著者の研究室での作業は、ドイツ・フォルシュングスゲミンシャフト(http://www.dfg.de によってサポートされました。CO 1139/1-1)、フォンズ・デア・ケミッシェン・インダストリー(http://www.vci.de/fonds)、ゲッティンゲン分子生物学センター(GZMB)。著者たちは、ヨルグ・シュテルケに対する有益なコメントと原稿の批判的な読み取りを認めたいと考えています。

寒天プレート、培養メディア、クライオストック、プレカルチャーの製造
<ol><li>成長培地と必要な試薬を準備します(材料および試薬の表を参照)。</li>
	<li>B.サチリス株をストリーク(例えば.BP40(rocG+ gudBCR amyE::PgudB-yfp)および BP52(rocG+ gudB+ amyE::PgudB-cfp) は、それぞれ1つと2つのアクティブなGDHを発現します)2 SPミディアガルプレートの競技実験で使用される単一コロニーを得る。プレートを一晩37°Cでインキュベートする。</li>
	<li>単一コロニーを取り、4 ml LB液体培地を含む滅菌培養管を接種する。28°Cおよび220のrpmで一晩細菌を成長させる。</li>
	<li>細胞がライスまたは胞子を避けるために28°Cで一晩培養を成長させます。</li>
	<li>標準分光光度計を用いて、波長600nm(OD600)で培養液の光学濃度を測定します。</li>
	<li>培養物が2.0のOD600 に達した場合、1.5ml反応チューブ中の無菌50%グリセロール溶液の0.75mlと培養物の0.75mlを混合する。最終OD600 は、約108 細胞/mlを含む凍結ストックを得るために1.0でなければなりません。</li>
	<li>チューブは-80°Cで保管してください。</li>
	<li>フルオロフォア遺伝子をコードする cfp および yfp で標識された各株の3つの前培養を行う。-80°Cのクライオストックから1μl細胞で前培養(4ml LB液体培地を含む無菌培養チューブ)を接種する。培養物を28°Cおよび220rpmで一晩インキュベートする。</li>
</ol>2. 細菌の共同栽培、サンプル採取、試料保存
<ol><li>100 ml の C-Glc および CE-Glc の最小限の培地 (試薬と材料の表を参照) を準備し、各培地の 20 ml を滅菌 100 ml の振るフラスコに移します。</li>
	<li>一晩栽培したプレカルチャーの0.1mlを取り、1.5mlキュベットで0.9ml LB培地で希釈し、OD600を決定する。</li>
	<li>競技実験では、1.0~1.5の間に類似のOD600 を有する異なる株の前培養を行う。</li>
	<li>混合細胞集団を得るために、適切なOD600 を有した培養前培養の細胞を、20mlC-GlcおよびCE-Glc最小培地中の0.05のOD600 に希釈し、100mlの揺れフラスコで補充した。競技実験では、2つの株を1:1の比率で混合する必要があります。</li>
	<li>フラスコから10mlのサンプルを取り、15mlのプラスチックチューブに移し、標準的なテーブル上部遠心分離機で4,000 x gで10分間遠心分離して細胞を収穫し、上清を捨てます。</li>
	<li>0.5 ml新鮮なLB培地で細胞を再懸濁し、細胞を無菌1.5 ml反応カップに移します。0.5 mlの50%無菌グリセロールを加え、厳密なボルテックスで懸濁液を混合し、さらに処理するまで-80°Cの冷凍庫に保存します。</li>
	<li>37°Cおよび220rpmで最大24時間、振るフラスコに残された細胞をインキュベートする。蛍光色素の写真の漂白を防ぐために、暗闇の中で揺れるフラスコを保ちます。</li>
	<li>7時間および24時間の成長の後に0.1 mlのさらなるサンプルを取る。サンプルの1:10希釈液(C-GlcまたはCE-Glc最小媒体)のOD600 を測定して注意してください。</li>
	<li>各培養物から適切な量の細胞を取り出し、OD600 が1.0の凍結ストックを作ります。さらに処理するまで-80°Cでサンプルを保管してください。</li>
</ol>3. 定量分析のためのサンプル処理、めっき、インキュベーション
<ol><li>すべてのサンプルを採取した後、凍結ストックを解凍し、細胞を0.9%の生理食塩水(試薬および材料の表を参照)で10-3まで希釈した。</li>
	<li>SP培地寒天プレート上の10-3 希釈液の0.1mlをプレートし、滅菌ガラスピペットを使用して細胞を分配する。</li>
	<li>単一のコロニーが出現するまで、暗闇の中で37°Cで一晩プレートをインキュベートします。</li>
</ol>4. 定量分析のための立体蛍光顕微鏡による被爆者数
<ol><li>ステレオ蛍光顕微鏡の下で生存者を数えながら、より良い方向のために4つの部分に黒いペンで寒天プレートの底を分割します。</li>
	<li>プレートを顕微鏡の下に逆さまに置き、冷たい光源を使用してコロニーを焦点にします。</li>
	<li>コロニーが焦点を合わせられたら、CFP用に適切なフィルタセットに切り替えて 、cfp標識株の生存細胞を視覚化します。ペンでコロニーにラベルを付けることによって、この株の生存者を数え、数をメモします。</li>
	<li>エタノールでラベルを取り除き、YFPフィルタセットに切り替えて 、yfpラベル付き歪みの生存細胞を可視化します。ペンでコロニーにラベルを付けることによって、再び生存者を数え、番号をメモします。</li>
</ol>5. 半定量分析のためのサンプル処理と顕微鏡
<ol><li>例示的な図では、SP寒天プレート上のステップ3.1から10-4 希釈液(約100細胞)の10μlをスポットする( 図3を参照)。</li>
	<li>単一のコロニーが出現するまで、暗闇の中で37°Cで一晩プレートをインキュベートします。</li>
	<li>顕微鏡の下に蓋をせずにペトリ皿を置き、冷たい光源を使用してスポットを焦点にします。</li>
	<li>例示の図のためのスポットの写真を撮ります。冷たい光源でコロニーの写真を撮るための適切な露出時間を選択してください。</li>
	<li>プレートを動かさずに、CFPフィルタセットに変更し、露光時間を調整して写真を撮ります。YFP フィルタセットでも同じ操作を行い、画像を保存してさらに解析を行います。</li>
</ol>6. データ分析
<ol><li>定量的データ分析には、Excel などのソフトウェアを使用します。カウントされたコロニーに基づいて、100%に設定されたコロニーの総数に対する黄色と青のコロニーの割合を計算します。</li>
	<li>計算された数値を使用して、積み上げバー図を作成します ( 図 4 および図 5を参照)。Adobe Photoshop などの画像処理プログラムを使用して、さまざまな場所から撮影した写真のマージされた画像を作成します。あるいは、自由に入手可能なソフトウェアImageJは <a href="http://rsbweb.nih.gov/ij/">、http://rsbweb.nih.gov/ij/</a> からダウンロード可能な画像処理に使用することができる。</li>
	<li>CFP フィルタ セットと YFP フィルタ セットで撮影した 1 つの場所から画像を開きます。メディアから任意のバックグラウンド蛍光を低減するためにコントラストと明るさを最適化します。</li>
	<li>いずれかの画像に移動し、すべてを選択します。図をコピーして、他の図に貼り付けます。</li>
	<li>「カラードッジ」機能を使用して画像をマージするか、チャンネルタブを使用して蛍光画像をオーバーレイします。異なる媒体および異なる時点からのコロニーの結合された写真は、液体培養中の異なる株の成長を表す。</li>
</ol>7. 具体的なヒント:色素スイッチ実験とcfpとyfpで標識された同種株の共同栽培
B.サブティリスにおける2つのフルオロフォアコード遺伝子のどちらかの発現は、適性および細菌の増殖速度に影響を与える可能性がある。したがって、栽培中の細胞集団からの1つの競合株の排除が単にフルオロフォアの悪影響によるものであることを排除するために、以下の実験を行うことを推奨する。
<ol><li>実験全体を繰り返し、逆に標識された株を共同栽培する(例えば、以前の cfp標識株は yfp 遺伝子で標識され、 その逆も同様である)。逆であるが、得られた結果は、一方の株が他の株よりも選択的な成長優位性を有するという以前の観察と同等であるべきである。</li>
	<li>実験全体を繰り返し、yfpおよびcfpでラベル付けされた同等体性株(例えばBP40(rocG+ gudBCR amyE::PgudB-yfp)およびBP41(rocG+ gudBCR amyE::PgudB-cfp))を共同栽培する。時間の経過に伴う細胞集団の組成に従うことによって、細菌の適合性に対する2つのフルオロフォアのどちらかの負の効果を推定する。</li>
</ol>

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著者らは、競合する財政的利益はないと宣言している。

細菌の競争力のある適合性を分析するためにいくつかの方法が開発されています16.多くの場合、細菌は異なる抗生物質耐性カセット17で標識された。我々のアプローチと同様に、抗生物質耐性カセットを用いた細胞の標識は、定義された成長条件下での共培養中の細菌の競争力の適合性の評価を可能にする。さらに、この方法は、染色体17上の特定の軌跡において互い...

土壌細菌は、通常、柔軟な規制ネットワークと広範な代謝能力に恵まれています。両方の特徴は、細胞が与えられた生態学的ニッチ1で利用可能である栄養素のために彼らの仲間や他の微生物と競合するために、それらの同化および同化経路を調整することを可能にする。しかし、細菌が環境に適応できない場合、他のメカニズムは種の生存を説明する可能性があります。確かに、多くの細菌が急速に増殖し、集団が高い細胞密度に達することができるように、亜集団は自発的に細胞に選択的な成長の利点を提供し、したがって、その適性を高める有益な突然変異を蓄積している可能性があります。さらに、突然変異ホットスポットおよびストレス誘発適応性変異発生は、不適合細菌2,3の進化を促進することができる。したがって、連続選択下での突然変異および増殖の蓄積は、同一属4,5内であっても、巨大な微生物多様性の起源である。自然界と同様に、細菌ゲノムの形成は、選択下の連続栽培のために実験室でも起こる。これは、基礎研究および産業界で世界的に使用されているグラム陽性細菌B.サブティリスの家畜化によって例示される。1940年代にB.サチリスをDNA損傷X線で処理し、その後、特定の成長条件6で培養した。それらの家畜化中に細菌に蓄積された突然変異は、多くの成長特性の喪失を占める、すなわちB.サブチリス実験室株168は、複雑なコロニー7,8を形成する能力を失った。
今日では、最もよく研究されたモデル細菌 大腸菌 と B.サブティリスのために、特定の科学的な質問に対処するためにゲノムを遺伝的に操作するための様々な強力なツールが利用可能です。時には、目的の遺伝子の不活性化は、重篤な成長欠陥を引き起こし、その後、標準的な成長培地9ではっきりと見える。対照的に、弱い成長欠陥を引き起こし、ひずみの適性にわずかに影響を与える突然変異はしばしば無視される。しかしながら、いずれの場合も、数世代にわたって変異株の培養および経架を長時間化すると、通常は親株2,9の表現型を回復したサプレッサー変異体の蓄積をもたらす。サプレッサー変異体の特徴付けと、親突然変異株の成長欠陥を回復した突然変異の同定は、重要かつしばしば新しい細胞プロセス10,11の解明を可能にする非常に有用なアプローチである。
我々はB.サチリス12におけるグルタミン酸恒常性の制御に興味を持っている。大腸菌と同様に、B.サチリスは、サプレッサー突然変異体の蓄積によるグルタミン酸恒常性(すなわちグルタミン酸分解2のブロック)の摂動に応答する。自発的突然変異によって獲得されたこれらのサプレッサー変異体におけるゲノム変化は、グルタミン酸恒常性9,13を迅速に回復させることが示された。したがって、細菌の家畜化中の特定の成長条件に対するB.サチリスの適応が酵素合成および進化した酵素活性においてミラーリングされ、グルタミン酸代謝12に関与していることは驚くべきことではない。家畜化過程中の成長培地における外因性グルタミン酸の欠如が、実験室株1682,14における暗号性グルタミン酸脱水素酵素(GDH)gudB CR遺伝子の出現および固定化の原動力であったことが示唆されている。この仮説は、実験室株におけるGDH活性の減少量が外因性グルタミン酸が不足しているときに選択的増殖の利点を細菌に提供するという我々の観察によって支持される2。また、B.サチリス株の培養は、GDH GudBを合成し、外因性グルタミン酸がない場合には、gudB遺伝子2を不活性化したサプレッサー変異体の蓄積をもたらす。明らかに、同化活性GDHの存在は、他の方法で使用できる内因的に産生されたグルタミン酸がアンモニウムおよび2-オキソグルタル酸に分解されるため、細胞にとって不利である(図1)。対照的に、グルタミン酸が培地によって提供される場合、高レベルGDH活性を有するB.サチリス株は、1つの機能GDHのみを合成する株に対して選択的な増殖性を有する。高レベルのGDH活性により、細菌は培地2によって提供される他の炭素源に加えて、第2の炭素源としてグルタミン酸を利用することを可能にする(図1を参照)。したがって、GDH活性は、外因性グルタミン酸の入手可能性に応じて、細菌の適性に強く影響を及ぼす。
ここでは、染色体上の単一の遺伝子座で異なる2つの B.サチリス 株間の種内競争を監視し、可視化するための非常に例示的な方法を提示する(図2)。この2株を、フルオロフォアYFPおよびCFPをコードする yfp および cfp 遺伝子で標識し、異なる栄養条件下で共栽培した。時間をかけてサンプリングし、寒天プレートに適切な希釈液をめっきすることにより、各培養物の生存者は、一般的なステレオ蛍光顕微鏡を使用して容易に監視することができます。この論文に記載されている手順は、細胞集団における有益および有害な突然変異の急速なクローン拡張および除去を、時間の経過とともに容易に行い、視覚化するのに適している。

<table border="0" cellpadding="0" cellspacing="0" width="814">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">(NH4)2SO4&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">3746</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Agar</td>
			<td style="width:175px;">Difco, USA</td>
			<td style="width:111px;">214010</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="40">
			<td height="61" rowspan="2" style="height:61px;width:363px;">Ammonium ferric citrate (CAF)</td>
			<td rowspan="2" style="width:175px;">Sigma-Aldrich, Germany</td>
			<td rowspan="2" style="width:111px;">9714</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">CaCl2&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">5239</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Glucose</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3617</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Glycerol</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">4043</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">K2HPO4 x 3 H2O</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">6878</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">KCl</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3582</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">KH2PO4</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">3904</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">KOH</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">6751</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">MgSO4 x 7 H2O&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">P027</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">MnCl2&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">T881</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="40">
			<td height="61" rowspan="2" style="height:61px;width:363px;">MnSO4 x 4 H2O</td>
			<td rowspan="2" style="width:175px;">Merck Millipore, Germany</td>
			<td rowspan="2" style="width:111px;">102786</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">NaCl</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">9265</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Nutrient broth</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">X929</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Potassium glutamate</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3712</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Tryptone</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">8952</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Tryptophan</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3445</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Yeast extract</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">2363</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">1.5 ml Reaction tubes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">72,690,001</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">2.0 ml Reaction tubes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">72,691</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">15 ml Plastic tubes with screw cap</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">62,554,001</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Petri dishes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">82.1473</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">1.5 ml Polystyrene cuvettes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">67,742</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">15 ml Glass culture tubes&#160;</td>
			<td rowspan="2" style="width:175px;">Brand, Germany</td>
			<td rowspan="2" style="width:111px;">7790 22</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">with aluminium caps</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">100 ml Shake flasks with aluminium caps</td>
			<td style="width:175px;">Brand, Germany</td>
			<td style="width:111px;">928 24</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Sterile 10 ml glass pipettes</td>
			<td style="width:175px;">Brand, Germany</td>
			<td style="width:111px;">278 23</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Incubator (28 and 37 &#176;C)</td>
			<td style="width:175px;">New Brunswick</td>
			<td style="width:111px;">M1282-0012</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="42">
			<td height="85" rowspan="3" style="height:85px;width:363px;">Standard pipette set (2-20 &#956;l, 10-100 &#956;l, 100-1000 &#956;l)</td>
			<td rowspan="3" style="width:175px;">Eppendorf, Germany</td>
			<td style="width:111px;">4910&#160;000.034, 4910 000.042,&#160;&#160;</td>
			<td rowspan="3" style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">4910 000.042,</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:111px;">4910 000.069&#160;</td>
		</tr>
		<tr height="80">
			<td height="101" rowspan="2" style="height:101px;width:363px;">Table top centrifuge for 1.5 and 2 ml reaction tubes</td>
			<td rowspan="2" style="width:175px;">Thermo Scientific, Heraeus Fresco 21, Germany</td>
			<td rowspan="2" style="width:111px;">75002425</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="60">
			<td height="81" rowspan="2" style="height:81px;width:363px;">Table top centrifuge for 15 ml plastic tubes</td>
			<td rowspan="2" style="width:175px;">Heraeus Biofuge Primo R, Germany</td>
			<td rowspan="2" style="width:111px;">75005440</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="80">
			<td height="101" rowspan="2" style="height:101px;width:363px;">Standard spectrophotometer</td>
			<td rowspan="2" style="width:175px;">Amersham Biosciences Ultrospec 2100 pro, Germany</td>
			<td rowspan="2" style="width:111px;">80-2112-21&#160;</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Stereofluorescence microscope&#160;</td>
			<td style="width:175px;">Zeiss SteREO Lumar V12, Germany</td>
			<td style="width:111px;">495008-0009-000</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Freezer (-20 and -80 &#176;C)</td>
			<td style="width:175px;">-</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Fridge (4 &#176;C)</td>
			<td style="width:175px;">-</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Autoclave</td>
			<td style="width:175px;">Zirbus, LTA 2x3x4, Germany</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">pH meter</td>
			<td style="width:175px;">pH-meter 766, Calimatic, Knick, Germany</td>
			<td style="width:111px;">766</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="40">
			<td height="61" rowspan="2" style="height:61px;width:363px;">Vortex</td>
			<td rowspan="2" style="width:175px;">Vortex&#160; 3, IKA, Germany</td>
			<td rowspan="2" style="width:111px;">3340000</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="21">
			<td height="64" rowspan="3" style="height:64px;width:363px;">Balance</td>
			<td rowspan="3" style="width:175px;">CP2202S, Sartorius, Germany</td>
			<td style="width:111px;">replaced by</td>
			<td rowspan="3" style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">CPA2202S</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:111px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Black pen (permanent marker)</td>
			<td style="width:175px;">Staedler, Germany</td>
			<td style="width:111px;">317-9</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Powerpoint program</td>
			<td style="width:175px;">Microsoft, USA</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Office Excel program</td>
			<td style="width:175px;">Microsoft, USA</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">Program for data processing</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Adobe Photoshop CS5</td>
			<td style="width:175px;">Adobe, USA</td>
			<td style="width:111px;">replaced by CS6, download</td>
			<td style="width:167px;">Computer program for image processing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Computer</td>
			<td style="width:175px;">PC or Mac</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">ZEN pro 2011 software for the stereofluorescence microscope</td>
			<td style="width:175px;">Zeiss, Germany</td>
			<td style="width:111px;">410135 1002 110</td>
			<td style="width:167px;">AxioCam MRc Rev. Obtained through Zeiss</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:815px;">Specific solution recipes</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">SP medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">8 g Nutrient broth</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">0.25 mg MgSO4 x 7 H2O</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">1 g KCl</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">if required, add 15 g agar for solid SP medium</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">1 ml CaCl2 (0.5 M), sterilized by filtration</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">1 ml MnCl2 (10 mM) sterilized by filtration</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:815px;">2 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">LB medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 g Tryptone</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">5 g Yeast extract</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 g NaCl</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">if required, add 15 g agar for solid LB medium</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">C-Glc minimal medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">200 ml 5 x C salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml III&#8217; salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">25 ml Glucose (20%), autoclaved for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">CE-Glc minimal medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">200 ml 5 x C salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml III&#8217; salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">20 ml Glutamate (40%)</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">25 ml Glucose (20%), autoclaved for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">5 x C salts&#160;</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">20 g KH2PO4</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">80 g K2HPO4 x 3 H2O</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">16.5 g (NH4)2SO4</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">III&#8217; salts</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">0.232 g MnSO4 x 4 H2O</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">12.3 g MgSO4 x 7 H2O</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">40% Glutamate solution</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">200 g L-Glutamic acid</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">adjust the pH to 7.0 by adding approximately 80 g KOH</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 0.5 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">0.9% Saline (NaCl) solution</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">50% Glycerol solution</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">295 ml Glycerol (87%)</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 0.5 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:815px;">Bacteria (All strains are based on the Bacillus subtilis strain 168)</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP40 (rocG+ gudBCR amyE::PgudB-yfp)&#160;</td>
			<td rowspan="4" style="width:167px;">Laboratory strain collection</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP41 (rocG+ gudBCR amyE::PgudB-cfp)&#160;</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP52 (rocG+ gudB+ amyE::PgudB-cfp)</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP156 (rocG+ gudB+ amyE::PgudB-yfp)</td>
		</tr>
	</tbody>
</table>

monitoring intraspecies competition in a bacterial cell population by cocultivation of fluorescently labelled strains

細菌などの多くの微生物は非常に急速に増殖し、集団は高い細胞密度に達する可能性があります。集団の細胞の小さい分数は、常に細胞にとって有害または有益な突然変異を蓄積している。突然変異の適性効果がサブ人口に強力な選択的成長優位性を提供するならば、この亜集団の個体は急速に競争し、さらには彼らの直近の仲間を完全に排除するかもしれない。したがって、有益な突然変異を獲得した細胞の小さな遺伝的変化および選択主導的蓄積は、細胞集団の遺伝子型の完全なシフトにつながる可能性がある。ここでは、グラム陽性モデル 細菌サブティリスの蛍光標識体の共培養によって、時間の経過とともに細菌細胞集団における有益および有害な突然変異の迅速なクローン拡張および消失を監視する手順を提示する。この方法は、細菌細胞集団における個体間の種内競争を表示する方法が容易かつ非常に例示的である。

ここで説明した方法は、フルオロフォアCFPおよびYFPをコードするcfp遺伝子およびyfp遺伝子で標識されたB.サチリス株からなる細胞集団における種内競争を可視化するために、それぞれうまく適用された。図3に示すように、この方法は、非常に例示的な方法で種内競争を視覚化するために使用することができる。小さな領域にサンプルを見つけることによって?...

Watch this Scientific Journal Video about Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains at JoVE.com

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

細菌は、その生涯の間に有害または有益な突然変異のいずれかを蓄積することができます。細胞の集団では、有益な突然変異を蓄積した個体は、急速に仲間を上回る可能性があります。ここでは、蛍光標識された個体を用いて、時間の経過とともに細菌細胞集団における種内競争を可視化する簡単な手順を提示する。

蛍光標識株の共同栽培による細菌細胞集団における種内競争のモニタリング

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

monitoring-intraspecies-competition-bacterial-cell-population

Research

JoVE Journal

Biology

8.4K ビュー.  Georg-August University. 細菌は、その生涯の間に有害または有益な突然変異のいずれかを蓄積することができます。細胞の集団では、有益な突然変異を蓄積した個体は、急速に仲間を上回る可能性があります。ここでは、蛍光標識された個体を用いて、時間の経過とともに細菌細胞集団における種内競争を可視化する簡単な手順を提示する。

ビデオ: Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Title Cell Encapsulation by Droplets

滴タイトルセルのカプセル化

生物学

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

免疫手術でヒト胚性幹細胞の導出

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

遺伝的にコードされたカルシウムのセンサを用いたアポトーシス時のミトコンドリア内カルシウム濃度の動的変更の監視

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

シトクロムの細胞質マイクロインジェクションによるアポトーシスの活性化 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

FRETはレシオメトリックで細胞周期のキナーゼおよびホスファターゼの活動を監視

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking1,2 but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases3.
Yet their existence is still a matter of controversy4,5. Indeed, lipid raft size has been estimated to be around 20 nm6, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized7,8. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells.
Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts9,10. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models11.
FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently12. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change12.

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.

脂質ラフトの決定は、蛍光相関分光法（FCS）による生細胞の蛍光タグ付きプローブの分割

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

甲殻類との長期致死毒性試験アルテミアfranciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

定量的RT-PCRによって個々のヒト胚性幹細胞のプロファイリング

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

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, ...

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and product development. Stress-strain relationships are typically obtained from mechanical testing data to enable comparative assessment among samples and in some cases identification of constitutive mechanical properties. However, errors may be introduced through the use of average strain measures, as significant heterogeneity in the strain field may result from geometrical non-uniformity of the sample and stress concentrations induced by mounting/gripping of soft tissues within the test system. When strain field heterogeneity is significant, accurate assessment of the sample mechanical response requires measurement of local strains. This study demonstrates a novel biomechanical testing protocol for calculating local surface strains using a mechanical testing device coupled with a high resolution camera and a digital image correlation technique. A series of sample surface images are acquired and then analyzed to quantify the local surface strain of a vascular tissue specimen subjected to ramped uniaxial loading. This approach can improve accuracy in experimental vascular biomechanics and has potential for broader use among other native soft tissues, engineered soft tissues, and soft hydrogel/polymeric materials. In the video, we demonstrate how to set up the system components and perform a complete experiment on native vascular tissue.

We describe the use of digital image correlation to characterize the local surface strain field on vascular tissue samples subjected to uniaxial tensile testing. These measurements facilitate precise quantification of the sample mechanical response and the generation of constitutive stress-strain relations.

デジタル画像相関を使用すると、血管の組織標本のローカル株を特徴づけるために、

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...