Name: simple detection of primary cilia by immunofluorescence
Uploaded: 2020-05-15T14:00:00
Description: Watch this Scientific Journal Video about Simple Detection of Primary Cilia by Immunofluorescence at JoVE.com

この作業は、チェコ共和国の防衛省によって支援されました - 長期組織開発計画軍事保健科学部の大量破壊兵器の医療側面、防衛大学;チェコ共和国教育・青年スポーツ省(特定研究プロジェクト第一:SV/FVZ201703)とPROGRES Q40/06また、英語の改訂で彼の親切な支援のためのダニエル・ディアスに感謝します。

1. 文化メディア、ソリューション、料理の準備
<ol><li>カバーリップをオートクレーブ(22 x 22 mm)にします。6つのウェルプレートを準備します。ウシ胎児血清(FBS)および抗生物質ペニシリン/ストレプトマイシンを解凍し、培養培地を室温(RT)まで温める。トリプシン-EDTAを使用する (0.25%)1x PBS(リン酸緩衝食塩分カルシウムとマグネシウム)を細胞を通過させる。</li>
	<li>新鮮な4%パラホルムアルデ?...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+levels+of+cytosolic+tubulin+regulate+ciliary+length+control.">Soluble levels of cytosolic tubulin regulate ciliary length control.</a> Molecular Biology of the Cell. 22 (6), 806-816 (2011).</li><li>Filipov&#225;, 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=Ionizing+radiation+increases+primary+cilia+incidence+and+induces+multiciliation+in+C2C12+myoblasts.">Ionizing radiation increases primary cilia incidence and induces multiciliation in C2C12 myoblasts.</a> Cell Biology International. 39 (8), 943-953 (2015).</li><li>Filipov&#225;, 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=The+toxic+effect+of+cytostatics+on+primary+cilia+frequency+and+multiciliation.">The toxic effect of cytostatics on primary cilia frequency and multiciliation.</a> Journal of Cellular and Molecular Medicine. 23 (8), 5728-5736 (2019).</li><li>Gadadhar, S., 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=Tubulin+glycylation+controls+primary+cilia+length.">Tubulin glycylation controls primary cilia length.</a> The Journal of Cell Biology. 216 (9), 2701-2713 (2017).</li><li>Shamloo, K., 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=Chronic+Hypobaric+Hypoxia+Modulates+Primary+Cilia+Differently+in+Adult+and+Fetal+Ovine+Kidneys.">Chronic Hypobaric Hypoxia Modulates Primary Cilia Differently in Adult and Fetal Ovine Kidneys.</a> Frontiers in Physiology. 8, 677 (2017).</li><li>Malone, A. M. D., 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=Primary+cilia+mediate+mechanosensing+in+bone+cells+by+a+calcium-independent+mechanism.">Primary cilia mediate mechanosensing in bone cells by a calcium-independent mechanism.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (33), 13325-13330 (2007).</li><li>Luesma, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+neurons+show+a+primary+cilium.">Enteric neurons show a primary cilium.</a> Journal of Cellular and Molecular Medicine. 17 (1), 147-153 (2013).</li><li>Pugacheva, E. N., Jablonski, S. A., Hartman, T. R., Henske, E. P., Golemis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEF1-dependent+Aurora+A+activation+induces+disassembly+of+the+primary+cilium.">HEF1-dependent Aurora A activation induces disassembly of the primary cilium.</a> Cell. 129 (7), 1351-1363 (2007).</li><li>Li, 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=Ciliary+transition+zone+activation+of+phosphorylated+Tctex-1+controls+ciliary+resorption,+S-phase+entry+and+fate+of+neural+progenitors.">Ciliary transition zone activation of phosphorylated Tctex-1 controls ciliary resorption, S-phase entry and fate of neural progenitors.</a> Nature Cell Biology. 13 (4), 402-411 (2011).</li><li>Spalluto, C., Wilson, D. I., Hearn, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+reciliation+of+RPE1+cells+in+late+G1+phase,+and+ciliary+localisation+of+cyclin+B1.">Evidence for reciliation of RPE1 cells in late G1 phase, and ciliary localisation of cyclin B1.</a> FEBS Open Bio. 3, 334-340 (2013).</li><li>Malicki, J. J., Johnson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cilium:+Cellular+Antenna+and+Central+Processing+Unit.">The Cilium: Cellular Antenna and Central Processing Unit.</a> Trends in Cell Biology. 27 (2), 126-140 (2017).</li><li>Morleo, M., Franco, 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+Autophagy-Cilia+Axis:+An+Intricate+Relationship.">The Autophagy-Cilia Axis: An Intricate Relationship.</a> Cells. 8 (8), 905 (2019).</li><li>Ott, C., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+live+primary+cilia+dynamics+using+fluorescence+microscopy.">Visualization of live primary cilia dynamics using fluorescence microscopy.</a> Current Protocols in Cell Biology. , (2012).</li><li>Sun, S., Fisher, R. 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H., 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=Genome-wide+screen+identifies+novel+machineries+required+for+both+ciliogenesis+and+cell+cycle+arrest+upon+serum+starvation.">Genome-wide screen identifies novel machineries required for both ciliogenesis and cell cycle arrest upon serum starvation.</a> Biochimica et Biophysica Acta. 1863 (6), 1307-1318 (2016).</li><li>Yuan, K., 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=Primary+cilia+are+decreased+in+breast+cancer:+analysis+of+a+collection+of+human+breast+cancer+cell+lines+and+tissues.">Primary cilia are decreased in breast cancer: analysis of a collection of human breast cancer cell lines and tissues.</a> The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society. 58 (10), 857-870 (2010).</li><li>Smith, Q., 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=Differential+HDAC6+Activity+Modulates+Ciliogenesis+and+Subsequent+Mechanosensing+of+Endothelial+Cells+Derived+from+Pluripotent+Stem+Cells.">Differential HDAC6 Activity Modulates Ciliogenesis and Subsequent Mechanosensing of Endothelial Cells Derived from Pluripotent Stem Cells.</a> Cell Reports. 24 (4), 895-908 (2018).</li><li>Mirvis, M., Siemers, K. 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いくつかの,著者は、プライマリ繊毛の検出のための多様な方法を説明し、時には検出6、20、21、2220,に影響を与えることができる様々な固定方法も記述している。62122いずれにせよ、検出のための完全でわかりやすいプロトコルを見つけることは困難です。このような方法の準備ができたのは、特...

原性繊毛は、細胞の母中心に関連する感覚的、孤独な、膜結合性、非モタイル構造である。原発性繊毛は、赤血球、血液胞子1、および肝細胞2を除くほとんどの脊椎動物細胞に見られる。原繊毛は、細管によって構成される細長い軸索として形成され、その主成分はαチューブリンである。アキソンムは、γチューブリンから構成される基底体から成長する。プライマリ繊毛の長さは2~10μmの間で変化する。しかし、その寸法は、グリチル化、飢餓、低酸素、細胞毒性ストレス、または電離放射線33、4、5、6、74,5,6,7への暴露後に変化する可能性があります。通常、細胞は一次シリウムを1つしか持っていないが、これは形態形成および細胞シグナル伝達経路に関与し、細胞増殖および分化に重要な8,99である。
一次繊毛は、細胞周期進行中、特にG0/G1相中に動的に調節され、HDAC6(ヒストンデアセチルラーゼ6)10によって媒介される管内脱アセチル化に関連する過程で有糸分裂に入る前10にリソードされる。一次繊毛吸収の正確な瞬間は,、オーロラA、Plk1、TcTex-1 11、12、1311,など、このプロセスに直接関与する細胞の種類と遺伝子の発現に依存する。1213細胞の種類に応じて、プライマリ繊毛は異なるタイプの受容体、イオンチャネル、および活性シグナル伝達経路を発現する。これらには、増殖および生存に影響を及ぼす最も重要なシグナル伝達受容体、EGFR、PDGFR、およびFGFRが含まれる。また、ヘッジホッグ、ノッチ、Wntを含む1つ以上の器官の機能に影響を与える可能性のあるシグナル伝達経路のいくつかも含まれています。この機能により、原発性繊毛は、ノッチ、ホルモン、セロトニンまたはソマトスタチンなどの生物学的に活性な物質に対する特異的なリガンドを検出することができます。異なる長さの一次繊毛によって示される他の特定の機能は、温度、重力、および浸透病性14の変化に対する反応を含む。
一次繊毛は、ライブビジュアライゼーション、透過電子顕微鏡、3Dイメージング、または一,次繊毛5、15、16、1715の5自動検出のためのソフトウェアなど、さまざまな方法を通じて可視化することができる。,1617しかし、これらの方法は高度に専門化されており、研究のあらゆる段階で原型繊毛を染色するための基本的で迅速で簡単な方法が必要です。記載は、培養細胞における一次繊毛の検出に対する簡易で有用な方法である。

<table border="0" cellpadding="0" cellspacing="0" style="width:1301px;" width="1301">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">6-well plate</td>
			<td style="width:217px;">TPP</td>
			<td style="width:119px;">92406</td>
			<td style="width:581px;">Dimensions 128x86x22 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Alexa Fluor488</td>
			<td style="width:217px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">111-546-047</td>
			<td>AffiniPure F(ab&#39;)&#8322; Fragment Goat Anti-Rabbit IgG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Anti-Tubulin &#947;</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">T5192</td>
			<td>Polyclonal Rabbit anti-Mouse IgG2a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">C2C12</td>
			<td style="width:217px;">ATCC</td>
			<td style="width:119px;">CRL-1772</td>
			<td>Myoblast (mouse)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Cy3</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">C2181</td>
			<td>Anti-Mouse IgG (whole molecule) F(ab&#8242;)2 fragment&#8211;Cy3 antibody produced in sheep</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Dapi (4&#8242;,6-Diamidino-2-phenylindole dihydrochloride)</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Dulbecco&#180;s Modified Eagle&#180;s medium</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">11960044</td>
			<td>High glucose, No glutamine, Gibco</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:384px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8662</td>
			<td>With MgCl2 and CaCl2, Sterile-filtered, Suitable for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Fetal Bovine Serum</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">16000044</td>
			<td>Sterile-Filtered, Gibco</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">L-Glutamine</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">MEF</td>
			<td style="width:217px;">ATCC</td>
			<td style="width:119px;">SCRC-1039</td>
			<td>Mouse embryonic fibroblast</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Monoclonal Anti-Acetylated Tubulin</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">T7451</td>
			<td>Monoclonal Anti-Acetylated Tubulin antibody produced in mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">NHLF</td>
			<td style="width:217px;">Lonza</td>
			<td style="width:119px;">CC-2512</td>
			<td>Primary lung fibroblasts (human)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Normal Goat Serum</td>
			<td style="width:217px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">005-000-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Paraformaldehyde</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">158127-500G</td>
			<td>Powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Penicillin-Streptomycin</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">P0781</td>
			<td>10,000 units penicillin and 10 mg streptomycin per mL in 0.9% NaCl, Sterile-Filtered</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">ProLong Diamond Antifade Mountant</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">P36961</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:384px;">Skin fibroblasts</td>
			<td style="width:217px;">Kindly gifted from Charles University, Faculty of Medicine in Hradec Kr&#225;lov&#233;.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Square Cover Slips</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">22X22-1.5</td>
			<td>Borosilicate glass, 22x22mm, Square</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Triton X-100</td>
			<td style="width:217px;">Sigma-Aldrich</td>
			<td style="width:119px;">11332481001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">25200072</td>
			<td>Sterile-Filtered, Gibco</td>
		</tr>
	</tbody>
</table>

simple detection of primary cilia by immunofluorescence

一次繊毛は、細胞周期進行中、特に細胞周期のG0/G1段階の間に動的に調節され、有糸分裂の前に再調節される。一次繊毛は、透過型電子顕微鏡、3Dイメージング、または一次繊毛の自動検出のためのソフトウェアを使用するなど、高度に洗練された方法で視覚化することができます。しかしながら、これらの方法を行うためには、一次繊毛の免疫蛍光染色が必要である。本書では、アセチル化アルファチューブリン(axoneme)とガンマ管(基底体)を染色することにより、インビトロで一次繊毛を容易に検出するためのプロトコルについて説明する。この免疫蛍光染色プロトコルは比較的単純であり、高品質の画像をもたらします。本プロトコルは、原性繊毛を発現する4つの細胞株(C2C12、MEF、NHLF、および皮膚線維芽細胞)を、どのように固定し、免疫染色し、蛍光顕微鏡または共焦点顕微鏡で画像化した方法を記述する。

原繊毛の免疫蛍光染色は、高品質の画像をもたらす比較的簡単な手順です。これらの実験では、原発繊毛を発現する線維芽細胞を固定し、免疫染色し、上記のプロトコルに従って蛍光顕微鏡または共焦点顕微鏡で画像化した。一次シリウムはアセチル化αチューブリンおよびγチューブリンを用いて検出した。原繊毛の評価は、種々のレベルで行うことができるし、この点に関する任意の変化...

Watch this Scientific Journal Video about Simple Detection of Primary Cilia by Immunofluorescence at JoVE.com

Simple Detection of Primary Cilia by Immunofluorescence

一次繊毛は、中心に関連する細胞外構造である。免疫蛍光染色による一次繊毛検出は、非常に高品質な画像をもたらす比較的簡単な手順です。このプロトコルでは、原発繊毛を発現する線維芽細胞を固定し、免疫染色し、蛍光顕微鏡または共焦点顕微鏡で画像化した。

免疫蛍光による原発性繊毛の簡易検出

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to ...

このプロトコルは、様々な研究努力の中で、培養細胞内の原発繊の迅速かつ容易な検出を可能にする。この方法は、原発繊毛の基底体に影響を与える障害で利用することができる。この手順は、パラフィン埋め込み組織での原発繊毛の染色や、蛍光が必要な他の実験にも適用できます。22 x 22ミリメートルのカバースリップをオートクレーブし、実験用の材料を準備します。FBSおよびペニシリンストレプトマイシンを解凍し、培養培地を室温に温める。トリプシンEDTAおよびPBSで細胞を通過させる。原稿の指示に従って、新鮮な4%パラホルムアルデヒド、培養培地、および1%ゼラチン溶液を調製する。その後、70%エタノールで層流フードを洗浄し、すべての必要な材料を内部に入れます。パラホルムアルデヒドは非常に危険です。適切なPPEは常に着用する必要があり、それは化学フードでのみ処理する必要があります。所望の細胞を70%合流に成長した後、インキュベーターからそれらを取り除き、層流フードに入れます。培養液を取り出し、PBSで細胞を2回リンスします。培養フラスコに0.25%トリプシンEDTAの約2ミリリットルを加え、摂氏37度で5分間インキュベートします。反転顕微鏡で細胞を定期的にチェックして、剥離を監視します。細胞が10ミリリットルの培養培地で穏やかに再懸濁した場合、慎重にピペット処理して単一の細胞懸濁液を作り出す。細胞懸濁液を50ミリリットル円錐形チューブに移し、200倍G'5分間遠心分離します。上清をデカントし、10ミリリットルの培養培地を加え、ペレットを軽く再懸濁します。細胞懸濁液を20マイクロリットル取り、1対1の体積比でトリパンブルーと混ぜます。次に、標準的なヘモサイトメトリーを使用して細胞を数えます。6つのウェルプレートの各ウェルの中にカバースリップを置き、カバースリップに2ミリリットルのゼラチン溶液をコーティングします。ゼラチンを取り出し、プレートを数分間空気乾燥させます。種子100,000の線維芽細胞を各ウェルに入れ、2ミリリットルの培養培地を添加する。その後、セルを摂氏37度、二酸化炭素5%、相対湿度90%で24時間インキュベートします。培養後、毛様体化を誘導するために細胞を治療する。4%パラホルムアルデヒドを室温まで温め、牧草地のピペット、ABS、廃容器、15ミリリットルの円錐管、マイクロピペット、および先端を準備します。インキュベーターから細胞を取り出し、ラボベンチに置きます。カバースリップを残して、各井戸からメディアを取り外します。2ミリリットルのPBSで細胞を3回軽く洗います。その後、細胞を固定するために各ウェルに4%PFAの2ミリリットルを追加します。プレートを室温で10分間インキュベートし、PFAを取り出し、PBSで洗い洗いを繰り返します。各ウェルに0.5%トリトンX-100の2ミリリットルを加え、プレートを15分間インキュベートします。その後、PBSで井戸を4回洗います。ブロッキング溶液を作るために、ヤギの血清を5分間解凍し、PBSで1〜20の比率で希釈します。各カバースリップにこの溶液の150マイクロリットルを加え、20分間プレートをインキュベートします。一次抗体を5分間解凍し、テキスト原稿に記載されているようにPBSで別々に希釈する。カバースリップからブロッキング溶液を取り除き、両方の抗体溶液の150マイクロリットルを加えます。その後、プレートを室温で60分間インキュベートします。一次抗体を取り除き、カバースリップを2ミリリットルのPBSで3回軽く洗います。希釈Cy3羊抗マウスとアレクサフルーター488ヤギ抗ウサギをPBSで1〜300の比率で、カバースリップに両方の二次抗体溶液の150マイクロリットルを追加します。50ミリリットルのPBSで10マイクロリットルのストックを希釈して作業DAPI溶液を調製し、この希釈液をカバースリップに2ミリリットル加えます。室温で、暗闇の中で5分間プレートをインキュベートします。スライドにカバースリップを配置する前に、2本の針、ピンセット、取り付けメディアを準備し、すべてのスライドにラベルを付けます。ウェルからDAPI溶液を取り出し、PBSで3回洗浄します。各スライドに取り付けメディアを1滴置きます。針を使って、カバースリップをウェルの底からそっと持ち上げます。その後、それを反転し、ピンセットを使用して取り付けメディアのドロップの上にそっと置きます。気泡を慎重に取り除きます。スライドを光から保護し、摂氏4度で一晩保管してください。翌日には蛍光顕微鏡または共焦点顕微鏡を使用して、高倍率で一次繊毛を可視化します。このプロトコルは、免疫染色体を固定するために使用され、一次繊毛を発現する様々な細胞株を画像化した。マウス筋芽細胞が様々な線量で電離放射線にさらされたとき、高用量が複数の一次繊毛の出現を誘発することを発見した。低用量では、単一の一次繊毛の出現をもたらした.同様に、ヒト肺線維芽細胞を低用量で放射した場合、単一の原発繊毛は免疫蛍光によって検出された。飢えたマウス胚性線維芽細胞では原発繊の増加が観察され、代謝ストレスが原発性繊毛の事件に影響を及ぼすことが示された。皮膚線維芽細胞を120ナノモルドキソルビシンで処理したところ、単一の一次シリウムを発現した。高用量は、複数の一次繊毛の出現を誘発する。1.25ナノモルタキソールで処理された線維芽細胞も単一の一次シリウムを発現した。しかし、高用量で治療した後、複数の繊毛は検出されなかった。このプロトコルを開く際は、すべてのPPE規制に従ってください。選択した細胞ラインの培養ニーズを念頭に置き、抗体を慎重に選択してください。細胞は取り返しのつかないため、この手順を他の手順で直接実行することはできません。代わりに、並列実験をプログラムする必要があります。

simple-detection-of-primary-cilia-by-immunofluorescence

Research

JoVE Journal

Biology

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxy&#64258;uorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.

一次組織の二つのチミジン類似体の免疫蛍光検出（CldUとIDU）

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

生物学

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

ザイモグラフィーによる機能マトリックスメタロプロテイナーゼの検出

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

ELISAによるネイティブ無傷ヌクレオソーム上の翻訳後修飾の検出

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

蛍光によるウイルスRNAの検出<em&gt;その場で</em&gt;ハイブリダイゼーション（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Internal organs such as the heart, brain, and gut develop left-right (LR) asymmetries that are critical for their normal functions1. Motile cilia are involved in establishing LR asymmetry in vertebrate embryos, including mouse, frog, and zebrafish2-6. These 'LR cilia' generate asymmetric fluid flow that is necessary to trigger a conserved asymmetric Nodal (TGF-&beta; superfamily) signaling cascade in the left lateral plate mesoderm, which is thought to provide LR patterning information for developing organs7. Thus, to understand mechanisms underlying LR patterning, it is essential to identify genes that regulate the organization of LR ciliated cells, the motility and length of LR cilia and their ability to generate robust asymmetric flow.
In the zebrafish embryo, LR cilia are located in Kupffer's vesicle (KV)2,4,5. KV is comprised of a single layer of monociliated epithelial cells that enclose a fluid-filled lumen. Fate mapping has shown that KV is derived from a group of ~20-30 cells known as dorsal forerunner cells (DFCs) that migrate at the dorsal blastoderm margin during epiboly stages8,9. During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo10,11. The ability to identify and track DFCs&mdash;in combination with optical transparency and rapid development of the zebrafish embryo&mdash;make zebrafish KV an excellent model system to study LR ciliated cells.
Interestingly, progenitors of the DFC/KV cell lineage retain cytoplasmic bridges between the yolk cell up to 4 hr post-fertilization (hpf), whereas cytoplasmic bridges between the yolk cell and other embryonic cells close after 2 hpf8. Taking advantage of these cytoplasmic bridges, we developed a stage-specific injection strategy to deliver morpholino oligonucleotides (MO) exclusively to DFCs and knockdown the function of a targeted gene in these cells12. This technique creates chimeric embryos in which gene function is knocked down in the DFC/KV lineage developing in the context of a wild-type embryo. To analyze asymmetric fluid flow in KV, we inject fluorescent microbeads into the KV lumen and record bead movement using videomicroscopy2. Fluid flow is easily visualized and can be quantified by tracking bead displacement over time.
Here, using the stage-specific DFC-targeted gene knockdown technique and injection of fluorescent microbeads into KV to visualize flow, we present a protocol that provides an effective approach to characterize the role of a particular gene during KV development and function.

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer&#039;s Vesicle

Cilia-generated fluid flow in Kupffer&#x2019;s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.

クッパー胞における遺伝子機能と繊毛発生する流体の流れの可視化の分析

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

ライブの検出<em&gt;エシェリヒア·コリ</em&gt; O157：PMA-qPCRによりH7細胞

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

活性化カスパーゼ3を検出するために、ホールマウント免疫蛍光法によりゼブラフィッシュ胚におけるアポトーシスの分析

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糞便グルココルチコイド分析：ウマにおける非侵襲性副腎モニタリング

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

共焦点顕微鏡により、リガンド活性化Gタンパク質共役型受容体内在化の検出

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...