研究は、NIH 1R15GM094770によって資金を供給された。

1。コロニーの単離と固定<ol><li>指定された時間のために寒天培地に300コロニーをインキュベートする（孤立コロニーは直径に1-2 mmにしてください）​​。 </li><li>コロニー（顔のアップ）と狭いヘラを使用して、基礎となる培地を削除します。 </li><li> 2％寒天、42℃、それが固化する前に、寒天の表面に1 mLのpipetmanの先端と、すぐに場所のコロニーを用いて顕微鏡スライド上にCを数滴を置きます。 </li><li> 42 ° Cコロニーに2％の寒天を数滴を配置し、固化することができます。 </li><li>このプロトコルの残りの部分を通じてすべてのステップのための手袋を着用してください</li><li>かみそりの刃を持つブロックをトリムし、4℃で7日間、2％パラホルムアルデヒド/ 2％グルタルアルデヒド固定液を含む3.5mLのホウケイ酸塩のスクリューキャップ付きバイアルの中に置いてください</li></ol> 2。洗浄およびオスミウム処理<ol><li>約1週間後、完全に同じボリュームで5分間、その後、植民地（約1.5mL）をさらに2回をカバーするのに十分な0.15Mカコジル酸ナトリウム（pH7.2）で15分間二回インキュベートすることにより、化学煙フードの下に氷の上の寒天ブロックを洗う1X OSバッファー（100mMのKH 2 PO 4 10mMのMgCl 2、pH6.0）での。 </li><li> [2 mLのOSO 4（2％）+ 2 mLの2倍のOSのバッファ] 1％の同じ体積OSO の追加4バイアル（氷上で）に1時間化学煙フード下の寒天ブロックをカバーするために、同じ金額で2回洗浄10分ごとに1X OSバッファの。 </li><li> 4℃バイアルは一晩おきます° 1X OSのバッファでC。 </li><li>固定し、このステップですべての洗浄は、危険廃棄物として廃棄されています。 OSO 4を含むすべてのピペットは、植物油と3Xすすいだ。 </li></ol> 3。洗浄と脱水<ol><li>翌朝は、パスツールピペットを用いて、10分ごとに冷たい水の上と同じボリュームで氷の上で二回寒天ブロックを洗う。 </li><li> 25％の同じ体積で順次洗浄、10分ごとに50％、75％、95％エタノールで10分間100％エタノールで2回洗浄した。 4℃で一晩おきます〜100％エタノールでCを。 </li><li>このステップのすべての洗浄は、危険廃棄物として廃棄されています。 </li></ol> 4。スパーの準備<ol><li>次の朝は、（可能な限り早期）スパーの試薬を準備するヒュームフードの下で使い捨てのプラスチック製ビーカーに入れ、以下のソリューションを（EMサイエンス）重量を量る。この試薬を使用する後続のすべての作業では、ヒュームフードを使用し続ける。 （ERL 4221溶液5グラム） （DER 736溶液4グラム） （NSAの溶液13グラム） </li><li>これらのソリューションは、（ゆっくり）攪拌棒を使用してフードで20分間混ぜる</li><li> DMAEのソリューションを0.15グラムを追加し、上記のような20分をかき混ぜる。 </li><li>フード内の1〜2時間のためのドガ上記の混合物。 </li></ol> 5。スパーの浸透<ol><li>スパーのが上記の10分ごとに100％の室温のエタノールのと同じボリュームで寒天ブロックを5回洗浄なされるとすぐに。最後にエタノールで洗浄後に残っているエタノールは、ちょうど寒天ブロックを覆うように、パスツールピペットを用いてエタノールを除去します。 </li><li>約スパーの試薬（約0.5mL）を同量を追加し、30分間放置するし、室温で15分間ホイール上にバイアルを回転させると。 30分は完全にパスツールピペットを用いて溶液を除去した後、追加スパーは、寒天ブロック（約1.5 mL）をカバーするもの、室温で15分間ホイールにバイアルを回転させ、30分間放置する。スパーの交換、回転、およびインキュベーションさらに3回繰り返します。 </li><li>最後の30分後、スパーの試薬を除去し、ブロックをカバーするために新鮮なスパーのを追加します。寒天ブロックは4時間室温で放置する。 </li><li>スパーのを交換し、一晩回転する。 </li><li>朝にはスパーのを交換し、翌日になるまで回転しておきます。 </li><li>単に金型の底をカバーするために番号のシリコンモールドで翌朝の場所のスパーの（フィッシャーサイエンティフィック）は、その後、金型に寒天ブロックを追加します。 4時間60℃でインキュベートする</li><li>より多くのスパーのインキュベート、60℃で一晩でカビオフ上部後4時間、、。 </li><li>金型からブロックを削除した後のブロックはまだわずかに柔軟である場合、金型に戻り、追加の2日間インキュベートする</li></ol> 6。セクショニング<ol><li>ライカUltracut Sミクロトームは、光学顕微鏡では0.5μ厚の切片を0.1μ伝送EM用薄切片を切断するために使用された。 </li><li>セクションがカットされたとして、光学顕微鏡の場合は、、それらはのdH 2 Oのドロップで配置され、そして52℃のヒートブロック上で乾燥。乾燥のセクションは、1.0パーセントトルイジンブルー、5-15秒1％ホウ酸ナトリウムの滴で染色した。染色後、切片をすぐに、水の流れの下で洗浄し、乾燥し、マウントメディア（KPL）に覆われており、カバースリップは、試料上密封した。 </li></ol>酵母や他の微生物のパターン形成を測定するの広範な関連性は、以前は1日している。特に注目すべきは、無秩序な社会への相対的な組織的な微生物のコロニーの増加機能があります。 記載されている方法は大きく専門的なコロニー2を埋め込 ​​むための方法の変更であり、そして私達の変更の簡単な説明は、3を発表されている。 野生S.の植民地の中心を通ってセクションの光顕微鏡写真の例出芽酵母の酵母のコロニーを図1に示されています。 ASCI、pseudohyphaeと卵酵母がわかりやすい、基礎となる寒天を侵略植民地の地域にも明らかである。 酵母の実験室株（W303背景）の電子顕微鏡写真の例を図2に示されています。画像は、胞子形成細胞の高周波を含むコロニーの地域からです。 <img src="/files/ftp_upload/2510/2510fig1.jpg" alt="図1" /> 図1。野生酵母のコロニーの光学顕微鏡。この酵母（YPS133）は、ツリーの滲出液4から最近単離された。コロニーは、前の切片にYNA媒体5の6日間インキュベートした。オープン矢印は、塗りつぶされた矢印は、細長い細胞のチェーン（pseudohyphae）とASCIを示す、塗りつぶされた矢印は代表的な卵形の栄養細胞を示す、代表的なASCIを示している。 <img src="/files/ftp_upload/2510/2510fig2.jpg" alt="図2" /> 図2実験室酵母株の電子顕微鏡。 SH1020（W303背景）のコロニーが3切片の前にYNAの培地上で6日間インキュベートした。画像は、ASCIの高い周波数を持つセクションの領域を示しています。矢印は胞子壁の二層構造を示している。 <img src="/files/ftp_upload/2510/2510fig3.jpg" alt="図3" /> 図3。コロニーにおける胞子形成細胞の分布の定量化：）その長辺に沿って積み重ね15に隣接する矩形を含むグリッドは、コロニーのセクションの画像の中央部に重畳する。その比率を一定に保ちながらグリッドは単に植民地の上部と下部をカバーするために、スケーリングされます。 （B＆C）その長方形の全細胞に対する各長方形の胞子形成細胞の割合は、4日齢（B）、8日齢（C）コロニーからの画像の目視検査によって決定されます。例えば、植民地の上部にある長方形は（&quot;1&quot;とラベルされた）のグラフの左側に対応しています。 4コロニーの平均値が表示され、エラーバーはSTDを表示します。エラー。

<ol>
	<li>Kimmel, A. R., Firtel, 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=Breaking+symmetries:+regulation+of+Dictyostelium+development+through+chemoattractant+and+morphogen+signal-response.">Breaking symmetries: regulation of Dictyostelium development through chemoattractant and morphogen signal-response.</a> Curr Opin Genet Dev. 14, 540-540 (2004).</li><li>Palkova, Z., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+within+a+community:+benefit+to+yeast+long-term+survival.">Life within a community: benefit to yeast long-term survival.</a> FEMS Microbiol Rev. 30, 806-806 (2006).</li><li>Shapiro, J., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significances+of+bacterial+colony+patterns.">The significances of bacterial colony patterns.</a> Bioessays. 17, 597-597 (1995).</li><li>Shapiro, J., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinking+about+bacterial+populations+as+multicellular+organisms.">Thinking about bacterial populations as multicellular organisms.</a> Annu Rev Microbiol. 52, 81-81 (1998).</li><li>Engelberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+stalk-like+structures+in+Saccharomyces+cerevisiae.">Multicellular stalk-like structures in Saccharomyces cerevisiae.</a> J Bacteriol. 180, 3992-3992 (1998).</li><li>Scherz, R., Shinder, V., Engelberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+analysis+of+Saccharomyces+cerevisiae+stalk-like+structures+reveals+spatial+organization+and+cell+specialization.">Anatomical analysis of Saccharomyces cerevisiae stalk-like structures reveals spatial organization and cell specialization.</a> J Bacteriol. 183, 5402-5402 (2001).</li><li>Piccirillo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rim101p/PacC+pathway+and+alkaline+pH+regulate+pattern+formation+in+yeast+colonies.">The Rim101p/PacC pathway and alkaline pH regulate pattern formation in yeast colonies.</a> Genetics. 184, 707-707 (2010).</li><li>Sniegowski, P. D., Dombrowski, P. G., Fingerman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+cerevisiae+and+Saccharomyces+paradoxus+coexist+in+a+natural+woodland+site+in+North+America+and+display+different+levels+of+reproductive+isolation+from+European+conspecifics.">Saccharomyces cerevisiae and Saccharomyces paradoxus coexist in a natural woodland site in North America and display different levels of reproductive isolation from European conspecifics.</a> FEMS Yeast Res. 1, 299-299 (2002).</li><li>Piccirillo, S., Honigberg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporulation+patterning+and+invasive+growth+in+wild+and+domesticated+yeast+colonies.">Sporulation patterning and invasive growth in wild and domesticated yeast colonies.</a> Res Microbiol. 161, 390-390 (2002).</li><li>Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+developing+multicellular+yeast+colony:+spatio-temporal+expression+of+Ato1p+ammonium+exporter.">Architecture of developing multicellular yeast colony: spatio-temporal expression of Ato1p ammonium exporter.</a> Environ Microbiol. , (2009).</li></ol>

利害の衝突は宣言されません。

提示方法は、コロニーの内部構造を明らかにする。方法は、Sの範囲内のセルのタイプのパターンを決定する上で効果的ですので、別のコロニーの形態を持つ、とも近縁種S. paradoxus 5の出芽酵母株は 、この方法はまた、真菌や他の微生物の広い範囲で動作する可能性があります。 メソッドの成功のための一つの重要なステップは、コロニーの?...

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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>
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<tr>
<td>Osmium tetroxide</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 19152</td>
<td>&nbsp;</td>
<tr>
<td>Silicone embedding molds</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>NC 9975029</td>
<td>&nbsp;</td>
<tr>
<td>Cycloaliphatic epoxide resin</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 15004</td>
<td>ERL 4221</td>
</tr>
<tr>
<td>Epoxy resin</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 13000</td>
<td>DER 736</td>
</tr>
<tr>
<td>Nonenyl Succinic Anhydride</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 19050</td>
<td>NSA</td>
</tr>
<tr>
<td>2-Dimethyl aminoethanol	</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 13300</td>
<td>DMAE</td>
</tr>
<tr>
<td>Mounting media</td>
<td>&nbsp;</td>
<td>KPL</td>
<td>71-00-16</td>
<td>&nbsp;</td>
<tr>
<td>Rotating wheel</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>Pelco 1055</td>
<td>&nbsp;</td>
<tr>
<td>Microtome</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>Ultracut S</td>
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yeast colony embedding method

胚の異なる細胞型のパターニングは、後生動物の発生において重要なメカニズムです。このようなコロニーやバイオフィルムのような微生物のコミュニティは、また、細胞の種類のパターンが表示されます。例えば、酵母のS.出芽酵母は 、胞子形成細胞と偽菌糸細胞が一様にコロニーで配布されていません。パターニングとこれらのパターンの根底にある分子機構の機能的重要性はまだよく理解されています。 真菌コロニーの細胞型のパターンを調査に関して、1つの課題は、後生動物の組織とは異なり、コロニー内の細胞は比較的弱く、お互いに接続されていることです。特に、真菌のコロニーはほとんどの組織に見られる細胞外マトリックスの同じ豊富なレベルが含まれていません。ここでは、これらのコロニーの細胞型の内部のパターンを明らかに酵母のコロニーを埋め込むとセクショニングの方法で報告する。方法は、光学顕微鏡および透過型電子顕微鏡に適した薄切片（0.1μ）のために有用（0.5μ）厚い切片を作製するために使用することができます。これらの細胞の内部構造はEMによって可視化することができますがASCIと偽菌糸の細胞が容易に、光学顕微鏡で卵形の酵母の細胞と区別することができます。 メソッドは、寒天でコロニーを囲むスパーの培地でそれらを浸潤し、切片に基づいています。 1〜2ミリメートルの範囲の直径を持つコロニーは、このプロトコルに適しています。コロニーの内部を可視化することに加えて、メソッドは基になる寒天に侵入するコロニーの領域を可視化できる。

Watch this Scientific Journal Video about Yeast Colony Embedding Method at JoVE.com

Yeast Colony Embedding Method

光および電子顕微鏡用切片可能に酵母のコロニーを埋め込むための方法。このプロトコルは、真菌、コミュニティ内での細胞型の組織を理解するための新しいツールを提供してコロ​​ニー内に胞子形成細胞と偽菌糸細胞の分布を決定することができます。

酵母のコロニーの埋め込み法

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Research

JoVE Journal

Biology

11.4K ビュー.  University of Missouri - Kansas City. 光および電子顕微鏡用切片可能に酵母のコロニーを埋め込むための方法。このプロトコルは、真菌、コミュニティ内での細胞型の組織を理解するための新しいツールを提供してコロ​​ニー内に胞子形成細胞と偽菌糸細胞の分布を決定することができます。

ビデオ: Yeast Colony Embedding Method

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

プラスチック製の埋め込みとアフリカツメガエル胚のセクショニング

生物学

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

出芽酵母の複製寿命の測定

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

酵母細胞からミトコンドリアの精製

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

ハイスループット酵母プラスミドの過剰発現の画面

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母のリボソームのクロマトグラフィー精製

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Microbes typically live in communities. The spatial organization of cells within a community is believed to impact the survival and function of the community1. Optical sectioning techniques, including confocal and two-photon microscopy, have proven useful for observing spatial organization of bacterial and archaeal communities2,3. A combination of confocal imaging and physical sectioning of yeast colonies has revealed internal organization of cells4. However, direct optical sectioning using confocal or two-photon microscopy has been only able to reach a few cell layers deep into yeast colonies. This limitation is likely because of strong scattering of light from yeast cells4.
Here, we present a method based on fixing and cryosectioning to obtain spatial distribution of fluorescent cells within Saccharomyces cerevisiae communities. We use methanol as the fixative agent to preserve the spatial distribution of cells. Fixed communities are infiltrated with OCT compound, frozen, and cryosectioned in a cryostat. Fluorescence imaging of the sections reveals the internal organization of fluorescent cells within the community.
Examples of yeast communities consisting of strains expressing red and green fluorescent proteins demonstrate the potentials of the cryosectioning method to reveal the spatial distribution of fluorescent cells as well as that of gene expression within yeast colonies2,3. Even though our focus has been on Saccharomyces cerevisiae communities, the same method can potentially be applied to examine other microbial communities.

We present a protocol for freezing and cryosectioning yeast communities to observe internal patterns of fluorescent cells. The method relies on methanol-fixing and OCT-embedding to preserve the spatial distribution of cells without inactivating fluorescent proteins within a community.

蛍光パターンを調査するための凍結セクショニング酵母コミュニティ

Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

軟寒天コロニー形成アッセイ

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

分裂酵母セクシャルライフサイクルの顕微鏡観察

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY：酵母増殖の多パラメータ定量化のための大規模法

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Pancreatic Islet Embedding for Paraffin Sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

パラフィン セクションの埋め込み膵島

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...

酵母のコロニーの埋め込み法

要約

さらに動画を探す

この動画の章

プラスチック製の埋め込みとアフリカツメガエル胚のセクショニング

出芽酵母の複製寿命の測定

酵母細胞からミトコンドリアの精製

ハイスループット酵母プラスミドの過剰発現の画面

高活性酵母のリボソームのクロマトグラフィー精製

蛍光パターンを調査するための凍結セクショニング酵母コミュニティ

軟寒天コロニー形成アッセイ

分裂酵母セクシャルライフサイクルの顕微鏡観察

ODELAY：酵母増殖の多パラメータ定量化のための大規模法

パラフィンセクションの埋め込み膵島

酵母のコロニーの埋め込み法

要約

さらに動画を探す

この動画の章

プラスチック製の埋め込みとアフリカツメガエル胚のセクショニング

出芽酵母の複製寿命の測定

酵母細胞からミトコンドリアの精製

ハイスループット酵母プラスミドの過剰発現の画面

高活性酵母のリボソームのクロマトグラフィー精製

蛍光パターンを調査するための凍結セクショニング酵母コミュニティ

軟寒天コロニー形成アッセイ

分裂酵母セクシャルライフサイクルの顕微鏡観察

ODELAY：酵母増殖の多パラメータ定量化のための大規模法

パラフィン セクションの埋め込み膵島

パラフィンセクションの埋め込み膵島