AQUAGAMETE FA 1205 コスト行動に感謝いたします。この作品は AGL201568330 C2 1 R プロジェクト (MINECO/フェダー) によって財政上支えられます。デイヴィッド g. Valcarce 資金が供給された軍事政権・ デ ・ カスティーリャ ・ イ ・ レオン (EDU1084/2012 年) とフォンド社会 Europeo。著者は、博士アナ リアサ Stolt 海ファーム社、博士パウリノ ・ デ ・ ラパス、博士・ イグナシオ ・ マルティネス モンテロとホセ ・ ラモン ・ Guiérrez を認めます。我々 はビデオ撮影にポーラ フェルナンデス Colado をありがとうも。

注: 螢光色素インキュベーションと共焦点解析コントロールと扱われたサンプルの少なくとも 2-3 時間になります。データ処理は、この時間の計算には含まれません。必要な材料は、材料テーブルで見つけることができます。このプロトコルは、新鮮なまたは凍結精子に適用できます。やっぱりアオモンイトトンボ冷たい水、冷たい条件 (4 〜 7 ° C) の下で常に仕事で産卵する魚の種であります。プロトコルの一般的なビューは、図 1を参照してください。
1 実験前に準備作業
<ol><li>ミトコンドリア染色 1 mM 原液分析学年 DMSO を準備します。</li>
	<li>1 μ g/mL 4 ', 6-diamidino-2-phenylindole 二塩酸塩 (DAPI) 脱イオン水で原液を準備します。</li>
	<li>200 mOsm/kg リンゲル液 (116 mM NaCl、KCl、2.9 mM 1.8 mM CaCl2, 5 mM HEPES、pH 7.7) の準備します。</li>
	<li>4 mM 2 ', 7 ' dichlorodihydrofluorescein ジアセテート (DCFH DA) 分析グレードのメタノールで原液を準備します。 注: は、-20 ° c まで必要な因数でストックのソリューションを維持します。</li>
	<li>4-7 ° C で、遠心を設定します。</li>
</ol>2. 螢光色素の孵化前にサンプル準備
注: ピペッティングと再、精子の穏やかな処理が望ましいです。
<ol><li>凍結保存した細胞を扱う場合水のお風呂が、やっぱりアオモンイトトンボ精子1040 ° C で融解することを備えることができます。7 槽のわらのサンプルを浸す s。</li>
	<li>カットするはさみを使用して、および microfuge の管にサンプルを空にして 4-7 ° C、1 分 1000 x g で遠心分離機します。</li>
	<li>汚損のプロトコルを妨げることができる任意のコンポーネントを破棄する分注して凍結サンプルから精液または凍結と精漿を削除します。</li>
	<li>50-100 μ L の 4 ° C 200 mOsm/kg リンガー液で細胞を再懸濁します。</li>
	<li>ノイバウアーや実体顕微鏡下で似たようなカウントの商工会議所と精子の濃度を決定します。</li>
	<li>1 - 2 106セル/mL 4 ° C リンガー液 0.5 mL の最終巻で x を細胞懸濁液を希釈します。</li>
</ol>3. 螢光色素インキュベーション
注: 螢は、低光の条件、特に溶液中で処理しなければなりません。
<ol><li>DCFH DA 原液の 3.125 μ L を追加 (サンプルの最終濃度: 25 μ M) 作業サンプル希釈します。4-7 ° C で闇10で 40 分間インキュベートします。</li>
	<li>30 分後の DAPI 原液 0.5 μ L を追加 (最終濃度サンプル: 2 μ M) とミトコンドリア染色原液 0.5 μ (サンプルの最終濃度: 100 nM) のサンプルに。暗闇の中で 10 分間両方の螢を孵化させなさい。</li>
</ol>4. 共焦点の顕微鏡検査のための試料
<ol><li>培養時間後 1000 x g、1 分の 4-7 ° C の細胞懸濁液を遠心します。</li>
	<li>ピペットで上澄みを慎重に廃棄します。</li>
	<li>200 mOsm/kg リンガー液の 10-20 μ L を追加します。</li>
	<li>濃縮細胞懸濁液の場所 5 μ L は、スライドにドロップします。</li>
	<li>慎重に、スライドを準備上のカバーを置く、ポーランド語でそれを封印します。ポーランドが乾燥して準備は共焦点の顕微鏡検査のための準備です。</li>
</ol>5 実験前共焦点のセットアップ
注: 顕微鏡によって DCFH DA が「焼かれる」。まず非貴重なサンプルに最適な条件を確立します。
<ol><li>顕微鏡、レーザー、カメラ、顕微鏡を実行しているコンピューターをオンにします。</li>
	<li>各螢光色素の励起と放射の最大値を考慮して励起レーザーを設定します。<ol>
<li>DAPI、用励振最大 358 nm、排出量最大 465 nm。</li>
		<li>ミトコンドリア染色用励振最大 644 nm、排出量最大 662 nm。</li>
		<li>DCFH-DA を使用して励起最大 504 nm、排出量最大 525 nm。</li>
	</ol>
</li>
	<li>精子に集中する 25 X 目的と作業を開始します。この螢光色素は、最高の強度を報告するために焦点を DAPI チャネルを選択します。セルを集中している、かつて用 63 X または 100 × 対物徹底的な評価ソレア アオモンイトトンボ精子サイズが約 2 μ m なので。</li>
	<li>各チャネルのピンホール、利得と電圧を最適化します。同じように、背景を減らすためにデジタル オフセットを調整します。すべてのパラメーターは、使用される螢光色素に最適な一度設定を保存します。単一のルーチン試験のソレア アオモンイトトンボ精子細胞を用いて (これらのパラメーターは、各実験で変化があります) 次: 1 の利得電圧 750 V のピンホール。</li>
</ol>6. 画像の取得
<ol><li>画像獲得の目的の品質条件を選択します。ビット深度、イメージ形式、光シート厚さのような取得設定を定義し、単一の両面照明を選択します。単一のルーチン試験のソレア アオモンイトトンボ精子使用 (これらのパラメーターは、各実験で変化があります) 次: フレーム; 1024 px のサイズビット/ピクセル 16;スキャン速度が 2-4)。</li>
	<li>開き、開始フィールドの画像を撮影してスキャン領域を中央にします。</li>
	<li>トリミング ツールを使用すると、関心領域を選択し、押しますライブ。</li>
	<li>範囲インジケーター ツールの助けを借りて、背景を減らすためにデジタル オフセットを調整します。チャンネルごとにこれを行うし、イメージを取る。</li>
	<li>各チャンネルごとの螢光と、その組み合わせの品質を確認します。</li>
</ol>7. 3 D イメージ ビデオを作成します。
<ol><li>Z スタック取得設定。<ol>
<li>ソフトウェアで Z スタック オプションを選択します。</li>
		<li>単一のセル、セルのグループまたは関心領域を選択し、フォーカスをします。</li>
		<li>Z スタック '最初スライス' と '最後のスライス' オプションを区切るし、良い焦点の助けを借りて、0.2 μ m 間隔に z ステップを確立します。最高の強度を報告ことがありますこの螢光色素と精子の頭全体を簡単に選択できるため、集中する DAPI チャネルを選択します。各チャンネルに最適なアクイジション ・ パラメーターを選択し、実験を実行します。</li>
	</ol>
</li>
	<li>Z スタック実験を実行します。チャンネルを分割し、各シグナルの細胞内の局在を観察します。</li>
	<li>Z スタック プロセス。<ol>
<li>ボリューム レンダリング: 実験が終わったら、Z スタック処理ソフトウェアのオプションを選択し、ボリュームのレンダリング オプションを選択します。ステップと回転度 (360 °) の数を選択します。動画の拡張子を持つファイルを保存します。</li>
		<li>ステレオ: オプションでステレオを選択プロセス ウィンドウ。このツールは、チャンネルの分割とビデオの 3 D 画像を作成することができます。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Amaral, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+functionality+and+chemical+compound+action+on+sperm+function.">Mitochondrial functionality and chemical compound action on sperm function.</a> Curr Med Chem. , (2016).</li><li>Morielli, T., O'Flaherty, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+impairs+function+and+increases+redox+protein+modifications+in+human+spermatozoa.">Oxidative stress impairs function and increases redox protein modifications in human spermatozoa.</a> Reproduction. 149 (1), 113-123 (2015).</li><li>G&#252;rler, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryopreservation+on+sperm+viability,+synthesis+of+reactive+oxygen+species,+and+DNA+damage+of+bovine+sperm.">Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species, and DNA damage of bovine sperm.</a> Theriogenology. , (2016).</li><li>Zhu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+E+Analogue+Improves+Rabbit+Sperm+Quality+during+the+Process+of+Cryopreservation+through+Its+Antioxidative+Action.">Vitamin E Analogue Improves Rabbit Sperm Quality during the Process of Cryopreservation through Its Antioxidative Action.</a> PLoS One. 10 (12), e0145383 (2015).</li><li>Fang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+ROS+production+through+mitochondria-targeted+antioxidant+and+mitochondrial+uncoupling+increases+post-thaw+sperm+viability+in+yellow+catfish.">Inhibition of ROS production through mitochondria-targeted antioxidant and mitochondrial uncoupling increases post-thaw sperm viability in yellow catfish.</a> Cryobiology. 69 (3), (2014).</li><li>Thomson, L. K., Fleming, S. D., Aitken, R. J., De Iuliis, G. N., Zieschang, J. A., Clark, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation-induced+human+sperm+DNA+damage+is+predominantly+mediated+by+oxidative+stress+rather+than+apoptosis.">Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis.</a> Hum Reprod. 24 (9), 2061-2070 (2009).</li><li>Kim, S. H., Yu, D. H., Kim, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryopreservation+on+phosphatidylserine+translocation,+intracellular+hydrogen+peroxide,+and+DNA+integrity+in+canine+sperm.">Effects of cryopreservation on phosphatidylserine translocation, intracellular hydrogen peroxide, and DNA integrity in canine sperm.</a> Theriogenology. 73 (3), (2010).</li><li>Guthrie, H. D., Welch, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+reactive+oxygen+species+on+sperm+function.">Effects of reactive oxygen species on sperm function.</a> Theriogenology. 78 (8), 1700-1708 (2012).</li><li>Aitken, R. J., Jones, K. T., Robertson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+and+sperm+function--in+sickness+and+in+health.">Reactive oxygen species and sperm function--in sickness and in health.</a> J Androl. 33 (6), (2012).</li><li>Valcarce, D. G., Robles, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+captivity+and+cryopreservation+on+ROS+production+in+Solea+senegalensis+spermatozoa.">Effect of captivity and cryopreservation on ROS production in Solea senegalensis spermatozoa.</a> Reproduction. 152 (5), (2016).</li><li>Gibb, Z., Lambourne, S. R., Aitken, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+paradoxical+relationship+between+stallion+fertility+and+oxidative+stress.">The paradoxical relationship between stallion fertility and oxidative stress.</a> Biol Reprod. 91 (3), (2014).</li><li>Cabrita, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+enhancing+fish+sperm+quality+and+emerging+tools+for+sperm+analysis.">Factors enhancing fish sperm quality and emerging tools for sperm analysis.</a> Aquaculture. 432, 389-401 (2014).</li></ol>

著者が明らかに何もありません。

精子の運動性と機能キー細胞小器官であるミトコンドリアが知られています。これらの細胞小器官は、同時に直接活性酸素産生に関与しています。興味深いことに、適切な精子機能1活性酸素の制御のレベルが必要です。肯定的な哺乳類11不妊と酸化ストレスとの関係が示されているが、過剰なレベルに影響を与える精子品質12。正または?...

活性酸素種の生産は、精子の質とリンクされている最近1。ミトコンドリアの活性酸素産生には、通常の生理学的なプロセスを考えることができる、さまざまなレベルで精子の損傷の明確な原因は活性酸素産生の過剰による酸化ストレス。人間、酸化ストレスは、男性不妊症、運動性や受精2を受けるための能力を変更することに関連付けられています。哺乳類で、凍結精子サンプルの DNA の完全性の変化は H2O23の合成にも関連しています。
凍結保存は養殖で銀行の遺伝子のための一般的な手法です。この技術はやっぱりアオモンイトトンボなど生殖器の問題を持つ種で特に重要です。市場でこの貴重な種は、求愛の不足のため飼育下で生まれた個体の生殖機能障害を示しています。この事実は、精子の凍結保存を人工受精精子の可用性を持っている必要になります。しかし、凍結は、酸化ストレスと抗酸化サプリメントの有益な効果が報告されての精子4有害なことができるのソース可能性があります。ミトコンドリアをターゲットとした抗酸化作用による活性酸素抑制は黄色いナマズ5精子凍結保存用伝え有益であった。
したがって、精子サンプル中の ROS のレベルは、これらの分子は、精子の生存と不妊治療8の欠点として認識されているので、特に凍結6、7の後知ることが重要。さらに、細胞内 ROS の分布を調査、潜在的な損傷のレベルを推測する重要な可能性があります。例として通常、精子機能と互換性のある、ミトコンドリアにおける活性酸素の低レベルを想定できるが、核内活性酸素の高レベルは、精子の DNA 損傷の指標かもしれない。H2O2は、ミトコンドリアから解放されることができる、小さなおよび電荷レス分子9だから核を浸透最も関連性の高い活性酸素の 1 つです。ジクロルフルオレッセン ジアセテート (DCFH DA) 具体的には緑色の蛍光を発光細胞内の過酸化水素を明らかにできます。この記事は、やっぱりアオモンイトトンボ精子共焦点顕微鏡を用いた H2O2細胞内の局在を検出するための詳しいプロトコルが表示されます。

<table border="0" cellpadding="0" cellspacing="0" width="790">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">2&#8242;,7&#8242;-Dichlorodihydrofluorescein diacetate (DCFH-DA)&#160;</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:119px;">D6883</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8242;,6-diamidino-2-phenylindole (DAPI)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C1016&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscopy</td>
			<td>Zeiss</td>
			<td>LSM800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover slips</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-541B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO, Analytical Grade</td>
			<td>Sigma-Aldrich</td>
			<td>W387520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol, Analytical Grade</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MitoTrackerDeep Red&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>M22426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge (refrigerated)</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neubauerchamber</td>
			<td>Sigma-Aldrich</td>
			<td>BR717810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>10143562BEF</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of intracellular location of reactive oxygen species in solea senegalensis spermatozoa

酸化ストレスは、精子の質を減少させる重要な要因の 1 つです。どんな種の重要度が高いは、精子の活性酸素種 (ROS) を検出するための効率的なプロトコルの開発が、これらのメソッドは、使用頻度の低いと硬骨魚も少ない。凍結保存は遺伝子銀行を含め、さまざまな目的のため養殖の便利なテクニックを年間を通じて精子可用性を保証します。凍結/融解手順や原因になる活性酸素産生精子細胞に損傷を与えます。考えると、将来の損害が活性酸素産生の過剰の原因、ローカリゼーション、ここによって精子の H2O2を検出し、共焦点顕微鏡による細胞内局在化を評価する詳細な方法について提供しています。この目的のため 3 螢 (2 ', 7 ' Dichlorodihydrofluorescein セルロースジ アセテート (DCFH DA)、ライブ ミトコンドリア染色と 4 ', 6-Diamidino-2-phenylindole 二塩酸塩 (DAPI)) の組み合わせは、2 H2O の共局在の評価に使用されます。精子核やミトコンドリアソレア senegalesis精子サンプル。

共焦点顕微鏡は、魚類の精子細胞内 ROS の評価のための理想的な方法です。(図 1) 本論文で示した 3 つの螢 (DAPI、ミトコンドリア染色、DCFH DA) の組み合わせは、基礎研究に適用することができますし、改善で使用されるプロシージャにアプリケーションを持つことができます多くの有用な情報を提供します凍結プロトコルなどの産業水産植物細胞...

Watch this Scientific Journal Video about Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa at JoVE.com

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

このプロトコルは、やっぱりアオモンイトトンボ精子核の ROS は、ミトコンドリアのライブ ミトコンドリア染色 DAPI の敏感な螢光色素 DCFH-DA を使用して内 H2O2の局在を検出するための詳細な方法についてを記述します。可視化、それぞれ。プロトコルは、新鮮なまたは解凍精子と 2 h 内で実行する設計されています。

やっぱりアオモンイトトンボ精子における活性酸素種の細胞内局在の評価

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) ...

evaluation-intracellular-location-reactive-oxygen-species-solea

Research

JoVE Journal

Developmental Biology

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

6.2K ビュー.  University of León. このプロトコルは、やっぱりアオモンイトトンボ精子核の ROS は、ミトコンドリアのライブ ミトコンドリア染色 DAPI の敏感な螢光色素 DCFH-DA を使用して内 H2O2の局在を検出するための詳細な方法についてを記述します。可視化、それぞれ。プロトコルは、新鮮なまたは解凍精子と 2 h 内で実行する設計されています。

ビデオ: Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

ひと多能性幹細胞からプリミティブかつ決定的な造血前駆細胞の分化を監督

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

発生生物学

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Heterologous IVF, a replacement for homologous IVF, could provide a means to test the sperm fertility potential; to study gamete physiology and early embryo development; and to avoid the use of valuable dolphin oocytes, which are difficult to obtain. Here, we present protocols that have been successfully used to collect and cryopreserve dolphin spermatozoa. The collection of semen is performed by manual stimulation on trained dolphins. Cryopreservation is accomplished using a TRIS egg-yolk based extender with glycerol. In addition, we present a protocol that describes heterologous IVF using dolphin spermatozoa and bovine oocytes and that verifies the hybrid nature of the resulting embryo using PCR. Heterologous fertilization raises questions on fertilization and can be used as a tool to study gamete physiology and early embryo development. In addition, the success of heterologous IVF demonstrates the potential of this technique to test dolphin sperm fertilizing capacity, which is worth further examination.

Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Here, we present protocols that have been successfully used for dolphin spermatozoa collection, cryopreservation, and heterologous IVF performance using bovine oocytes.

バンドウイルカ イルカ (バンドウイルカ) 精子: 異種には体外受精、凍結保存、コレクション

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

郭清ときのこ体の免疫蛍光染色と大人のショウジョウバエ脳光受容器ニューロン

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

傷害による老化と生体内での骨格筋におけるリプログラミングの評価

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

ライブショウジョウバエ卵巣組織の卵胞幹細胞系列のイメージング細胞内 pH の方法

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

線虫ニューロンの細胞内輸送を分析するための固定化

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

細胞分裂時の染色体分配の生きているセルイメージ投射

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. Recent studies have extended the role of cellular senescence to various physiological processes, including development, wound healing, immune surveillance, and age-related tissue dysfunction. Although cell cycle arrest is a critical hallmark of cellular senescence, an increased intracellular reactive oxygen species (ROS) production has also been demonstrated to play an important role in the induction of cellular senescence. In addition, recent studies revealed that senescent cells exhibit potent paracrine activities on neighboring cells and tissues through a senescence-associated secretory phenotype (SASP). The sharp increase in interest regarding therapeutic strategies against cellular senescence emphasizes the need for a precise understanding of senescence mechanisms, including intracellular ROS and the SASP. Here, we describe protocols for quantitatively assessing intracellular ROS levels during H-Ras-induced cellular senescence using ROS-sensitive fluorescent dye and flow cytometry. In addition, we introduce sensitive techniques for the analysis of the induction of mRNA expression and secretion of SASP factors. These protocols can be applied to various cellular senescence models.

Intracellular ROS has been shown to play an important role in the induction of cellular senescence. Here, we describe a sensitive assay for quantifying ROS levels during cellular senescence. We also provide protocols for assessing the senescence-associated secretory phenotype, which reportedly contributes to various age-related dysfunctions.

癌遺伝子が誘導する老化の中に活性酸素種および分泌老化関連の表現型正常ひと線維芽細胞の定量測定

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. ...

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy.

別の植物種から茎頂の共焦点のライブ イメージング

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses with an egg cell or a central cell. Thus, two independent fertilization events take place almost simultaneously. The fertilized egg cell and central cell develop into zygote and endosperm, respectively. Therefore, precise control of double fertilization is essential for the ensuing seed development. Double fertilization occurs in the female gametophyte (embryo sac), which is deeply hidden and covered with thick ovule and ovary tissues. This pistil tissue construction makes observation and analysis of double fertilization quite difficult and has created the present situation in which many questions regarding the mechanism of double fertilization remain unanswered. For the functional evaluation of a potential candidate for fertilization regulator, phenotypic analysis of fertilization is important. To judge the completion of fertilization in Arabidopsis thaliana, the shapes of fluorescence signals labeling sperm nuclei are used as indicators. A sperm cell that fails to fertilize is indicated by a condensed fluorescence signal outside of the female gametes, whereas a sperm cell that successfully fertilizes is indicated by a decondensed signal due to karyogamy with the female gametes&#8217; nucleus. The method described here provides a tool to determine successful or failed fertilization under in vivo conditions.

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

We demonstrate a method to determine successful or failed fertilization on the basis of sperm nuclear morphology in Arabidopsis double fertilization using an epifluorescence microscope.

アラビドプシス二重受精における精子核形態をたどる受精状態の評価

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses ...