性化筋線維芽細胞に限定されません。隔離された線維芽細胞は、その後することができます特性評価および腫瘍形成時にCAFSで発生し遺伝子発現の進化の比較のために使用される。実際、我々と他の人は16を細胞選別によって単離された線維芽細胞の発現プロファイリングを報告した。このプロトコルは正常皮膚、乳腺、膵臓、肺組織からの線維芽細胞の高度に濃縮された集団を分離し、プロファイリングを行った。また、我々の手法は、機能の実験を行うため、しばしば文化CAFSしようと、大きな障害となっている腫瘍細胞による汚染を避けるために、選別された細胞を培養することができます。

組織の線維芽細胞の高度に濃縮された集団の分離における線維芽細胞の結果のためのマーカーとしてPDGFRαを使用。ポスト·ソート解析（ 図2A）によって定量化と並べ替えの後に純度のレベルは、99％であった。細胞特異的な制御遺伝子の相対的発現（ 図2B）によって、非線維芽細胞を汚染の割合を推定することは、一般的に0.1から0.6パーセントの汚染を示しています?...

Watch this Scientific Journal Video about Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS at JoVE.com

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

癌関連線維芽細胞（CAFS）は増殖、血管新生、炎症を促進し、そのシグナル伝達を介して腫瘍の開始、成長と進行を容易にします。ここでは、表面マーカーとしてPDGFRαを使用して、細胞選別により新鮮なマウスおよびヒト組織由来正常線維芽細胞とCAFSの純粋な集団を単離するための方法を説明します。

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 ...

isolation-normal-cancer-associated-fibroblasts-from-fresh-tissues

Research

JoVE Journal

Biology

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

35.5K ビュー.  Tel Aviv University. 癌関連線維芽細胞（CAFS）は増殖、血管新生、炎症を促進し、そのシグナル伝達を介して腫瘍の開始、成長と進行を容易にします。ここでは、表面マーカーとしてPDGFRαを使用して、細胞選別により新鮮なマウスおよびヒト組織由来正常線維芽細胞とCAFSの純粋な集団を単離するための方法を説明します。

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

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

の単離と精製ショウジョウバエ</em磁気ビーズソート別>末梢ニューロン

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

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 ...

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 ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

ラットポータル線維芽細胞の分離により、<em&gt;その場</em&gt;肝灌流

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

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 ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

スペクトラルフローサイトメトリーにより固形組織から単離された細胞懸濁液の分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

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 ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

スクレラクチニアン細胞集団の分離のための蛍光活性化細胞選別

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

骨格筋線維芽細胞からのエクソソームの単離と特性評価

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...