Anyaporn SawasdichaiとNazefahアブドゥルハミドは、博士号のためにそれぞれタイ王国政府とマレーシア政府に感謝しています奨学金。この作品は、PSJとKGに授与ウェルカムトラストのプロジェクトの助成金によって賄われていた。我々はまた、ブリストルウォルフソンのバイオイメージング施設の大学に感謝しています。

分別のためのI.準備このセクションでは、ポリ- L -リジンコートした顕微鏡のカバーガラスと分画の前に細胞の添付ファイルの準備について説明します。必要に応じて細胞は一過性に付着前または後に、タンパク質の発現ベクターをトランスフェクトすることができます。 ポリ- L -リジンコートしたカバースリップのA.の準備<ol><li>蒸留水で1mg/mlポリ- L -リジンの溶液を調製します。 </li><li> 22にロッキングプラットフォーム上で少なくとも1時間溶液中でそれらをインキュベートすることによりポリ- L -リジンでコーティングクリーンカバースリップ℃に</li><li>二回滅菌蒸留水でコーティングされたカバーガラスを洗浄し、96％エタノールを持つ単一の洗浄に従ってください。 </li><li>空気はフィルター紙の上にコーティングされたカバーガラスを乾燥させ、将来の使用のためのドライコンテナ（一度乾燥、それらを積層することができます）に保管してください。 </li></ol>カバーガラスにB.添付細胞非接着細胞（ここでは、K562細胞を使用） <ol><li>上向きにコーティングされた表面を有する6ウェルプレートのウェル中のポリ- L -リジンコートしたカバースリップを配置。 </li><li> /よくダルベッコ変法イーグル10％ウシ胎児血清（FBS）を添加した培地（DMEM）、およびPS（ペニシリン100units/ml、ストレプトマイシン100mg/ml）の7 × 10 5細胞の密度でシードK562細胞。 K562細胞の場合には一過性トランスフェクションは、細胞を播種する前に、エレクトロポレーション（250V /975μFでのメディアの200μlの中の1 × 10 7細胞と0.4センチメートルエレクトロポレーションキュベット）を使用して実行することができます。 </li><li> 37℃、5％のC CO 2で24時間細胞をインキュベートする。 </li><li>培地を捨て、生理食塩水（PBS）バッファリングされた氷冷リン酸で細胞を2回洗浄する。 </li><li>細胞下分画プロトコルで従ってください。 </li></ol>接着細胞（ここでは、Cos - 7細胞を使用） <ol><li> 6ウェルプレートのウェルにコーティングされていないカバースリップを配置。 </li><li> PBSで2回洗浄は37℃で温した</li><li> 37℃でPBS中のトリプシン（0.03％EDTA、0.25％トリプシン）で消化して細胞を剥離℃で2〜5分間。細胞を過剰消化するために、主に個々の浮遊細胞が存在するとすぐに消化を停止しないことが重要です。 </li><li> /ウェル、10％FBSとPSG（ペニシリン100単位/ ml、ストレプトマイシン100ug/mlとL -グルタミン292ug/ml）を添加したDMEMで6穴プレートに3 × 10 5細胞の密度で細胞を播種による消化を停止する、通常のカバースリップ上で。接着細胞は、一般的に必要に応じしかし、ポリ- L -リジンコートしたカバースリップを使用することができる、通常の顕微鏡のカバースリップの上にも添付する。 </li><li> 37℃、5％CO 2で24時間細胞をインキュベートする。の場合Cos - 7細胞で一過性トランスフェクションは、トランスフェ6（Roche社）であれば、必要なと37℃でさらに24時間℃、5％のCO 2を回収するために左のセルを用いて行うことができる。 </li><li>培地を捨て、氷冷したPBSで細胞を2回洗浄する。 </li><li>細胞下分画プロトコルで従ってください。 </li></ol> II。細胞下分画 その場分数で回路図を図1に示すように、と変更以前に7と以下の詳細なプロトコールに記載してこれまで5に記載の方法に基づいているに応じて実行されます。それは、液体を除去する前にカバーを取り外し、各分画ステップの後、新鮮な6ウェルプレートにカバースリップを転送することをお勧めします。わずか4カバースリップが撮像が必要であるが、追加のカバースリップは、これは並列に実行する場合は、ウェスタンブロッティングのための全細胞抽出液と核マトリックスの分画を生成するために必要とされています。 <ol><li> 10mMのPIPES、300mmのスクロース、100mMの塩化ナトリウム、3mmののMgCl 2、1mMのEGTA：細胞骨格のバッファ（CSK）を準備し、フィルタ。 CSKバッファーは、新鮮な分別の日に準備する必要があります。 </li><li>必要な場合は、1つのカバースリップから細胞を穏やかに直接カバーガラスの上に200ulのTESバッファー（1％SDS、2mMのEDTA、20mMのトリス- HCl pH7.4）を配置し、1〜2分間インキュベートすることにより、ウェスタンブロッティングのために全細胞抽出液を調製するために使用することができます。 22℃全細胞抽出液を除去し、タンパク質は、1Mの250ul（NH 4）2 SO 4を添加すると、他の西部のサンプルが準備が整うまで氷上でインキュベートすることにより、沈殿させることができる。 </li><li> 22℃傾けて6ウェルプレートに2〜3回で二回1ミリリットルの氷冷PBSで残りのカバーグラスを洗ってください。 </li><li>細胞全体と新鮮な6 - ウェルプレートへの転送を表すカバーを取り外します。細胞の固定と免疫蛍光プロトコルに従ってください。 </li><li>静かに残りのウェルからPBSを取り除く。 </li><li> + 0.1％（V / V）トリトンX - 100は、それぞれの残りのカバーグラス上に直接静かに200ulのCSKのバッファを配置することにより、細胞質と緩く保有核タンパク質を抽出し、上でインキュベートする1分間氷。 </li><li>細胞質を削除し、緩く、ステップ2で説明した核抽出液と沈殿物を開催しました。 </li><li> 22で二回1ミリリットルの氷冷PBSでカバーグラスを洗う° Cを傾け6ウェルプレートで。 </li><li>しっかりと開催された核物質を表すカバーを取り外して、細胞の固定と免疫蛍光プロトコルに従ってください。 </li><li>優しく+ 0.5％（V / V）トリトンX - 100は、残りの各カバースリップ上に直接優しく200ul CSKのバッファを配置することによって、しっかりと保持しタンパク質を抽出し、20分間氷上でインキュベート後、残りのウェルからPBSを取り除く。 </li><li>しっかりと開催されたエキスを取り出して、ステップ2で説明したように沈殿する。 </li><li> 22で二回1ミリリットルの氷冷PBSでカバーグラスを洗う° Cを傾け6ウェルプレートで。 </li><li>クロマチン画分を表すカバーを取り外して、細胞の固定と免疫蛍光プロトコルに従ってください。 </li><li>そっとして場所200ul CSKバッファー+ 100ug/ml DNase Iの30分の残りのカバーガラスとインキュベート上で37℃、残りのウェルからPBSを削除する</li><li>クロマチンエキスを削除し、手順2で説明したように沈殿する。 </li><li> ° C傾け6ウェルプレート22に回氷冷PBSでカバーグラスを洗ってください。 </li><li>核マトリックス画分を表すカバーを取り外して、細胞の固定と免疫蛍光プロトコルに従ってください。 </li><li>必要に応じてマトリックスタンパク質は、ステップ2で説明したように200ulのTES緩衝液を用いてウェスタンブロットのための追加的な行列のカバースリップから削除することができます。 </li><li>ウェスタンブロット用サンプルは4℃ベンチトップ遠心機で13000rpmで遠心分離されるべき℃、40ul SDSローディングバッファー（62.5mMトリス- HCl、pH6.8、2％SDS（w / v）の、50mMのDTT、中に再懸濁したペレットSDS - PAGEによる分析の前に10％グリセロール、0.01％ブロモフェノールブルー（W / V））。 <img src="/files/ftp_upload/1958/1958fig1.jpg" alt="図1" /> 図1。回路図上皮細胞下分画での表現。細胞骨格のバッファ（CSK）は、10mMのPIPES、300mmのスクロース、100mMの塩化ナトリウム、3mmのMgCl 2を 、1mMのEGTAで構成されています。 TESバッファーは、1％SDS、2mMのEDTA、20mMのトリス- HCl pH7.4の構成されています。 </li></ol> III。細胞の固定と免疫蛍光<ol><li>ゆっくりと30分間、4℃でそれぞれカバーしてインキュベート° Cに4％ホルムアルデヒド/ PBSを1mlを加える。 </li><li>各カバースリップ22のPBSを1mlと軽く2〜3回℃を洗う</li><li> 10分間22℃でそれぞれカバーしてインキュベートして、0.1％トリトンX-100/PBSの400ulを追加。 </li><li>各カバースリップ22のPBSを1mlと軽く2〜3回℃を洗う</li><li>潜在的なバックグラウンド染色を低減するために20分間22℃で3％ウシ血清アルブミン（BSA）/ PBSとインキュベートの400ulを追加。 </li><li>各カバースリップ22のPBSを1mlと軽く2〜3回℃を洗う</li><li>直接1時間22℃でカバースリップとインキュベート上にPBSで一次抗体の代わりに100ul。一次および二次抗体は、所望の濃度にPBSで希釈してください。これは、使用される各抗体のために最適化する必要があります。 </li><li>各カバースリップ22のPBSを1mlと軽く2〜3回℃を洗う</li><li> 22のPBSとインキュベートにおける二次抗体の代わりに200ul℃で1時間離れて軽いからC。 </li><li>各カバースリップ22のPBSを1mlと軽く2〜3回℃を洗う</li><li> DAPI（4&#39;,6 -ジアミジノ-2 - フェニル塩酸塩）を含むVectashieldマウンティング培地を用いてガラスの顕微鏡スライド上に各カバースリップを（細胞がダウンして）マウントする。 </li><li>ペーパータオルを使用してカバースリップの周りから余分な封入剤をふき取り、少なくとも30分の光を避け、22℃でスライドをインキュベートする。 </li><li>透明なアクリルのマニキュアを使ってガラススライドにカバースリップを修正。 </li><li>蛍光顕微鏡による可視化する前に離れて光からスライドしてください。 </li></ol> IV。代表的な結果<img src="/files/ftp_upload/1958/1958fig2.jpg" alt="図2" /> 図2。非トランスフェクトしたCOS - 7細胞の細胞下分画。分別したCos - 7細胞をホルムアルデヒドで固定し、DAPIを含む封入剤で処理しTRITCに共役/ C抗体α-チューブリン、α-ヒストンH1またはα-ラミンを使用して免疫染色を行った。画像は、10倍接眼レンズとライカ共焦点顕微鏡を使用して63x対物レンズで取得した。細胞核は、マーカー抗体で染色した同じ分野の細胞中にDAPIフィルターセットを用いて可視化したTRITCフィルターセットを用いて可視化した。 <img src="/files/ftp_upload/1958/1958fig3.jpg" alt="図3" /> 図3。 GFP - 6E2融合タンパク質は核マトリックスに関連付けられています。ヒトパピローマウイルス（HPV）タイプ6に融合された緑色蛍光タンパク質（GFP）で構成される融合タンパク質を発現しているCOS - 7細胞の細胞下分画E2タンパク質（GFP - 6E2）。トランスフェクトされた細胞におけるGFP - 6E2の局在は、GFP蛍光を経由して直接視覚化された一方細胞核はDAPI染色を介して可視化した。ラミンA / Cは、α-ラミンTRITCおよびTRITCフィルターセットに結合したA / C抗体を用いて可視化した。マージのイメージは、GFP - 6E2タンパク質の一部は核マトリックス（右下のパネル）に関連付けられていることを明らかにする。図2で説明したように画像が得られた。

<ol>
	<li>Donaldson, M. M., Boner, W., Morgan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopBP1+regulates+human+papillomavirus+type+16+E2+interaction+with+chromatin.">TopBP1 regulates human papillomavirus type 16 E2 interaction with chromatin.</a> J Virol. 81, 4338-4342 (2007).</li><li>Javed, A., Guo, B., Hiebert, S., Choi, J. Y., Green, J., Zhao, S. C., Osborne, M. A., Stifani, S., Stein, J. L., Lian, J. B., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Groucho/TLE/R-esp+proteins+associate+with+the+nuclear+matrix+and+repress+RUNX+(CBF(alpha)/AML/PEBP2(alpha))+dependent+activation+of+tissue-specific+gene+transcription.">Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBF(alpha)/AML/PEBP2(alpha)) dependent activation of tissue-specific gene transcription.</a> J Cell Sci. 113, 2221-2231 (2000).</li><li>Kowalczyk, A. M., Roeder, G. E., Green, K., Stephens, D. J., Gaston, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+induction+or+inhibition+of+apoptosis+by+HPV+proteins.">Measuring the induction or inhibition of apoptosis by HPV proteins.</a> Methods Mol. Med. 119, 419-432 (2005).</li><li>McLarren, K. W., Theriault, F. M., Stifani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+with+the+nuclear+matrix+and+interaction+with+Groucho+and+RUNX+proteins+regulate+the+transcription+repression+activity+of+the+basic+helix+loop+helix+factor+Hes1.">Association with the nuclear matrix and interaction with Groucho and RUNX proteins regulate the transcription repression activity of the basic helix loop helix factor Hes1.</a> J Biol. Chem. 276, 1578-1584 (2001).</li><li>McNeil, S., Guo, B., Stein, J. L., Lian, J. B., Bushmeyer, S., Seto, E., Atchison, M. L., Penman, S., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+the+YY1+transcription+factor+to+the+nucleolus+and+the+nuclear+matrix+in+situ:+the+C-terminus+is+a+principal+determinant+for+nuclear+trafficking.">Targeting of the YY1 transcription factor to the nucleolus and the nuclear matrix in situ: the C-terminus is a principal determinant for nuclear trafficking.</a> J Cell Biochem. 68, 500-510 (1998).</li><li>Melan, M. A., Sluder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redistribution+and+differential+extraction+of+soluble+proteins+in+permeabilized+cultured+cells.+Implications+for+immunofluorescence+microscopy.">Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy.</a> J Cell Sci. 101, 731-743 (1992).</li><li>Zou, N., Lin, B. Y., Duan, F., Lee, K. Y., Jin, G., Guan, R., Yao, G., Lefkowitz, E. J., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hinge+of+the+human+papillomavirus+type+11+E2+protein+contains+major+determinants+for+nuclear+localization+and+nuclear+matrix+association.">The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association.</a> J. Virol. 74, 3761-3770 (2000).</li></ol>

利害の衝突は宣言されません。

一般的な問題や提案： すべてまたは細胞のほとんどは、洗浄中に失われます。洗浄ステップ中に液体がカバースリップを避ける6ウェルプレートの側に徐々に追加する必要があります。同様に液体を慎重に傾けプレートによって除去されるべきであると徐々に余分な水分をオフにピペッティング。細胞接着は、ポリ- L -リジンコートしたカバースリップを使用して増やすこ?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>BSA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>A9647-100G</td>
<td>&nbsp;</td>
<tr>
<td>DNase I</td>
<td>Chemical</td>
<td>Sigma</td>
<td>DN25-1G</td>
<td>&nbsp;</td>
<tr>
<td>poly-L-Lysine</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-8920</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>Chemical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EGTA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>E4378-25G </td>
<td>&nbsp;</td>
<tr>
<td>Fomaldehyde</td>
<td>Chemical</td>
<td>BDH</td>
<td>284216N</td>
<td>&nbsp;</td>
<tr>
<td>PIPES</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-6757</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>Chemical</td>
<td>Fisher Scientific</td>
<td>S/8600/53</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Chemical</td>
<td>Sigma</td>
<td>T-6876</td>
<td>&nbsp;</td>
<tr>
<td>Mounting Medium with DAPI</td>
<td>Chemical</td>
<td>Vector Laboratories</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Fugene 6 Transfection Reagent</td>
<td>Chemical</td>
<td>Roche </td>
<td>11814443001</td>
<td>&nbsp;</td>
<tr>
<td>Histone H1</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-8030</td>
<td>&nbsp;</td>
<tr>
<td>Lamin A/C</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-20681</td>
<td>&nbsp;</td>
<tr>
<td>Tubulin-&alpha;</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-32293</td>
<td>&nbsp;</td>		</tbody>
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in situ subcellular fractionation of adherent and non-adherent mammalian cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

Watch this Scientific Journal Video about In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells at JoVE.com

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

顕微鏡のカバーグラス上に、哺乳動物細胞の in situ細胞下分画ではタンパク質の局在を可視化できる。

接着および非接着性の哺乳動物細胞の in situ細胞下分画で

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

in-situ-subcellular-fractionation-adherent-non-adherent-mammalian

Research

JoVE Journal

Biology

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

15.7K ビュー.  University of Bristol. 顕微鏡のカバーグラス上に、哺乳動物細胞の in situ細胞下分画ではタンパク質の局在を可視化できる。

ビデオ: In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

哺乳動物細胞をトリプシン処理し、継代培養

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

生物学

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

哺乳動物細胞におけるRNAiスクリーニングのためのミッションesiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

異種哺乳動物細胞で発現したイオンチャネルの変異導入と機能解析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

クローン原性アッセイ：付着細胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

哺乳動物細胞における細胞周期の位置の解析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

哺乳動物細胞における小胞体ローカライズされたmRNAの可視化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

ゲノム規模で哺乳類Translatomesのソーム分画および分析

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

哺乳動物細胞におけるヘム合成レベルの測定

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

イメージングローカルCA<sup&gt; 2+</sup&gt;培養哺乳動物細胞内シグナル

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

動的細胞突起におけるソフトウェア支援トラッキングのためのグラフィカルユーザインタフェース

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...