この作品は、日の出 (R01GM11755) に P.W.J.、S.R.W.、j. h. 訓練助成フェローシップ国立がん研究所 (NIH) (CA009110) からサポートされていた

マウスの使用は、機関動物ケアとジョンズ ・ ホプキンス大学の使用・委員会によって承認されました。少年 (20-26 日産後、dpp) について実験を行った c57bl/6 j マウス、精子形成の準同期の最初の波の活用します。しかし、この手法も実行できますアダルト マウスを用いたします。
1. 解離とマウス精細管の分離
<ol><li>修正/溶解ソリューションと抗体希釈バッファー (ADB)の表 1と表 2に記載されているを準備します。</li>
	<li>リン酸緩衝生理食塩水 (1 x PBS、pH 7.4) x 1 の 3 ml 1 つ 35 mm シャーレとマウスあたり修正/溶解溶液 2 mL と別の 35 mm 皿を準備します。</li>
	<li>頚部転位または CO2窒息を介してマウスを犠牲に、70% エタノールで腹側腹部をスプレーします。V 字形の開口部を作る生殖不能のはさみを使って腹部骨盤腔を開きます。精巣、鉗子による副睾丸脂肪パッドを取り外し、逃げずを妨害しません。1x PBS を含む 35 mm のペトリ皿に精巣を配置します。 注:精子形成の最初の波を利用すると、金利14の富ませたセル人口を観察するために。精子形成サイクルの特定の段階は、年齢別のマウス (表 3) で濃縮されています。</li>
	<li>鋭い先端の鉗子で組織を穿刺によって精巣逃げずを削除し、緩やかな精細管を収集します。細管を修正/溶解液 2 mL を含む新しい 35 mm のペトリ皿に転送します。</li>
	<li>室温で 5 分間修正/溶解液尿細管を孵化させなさい。</li>
</ol>2. 精細管の準備
<ol><li>液体ブロッカー ペンでスライドのエッジのアウトラインによるポリ-L-リジン コーティング ガラス スライドを準備します。</li>
	<li>スライド ガラスの中央に修正/溶解溶液 100 μ L を適用します。</li>
	<li>滅菌の鉗子を用いたはさみはそっと離れて毛尾をいじめる。長い個々 の細管を切削セグメントの長さ約 20 mm。 注:中心などの長期固定露出に敏感な構造を視覚化する、1 × PBS の細管をまず分離し、直接表面に細管をミンチにポリ リジン スライドは修正溶解溶液 100 μ L でコーティング。</li>
	<li>5 つの 20 mm 長い精細管セグメントを含む修正/溶解液準備ポリ L リジン コーティング ガラス スライドに転送します。</li>
	<li>1.5 から 3.0 mm のセグメントに滅菌はさみミンチ精細管を使用しています。</li>
	<li>使用して滅菌鉗子がスライド ガラス上の尿細管のセグメントを配置組織が重なっていないと細管が均等に分散されます。 注:スライド ガラス上の尿細管のセグメントの最適数は 20 と 40 の間です。</li>
</ol>3. 退治精細
<ol><li>ベンチトップに分散した精細管セグメントを含むガラス スライドを転送します。スカッシュ ステップ中に組織を失うことを避けるために研究室のワイプを使用して余分な液体を削除します。</li>
	<li>スライド ガラスの上に観察 (22 × 60 1.5 mm) を適用し、10-20 秒の手のひらのかかとで圧力を適用します。精細管から精母細胞を分散させるのに十分な力を適用することが重要です。 注:尿細管のすべてのセグメントが中断されるので、退治の手順を最適化するために解剖顕微鏡を用いた尿細管を観察します。</li>
	<li>スライド鉗子を使用すると、すぐにフラッシュ凍結液 N2の小型のデュワーのガラス スライドを 15 秒間、または液体が泡を停止するまで。反応は、すぐにストレート エッジかみそりの刃で coverslip を削除する場合、は、先端の鉗子または 21 g 針を罰金します。 注:最適な結果を得る、immunolabel スライドをすぐに (手順 4 を参照)。ただし、この時点でスライドできる-80 ° C で 2 週間。タンパク質と細胞構造の寿命を最大にするには、すぐにドライアイスまたは-80 ° C の冷凍庫に転送でスライドを格納し、スライドに、coverslip を保管します。反応は、スライドを保存する場合は、手順 3.3 で説明した、15 秒間液体 N2のスライドを浸すことによって、coverslip を削除します。</li>
</ol>4. マウントした精細管の反応
<ol><li>1 × PBS と 3 回 1x PBS を含む 50 mL Coplin jar で 5 分間洗浄のスライドを浸します。 注:スライドを許可しない反応の間に完全に乾燥。</li>
	<li>1 mL の加湿チャンバー内で 1-2 時間のブロックをスライド ガラス上に抗体希釈バッファーを適用します。</li>
	<li>抗体希釈バッファーを削除し、加湿チャンバー内抗体希釈バッファーで希釈した一次抗体 100 μ。 注:最良の結果は、4 ° C で一晩一次抗体を孵化させなさい。Coverslip やパラフィルムでスライドを覆うことによって抗体 (例えば50 μ L) のより小さいボリュームを使用します。検証し、一次抗体15を最適化します。</li>
	<li>1x PBS を含む 50 mL Coplin jar に 3 回 5 分用のスライドをすすぐ。 注:洗浄を高めるためには、小さな電磁攪拌棒を使用して Coplin jar を扇動し低速で電磁攪拌プレート上に配置または卓上ミキサー低速での Coplin jar を配置します。</li>
	<li>室温で加湿チャンバーで 1 から 1.5 時間抗体希釈バッファーで希釈した二次抗体の 100 μ L を適用します。写真は共役分子蛍光抗体の漂白を避けるために暗いボックスにスライドを孵化させなさい。 注:Coverslip やパラフィルムでスライドを覆うことによって抗体 (例えば50 μ L) のより小さいボリュームを使用します。</li>
	<li>1x PBS を含む 50 mL Coplin jar に 2 回 5 分用のスライドをすすぐ。</li>
	<li>メディアを含んでいる 4', 6-diamidino-2-phenylindole マウント (DAPI、1.5 μ g/mL) のスライドをマウントし、coverslips (22 × 60 1.5 mm) を適用します。</li>
	<li>スライドを保持し、coverslips の移動を防ぐ明確なマニキュアでスライドに coverslips をシールします。</li>
</ol>5. 分析やイメージングの尿細管スカッシュ準備
<ol><li>正確な z 軸の動きと画像をキャプチャするための高解像度カメラにより自動ステージと落射蛍光顕微鏡を使用します。また、レーザー共焦点顕微鏡を使用します。 注:このステップのすべての利用可能なハードウェアとソフトウェアを使用します。たとえば、図 1および図 2で表示される画像は、ツァイス セル オブザーバー Z1 シャチ フラッシュ 4.0 CMOS カメラにリンクし、ツァイス禅 2012 青版画像ソフトウェアで解析を使用して捕獲されました。</li>
	<li>スカッシュの準備の質を決定する 20 × 対物を使用してスライドを評価します。良質のスライドが等間隔に核の分子があります。 注:スライドのいくつかのセクションは他の人より、さらなる高倍率を使用して分析のためのスライドの最高の領域を見つけることができますの座標をメモすると便利です。</li>
	<li>高い倍率目的 (例: 63 X または 100 X)、核の一貫した単分子膜を持つスライドのキャプチャ領域を使用しています。地域を選択すると、上限を設定、確実にすべてのフォーカスの光をキャプチャ Z スタックを下ろします。 注:Z スタックの上限と下限の制限の間に捕捉の最適数画像集録ソフトウェアが一般に指定し、は、それぞれの目的によって異なります。</li>
	<li>画像処理ソフトウェアを使用して、奥行きを拡張焦点画像をコンパイルします。ソフトウェアは各 Z スタックからフォーカスの光を組み合わせた、尿細管スカッシュ製剤の最適な画像のために不可欠です。</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+a+spermatogenic+wave+into+a+tsunami:+synchronizing+murine+spermatogenesis+using+WIN+18,446.">Turning a spermatogenic wave into a tsunami: synchronizing murine spermatogenesis using WIN 18,446.</a> Biol Reprod. 88 (2), 40 (2013).</li><li>Yoshida, 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=The+first+round+of+mouse+spermatogenesis+is+a+distinctive+program+that+lacks+the+self-renewing+spermatogonia+stage.">The first round of mouse spermatogenesis is a distinctive program that lacks the self-renewing spermatogonia stage.</a> Development. 133 (8), 1495-1505 (2006).</li><li>Kluin, P. M., Kramer, M. F., Rooij, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatogenesis+in+the+immature+mouse+proceeds+faster+than+in+the+adult.">Spermatogenesis in the immature mouse proceeds faster than in the adult.</a> Int J Androl. 5 (3), 282-294 (1982).</li><li>Rodriguez, I., Ody, C., Araki, K., Garcia, I., Vassalli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+early+and+massive+wave+of+germinal+cell+apoptosis+is+required+for+the+development+of+functional+spermatogenesis.">An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis.</a> EMBO J. 16 (9), 2262-2270 (1997).</li><li>Kangasniemi, M., 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=Modulation+of+basal+and+FSH+dependent+cyclic+AMP+production+in+rat+seminiferous+tubules+staged+by+an+improved+transillumination+technique.">Modulation of basal and FSH dependent cyclic AMP production in rat seminiferous tubules staged by an improved transillumination technique.</a> Anat Rec. 227 (1), 62-76 (1990).</li><li>Martianov, I., 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=Late+arrest+of+spermiogenesis+and+germ+cell+apoptosis+in+mice+lacking+the+TBP-like+TLF/TRF2+gene.">Late arrest of spermiogenesis and germ cell apoptosis in mice lacking the TBP-like TLF/TRF2 gene.</a> Mol Cell. 7 (3), 509-515 (2001).</li><li>Henriks&#233;n, K., Parvinen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-specific+apoptosis+of+male+germ+cells+in+the+rat:+Mechanisms+of+cell+death+studied+by+supravital+squash+preparations.">Stage-specific apoptosis of male germ cells in the rat: Mechanisms of cell death studied by supravital squash preparations.</a> Tissue Cell. 30 (6), 692-701 (1998).</li></ol>

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マウス精子形成時の減数分裂の進行を支配する細胞の活動を研究するための有用なモデル生物であると証明します。さらに、それはツールが終了するので、多くのイベントなど減数分裂前期から私の精子形成過程の研究に仕出し料理を開発する必要がある、性的に二形であります。このプロトコルでは、精細管スカッシュ法可視化とマウス精子形成サイクルのさまざまな段階の研究について?...

減数分裂は、細胞分裂の 2 つの連続したラウンドを DNA 複製の 1 つのラウンドの後、複雑な携帯電話イベントです。正確な染色体分離を確保するための減数分裂の初期の段階で減数分裂固有のいくつかのイベントを調整しなければなりません。これらのイベントは、相同組み換え、姉妹の共同向きの完了を含んで第一減数分裂と同族体の chiasmata を解決するコヒーシン複合体の段階的な損失の間に体。これらのプロセスの精密な規則は、豊饒を維持するために、発達障害の遺伝的および自然流産1をもたらす染色体紡錘イベントを防ぐために必要です。
減数分裂のキー イベントが行われる男性と女性の両方で、重要な時間および作用機構の違い精子と卵形成2間存在します。たとえば、減数分裂女性、前期私は萌芽期の開発中に発生した、思春期まで dictyate 段階で逮捕。対照的に、精子形成は思春期で逮捕なしの大人の生活の中での波の進行開始します。男性と女性の減数分裂の違いが具体的に精母細胞や卵母細胞でこれらのプロセスを評価するに向かって仕出し料理方法を開発する必要性を強調しています。現在、主として減数分裂の進行を評価するクロマチン スプレッド3,4、5の使用に依存します。クロマチン スプレッドは減数分裂期の染色体を調べる場合に役立ちますが、彼らは紡錘体微小管、中心、核膜、テロメアの添付ファイルなどの細胞構造の評価を防止する、細胞の完全性を維持するために失敗します。ライブ イメージングと長期培養技術が大きく進歩した女性減数分裂; の私達の理解ただし、そのままセル全体を視覚化する同様のアプローチについては、精子6、7の研究のため実装頻度。男性減数分裂中の動的イベントを視覚化するために急速にマウス精母細胞8,9の開発の細胞の機能を評価するために確立された尿細管スカッシュ技術を適応しています。ここで説明する方法は、精子形成サイクルのさまざまな段階で複数の細胞構造の研究を有効にするセルの整合性を維持します。
この尿細管スカッシュ技術は、蛍光顕微鏡による細胞構造の評価を可能にするセル全体アプローチです。ヘマトキシリン ・ エオシン染色パラフィン埋め込まれた精巣の凍結切片の免疫蛍光ラベリングなど男性マウスの減数分裂の進行を視覚化する一般的な組織学的アプローチは、減数分裂の進行の広範な概要。ただし、これらのテクニックは、単一細胞を減数分裂10,11全体で発生するイベントの詳細な分析に必要な範囲に解決する失敗します。減数分裂のプロセスを視覚化するための代替技術は、特定し、核物質の3,4,5を解決する状態の中断を重要な chemiosmotic に依存します。これらの化学治療は、一次精母細胞以外の細胞型の観測を妨げます。最近説明滑川による分離精母細胞の核の構造を維持する研究コミュニティが有効になって、cytospin およびいくつかの研究所の4にすぐに利用できない場合があります付属品の使用が必要です。対照的に、尿細管スカッシュ テクニックだけはほとんどの細胞生物学研究所で一般的に標準的な装置が必要です。
圏内には精細管、セルトリ細胞、精原細胞、第一次および二次精母細胞など多様な細胞型を表示するは、ここで説明した尿細管スカッシュ メソッドを使用できます。若年マウス精子形成の近く同期の最初の波でこの手法を結合することにより減数分裂12を操作するときに、精子細胞の豊かな人口を得ることが可能です。このプロセスは、精子形成、初期前期イベントなどを通してプロセスの詳細な分析、G2/MI と後期遷移、および精子形成に中期に許可されます。さらに、尿細管スカッシュ準備は染色体 (interchromatid ドメイン (Icd) や体) と (由来と一分素材/行列) 中心の細胞像を可視化する使用できます。スカッシュ メソッドは、クロマチン スプレッドと蛋白質の抽出など、他の実験的アプローチと並行で容易に実行できます。さらに、この手法は、直接可視化13スライドに精子細胞を入金する正常に変更されています。
ここで説明したメソッドには、野生型 c57bl/6 j マウスにおける G2/MI 転移を分析するセル全体精細管スカッシュ テクニックが含まれます。第一減数分裂に入る一次精母細胞の細胞学的特徴は、減数分裂のスピンドルを観察する蛍光顕微鏡で視覚化しました。この汎用性の高い手法は、他の減数分裂の段階と異なる種類の細胞を可視化する簡単に変更できます。技術はまた、DNA と RNA の魚のアプローチなど、代替の可視化戦略に従うです。

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			<td style="width:167px;">1 in 1000</td>
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		<tr height="21">
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			<td height="21" style="height:21px;width:251px;">Mouse anti-&#947;H2AX</td>
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			<td style="width:137px;">MA1-2022</td>
			<td style="width:167px;">1 in 500</td>
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			<td height="21" style="height:21px;width:251px;">Mouse anti-CENT3</td>
			<td style="width:247px;">Abnova</td>
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			<td height="21" style="height:21px;width:251px;">Rabbit anti-REC8</td>
			<td style="width:247px;">Courtesy of&#160; Dr. Karen Schindler</td>
			<td style="width:137px;">N/A</td>
			<td style="width:167px;">1 in 1000</td>
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</table>

a seminiferous tubule squash technique for the cytological analysis of spermatogenesis using the mouse model

雄の減数分裂の進行は、規制の厳しい携帯電話イベント数の協調的な行動を必要とするプロセスです。減数分裂の間に発生したエラーは不妊、妊娠の損失または遺伝的欠陥につながることができます。思春期と成人期にわたって継続の発症時、精母細胞の連続準同期波まで精子を受ける、最終的に半数体精子を形成します。マウスの精母細胞の減数分裂の開始を受けての最初の波は産後 10 日目 (10 dpp) で表示され、35 dpp で成熟した精子と精細管の内腔に放出されます。したがって、関心の高濃縮の人口を得るためにこの発達の時間ウィンドウ内でマウスを利用するため有利です。珍しいセル段階の分析は、細管内細胞集団の多様性を高める連続の精波の貢献のための古いマウスのより困難です。ここで説明する方法は、マウス精原細胞、精母細胞、細胞などの精細管内で見つかった細胞の細胞学的評価のため簡単に実装されている手法です。尿細管スカッシュ技術分離の雄性生殖細胞の整合性を維持でき、他の手法とは表現しにくいセル構造の検討。この尿細管スカッシュ手法の応用可能性を示すためには、紡錘アセンブリは精母細胞転移中期に前期を進める (G2/MI 転移) で監視されました。さらに、染色体花束形成、減数分裂の性染色体の不活性化 (MSCI)、中心体の複製は、この尿細管スカッシュ メソッドを使用して観察することができます細胞構造の例として評価されました。突然変異または外因性の摂動によって引き起こされる精子の中に特定の欠陥を特定するこの手法を使用でき、精子形成の分子理解に貢献。

ここでは、中期に前期を受けて一時的な細胞集団を視覚化する尿細管スカッシュ メソッドを使いましたが (G2/mi)、精子形成 (24 の最初の波を受けている少年の野生型マウスの精巣を収穫して濃縮した移行dpp)。図 1は、尿細管のスカッシュ メソッドを使用して視覚化することができますさまざまな細胞段階の代表的なイメージを示しています。中?...

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A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

この尿細管スカッシュ手法の目的は、急速に細胞の完全性を維持しながらマウス精母細胞の開発の細胞機能を評価するためです。このメソッドにより、精子形成のすべての段階の調査のため、マウスの減数分裂の研究のため他の生化学的な分子の生物学的アプローチと一緒に簡単に実装することができます。

精細管スカッシュ マウス モデルを用いた精子の細胞学的解析法

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

a-seminiferous-tubule-squash-technique-for-cytological-analysis

Research

JoVE Journal

Developmental Biology

14.8K ビュー.  Johns Hopkins Bloomberg School of Public Health. この尿細管スカッシュ手法の目的は、急速に細胞の完全性を維持しながらマウス精母細胞の開発の細胞機能を評価するためです。このメソッドにより、精子形成のすべての段階の調査のため、マウスの減数分裂の研究のため他の生化学的な分子の生物学的アプローチと一緒に簡単に実装することができます。

ビデオ: A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

同軸ノズルを用いて多能性幹細胞のアルギン酸カプセル化

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

発生生物学

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

の共焦点分析を用いました<em&gt;アフリカツメガエル</em&gt; WntおよびShhのモルフォゲン勾配の調節を調査するために、

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

ハイスループットマイクロプレート呼吸測定のためのマウス骨格筋の最小量から単離されたミトコンドリアを使用して

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

マウス胚中足骨の文化：軟骨内骨化の生理学的モデル

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

マウスの早い妊娠の特性の三次元の高周波超音波の新規な使用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

三次元レンダリングとカルビンディン抗体、解析人間の胎盤絨毛血管のネットワークを明らかにしました。

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

誘導多能性幹細胞由来肝細胞を用いた家族性高コレステロール血症ひと肝臓キメラマウス モデル

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to restrict knee joint mobility is a widely accepted model to generate experimental contracture. However, implanting application will inevitably cause surgical trauma to the animals. Aiming to develop a less invasive approach, we combined a muscle-gap separation modus with a previously reported mini-incision skill during the surgical procedure: Two mini skin incisions were made on the lateral thigh and leg, followed by performing muscle-gap separation to expose the bone surface. The rat knee joint was gradually immobilized by a preconstructed internal fixation at approximately 135&#176; knee flexion without interfering essential nerves or blood vessels. As expected, this simple technique permits rapid postoperative rehabilitation in animals. The correct position of the internal fixation was confirmed by an x-ray or micro-CT scanning analysis. The range of motion was significantly restricted in the immobilized knee joint than that observed in the contralateral knee joint demonstrating the effectiveness of this model. Besides, histological analysis revealed the development of fibrous deposition and adhesion in the posterior-superior knee joint capsule over time. Thus, this mini-invasive model may be suitable for mimicking the development of immobilized knee joint contracture.

Here, we present a protocol to describe a minimally invasive technique for knee joint immobilization in a rat model. This reproducible protocol, basing on muscle-gap separation modus and the mini-incision skill, is suitable for studying the underlying molecular mechanism of acquired joint contracture.

ラットの固定化誘発膝屈曲収縮を研究するための小型侵襲的内部固定技術

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to ...

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a differentiation state to sustain demands of the blood formation. However, it has been difficult to analyze the metabolic status (mitochondrial respiration and glycolysis) of HSPCs due to their limited numbers and lack of optimized protocols for non-adherent, fragile HSPCs. Here, we provide a set of clear, step-by-step instructions to measure metabolic respiration (oxygen consumption rate; OCR) and glycolysis (extracellular acidification rate; ECAR) of murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs. This protocol provides a higher amount of LSK HSPCs from murine bone marrow, improves the viability of HSPCs during incubation, facilitates extracellular flux analyses of non-adherent HSPCs, and provides optimized injection protocols (concentration and time) for drugs targeting oxidative phosphorylation and glycolytic pathways. This method enables the prediction of the metabolic status and the health of HSPCs during blood development and diseases.

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.

造血幹細胞細胞代謝の解析

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a ...

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I&#8211;XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

This protocol describes transillumination-assisted microdissection of segments of adult mouse seminiferous tubules representing specific stages of seminiferous epithelial cycle, and cell types therein, and subsequent immunostaining of squash preparations and intact tubule segments.

精細管スカッシュマウスモデルを用いた精子の細胞学的解析法

要約

さらに動画を探す

この動画の章

同軸ノズルを用いて多能性幹細胞のアルギン酸カプセル化

の共焦点分析を用いました

ハイスループットマイクロプレート呼吸測定のためのマウス骨格筋の最小量から単離されたミトコンドリアを使用して

マウス胚中足骨の文化：軟骨内骨化の生理学的モデル

マウスの早い妊娠の特性の三次元の高周波超音波の新規な使用

三次元レンダリングとカルビンディン抗体、解析人間の胎盤絨毛血管のネットワークを明らかにしました。

誘導多能性幹細胞由来肝細胞を用いた家族性高コレステロール血症ひと肝臓キメラマウスモデル

ラットの固定化誘発膝屈曲収縮を研究するための小型侵襲的内部固定技術

造血幹細胞細胞代謝の解析

下流免疫染色解析のためのマウス半離層上皮周期の特定段階のトランスイルミネーション支援解離

精細管スカッシュ マウス モデルを用いた精子の細胞学的解析法

要約

さらに動画を探す

この動画の章

同軸ノズルを用いて多能性幹細胞のアルギン酸カプセル化

の共焦点分析を用いました

ハイスループットマイクロプレート呼吸測定のためのマウス骨格筋の最小量から単離されたミトコンドリアを使用して

マウス胚中足骨の文化：軟骨内骨化の生理学的モデル

マウスの早い妊娠の特性の三次元の高周波超音波の新規な使用

三次元レンダリングとカルビンディン抗体、解析人間の胎盤絨毛血管のネットワークを明らかにしました。

誘導多能性幹細胞由来肝細胞を用いた家族性高コレステロール血症ひと肝臓キメラマウス モデル

ラットの固定化誘発膝屈曲収縮を研究するための小型侵襲的内部固定技術

造血幹細胞細胞代謝の解析

下流免疫染色解析のためのマウス半離層上皮周期の特定段階のトランスイルミネーション支援解離

精細管スカッシュマウスモデルを用いた精子の細胞学的解析法

誘導多能性幹細胞由来肝細胞を用いた家族性高コレステロール血症ひと肝臓キメラマウスモデル