This work was supported by grant from NIDDK (DK065949 for GG). We thank Dr. Brenda M. Jarvis and Jeff Duryea Jr. for reading the manuscript.

動物の使用は、プロトコルM /区のためのヴァンダービルト施設内動物管理使用委員会によって承認された181分の11で指定された手順に従います。 CD1またはCBA / BL6マウスは、市販業者から購入し、新生児マウスを得るために、ヴァンダービルトの動物施設で交配しました。 1.マウスの作製、ストックソリューション、および機器<ol><li>マウスのクロスの場合：マウスのクロスを設定し、実験計画を支援するために差し込む日付を記録します。 CD1マウスは、通常、交配後19日目の周りに出産します。使用CD1またはCBA / BL6は、それぞれ、CD1またはCBA / BL6新生児を得ることが横切ります。 2ライン間の交雑は、CD1雌及びCBA / BL6雄を利用します。 </li><li>コラゲナーゼ在庫について：高精密天秤で200mgのコラゲナーゼタイプIVの重量を量ります。 50 mL遠心管に移します。カルシウムと40 mLのHBSSに溶かし2+ / Mgの2+（それぞれ1.26および0.5 mMのは、）を5mg / mLのストック溶液を作製しました。 <ol><li>へのコラゲナーゼを許可します穏やかに振盪しながら〜30分間溶解します。少なくとも1年間有効のままになりストックとして-20℃で凍結させた溶液を、アリコートとキープ。 </li></ol></li><li>解剖および島ピッキングのためのピンセット、はさみ、およびP20のヒントのいくつかのペアを滅菌するオートクレーブ。 </li><li> 1 Mグルコース在庫について：グルコースを溶解するために50mlのRPMI 1640培地を追加し、50 mL遠心管に9グラムのグルコースを量ります。使用するまで4℃に保ち、ない6カ月以上。 </li></ol> 2.ワーキングソリューション注：膵島単離の日は、以下の試薬を準備します。 <ol><li>コラゲナーゼIV型作業溶液の場合：解凍し、氷上でコラゲナーゼの株式を希釈します。各1.7 mLのマイクロチューブに0.15 mLのストックを分注します。 Ca 2+ / Mgの2+で1.35 mLのHBSSを追加します。チューブを5回反転します。氷上で15分間のままにしておきます。 <ol><li>微量で3分間、高速で回転。上清を新しいチューブに移します。使用するまで氷上に残します。不溶性の破片でチューブを捨てます。 </li></ol></li><li>完全RPMI 1640培地の場合：500 mLのRPMI 1640培地に2.5 mLの1 Mグルコース、FBS不活化50 mLの熱、および5 mLの100倍ペン連鎖球菌の株式を追加します。使用するまで氷上におきます。 </li></ol> 3.膵臓の単離と消化<ol><li>承認されたプロトコルに従って断頭続いイソフルランと新生児を、安楽死させます。 </li><li>その腹を上に向けてマウスを置きます。殺菌のための70％エタノールでスプレーします。 </li><li>ピンセットで腹の皮膚を持ち上げ、陰部から胸郭に、ハサミで正中線に沿って縦方向に皮膚や筋肉層を切ります。これは、すべての内臓を露出させるために腹腔を開きます。 </li><li>ピンセットで100センチの皿にすべての内臓を転送します。胃と脾臓と十二指腸11にしがみつい腹部分にしがみつい背部分で散乱臓器である膵臓を、見つけます。 </李&gt; <li>臓器を沈めるために皿にHBSSを追加します。溶液中では、膵臓組織はもはや十二指腸、胃、または脾臓にしがみつくません。それは、その典型的な白い色の解剖範囲の下で容易に認識可能です。 HBSSで新しい60ミリメートル皿に離れて周囲の組織と転送から膵臓組織を剥離するためにピンセットを使用してください。 </li><li>任意のディメンション間5ミリメートルより小さな部分にハサミで各膵臓をカットします。 1.5 mLのマイクロ遠心チューブに移します。 5膵臓（P7より若い）まで1チューブ内で消化することができます。 P8-P16マウスの場合、膵臓あたり1チューブを使用しています。 </li><li>各チューブに1 mLのコラゲナーゼ作業溶液（それぞれP1-P7の膵臓用0.2 mLの各P8-P16膵臓のために0.5 mLのを使用してください。） - 0.5を追加します。最大15分間37℃のインキュベーター内でチューブを残します。チューブを2回毎分を反転。 </li><li> 〜5分後、膵臓組織の消化状況を監視するためにチューブをタップします。膵臓のほとんどまで消化プロセスを続行組織は、直径&lt;2ミリメートルで、断片化された細胞塊として表示されます。溶解物を、通常の腺房細胞の自己消化によるものである、曇って表示されます。 </li></ol> 4.溶解物の洗濯<ol><li>微量で10秒間500×gで溶解液をスピン。 P1,000ピペットマンで上澄み層を除去。上清は濁ったが、細胞クラスターを欠いて表示されます。すぐに起因するいくつかの粘着性のDNAの存在に下部の組織片を離れて吸引し得る、吸引を使用しないでください。 </li><li> 1 mLのRPMI 1640完全培地を追加します。断片化した溶解物を再懸濁するために静かにチューブをタップします。アップ裏返し、10回ダウン。繰り返し手順4.1。 </li><li>繰り返しステップ4.2をさらに2回または上清が透明になるまで。 1 mLの完全RPMI 1640培地で洗浄した膵臓の断片を再サスペンド。 </li></ol> 5.膵島単離注：膵臓の小さな番号については、直接手で摘み、以下のように（5.1）は、膵島ISOLのために使用することができますエーション。膵臓の多数のために（&gt; 6）、5.2に概説された方法が好ましいです。 <ol><li>直接手でピッキング： <ol><li> 60ミリメートル皿にステップ4.3からの溶解物を転送します。 5 mLの完全RPMI 1640培地を追加します。解剖顕微鏡下で持参してください。 </li><li>顕微鏡での明視野照明を設定します。最大電力の約50％の光量を調整します。この状態で、島は腺房細胞のような暗い不規則な形のクラスターのに対し（ 図1A）、など少しピンクのクラスタを表示されます。 （対物レンズと接眼のパワーを組み合わせる）〜50Xの倍率は、小島とピッキングに対して同時に開くピペットチップを可視化する最良の方法を提供することをお勧めします。 </li><li>腺房クラスタを避けながら、P20のピペットチップで膵島をピックアップ。 RPMI完全培地（ 図1B）を持つ新しい60ミリメートル皿に小島-濃縮された画分を分注します。 </li><li>二回以上手でピッキングプロセスを繰り返します。新しい60で純粋な島を残します4 mLの完全RPMI培地（ 図1C）とmmディッシュ。 </li></ol></li><li>勾配遠心分離： <ol><li> 1.077グラム/ mlの密度で2mLのポリスクロースおよびジアトリゾ酸ナトリウムがプリロードされた15 mL遠心チューブを準備します。使用するまで氷上でチューブを残します。 </li><li>パスツールピペットでポリスクロースおよびナトリウムジアトリゾ液の上部にステップ4.3からサスペンションを転送します。底層を乱さないでください。最大10の組織が新生または2 P17膵臓を各15mLの管に装填することができます。 </li><li> 4℃で15分間、スイングローターで〜600×gでチューブをスピン。ブレーキオフの設定を使用します。 </li><li>遠心分離した後、新しい15mLのチューブに（理想的な動作条件の下で、島は遠心分離後ポリスクロースと文化の媒体間の相間に配置されますことに注意してください）メディアと相間層を転送します。 10 mLのメディアを追加し、穏やかに、しかし完全に混合します。 </li><li>膵島濃縮画分をスピンダウン5分間、300×gで。洗浄培地を除去します。完全RPMI培地で洗浄をもう一度繰り返します。主に膵島およびダクト（ 図1D）が含まれている 4 mLのメディアへの再懸濁ペレット。 </li><li>セクション5.1に概説された膵島を手で選びます。 </li></ol></li></ol>単離された膵島6. GSISアッセイ<ol><li> 37℃の組織培養インキュベーター中で2時間、完全RPMI培地中でステップ5.1.4から膵島を培養します。 </li><li>解剖顕微鏡下でRPMIを削除するには、ピペットを使用してください。プレート3に2.8 mMグルコースと3 mLのKRBのソリューションを追加します。膵島を洗浄するために10回渦巻。 KRBソリューションを削除します。 <ol><li>一度洗浄工程を繰り返します。インキュベーター中で1時間、37℃で3 mLのKRB溶液で島をインキュベートします。 </li></ol></li><li> 12ウェルプレートの各ウェルにアリコート1mLのKRB。インキュベーター中で37℃にKRBストックとプレートを事前に温めます。 </li><li> 6.2のように予熱したKRBで二回の膵島を洗ってください。トランスFER予熱したプレートの各ウェルに10の島。 37℃のインキュベーター中でインキュベートします。 </li><li> 45分後、50μLの上清を撤回。これは、基礎グルコースの下で分泌されるインスリンのソリューションが含まれています。 </li><li> 32μL500 mMグルコースを追加します。溶液を混合するためにプレートを20回渦巻。 37℃のインキュベーター中でインキュベートします。プレートを5回ごとに5分を渦巻。 </li><li> 45分後、50μLの上清を撤回。これは、高グルコース下で分泌されるインスリンです。 </li><li> 1.5 mLのチューブに、すべての膵島を転送します。 30分間-20℃で凍結します。 5分間、室温で解凍します。凍結融解処理を繰り返します。 </li><li> 20mMのHClで0.5 mLの70％エタノールを追加します。一晩おきます。これは、ランゲルハンス島に残されたインスリンです。 </li><li>インスリンレベルをアッセイするために、従来のELISAを使用して、先に説明した3。 </li></ol>

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The authors declare no competing financial interests for the described work.

ここでは、従来の灌流のためには小さすぎる膵臓から膵島単離にステップバイステップのプロトコルを提供します。このような等 β細胞の精製、遺伝子発現解析、膵島β細胞の成熟、増殖、細胞ストレス応答、細胞生存率、代謝、機能GSISの維持、などのすべての島ベースの研究のための準備ができて膵島をもたらすことが期待されます。これは、我々の知る限りに、新生仔マウスから?...

Isolating pure pancreatic islets is essential for assaying glucose stimulated insulin secretion (GSIS) of beta cells and for islet transplantation from cadaveric donors 1-3. It is also necessary to establish endocrine gene expression in islet cells 4, 5. For this purpose, detailed protocols have been established to allow for isolation of pancreatic islets from large pancreata (6 and references therein). These methods are based on enzymatic perfusion to dissociate acinar from islet tissues, coupled with gradient separation and hand picking. Thus, islet isolation from large pancreas can be performed readily in most laboratories. On the other hand, no detailed step-by-step protocol exists to allow for the isolation of islets from pancreata that are too small to perfuse.
Studying gene expression and function of neonatal islets is important. Neonate islets have different properties from adults in insulin secretion and proliferation capability 7, 8. However, isolating islets from newly born animals, especially mice is challenging due to the small size of the newly born pancreas. The size prevents the usual perfusion process when collagenase is injected though the pancreatic duct. Indeed, several papers have presented studies along these lines, with enzyme or non-enzyme aided isolation procedures 7, 9, 10. However, detailed description of the islet isolation process with visual aid is lacking 7, 9, making it a challenge for most researchers to perform similar studies. 
We have explored several different conditions that yield high quality islets from neonatal mice. Here we present a protocol that is expected to help researchers learn the key details in the islet isolation process. This protocol is applicable to mouse pancreas up to two weeks of age, after which perfusion can be performed for routine islet isolation. Islets can be directly used for insulin secretion and gene expression assays.

<table border="0" cellpadding="0" cellspacing="0" width="769">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Collagenase&#160; Type IV</td>
			<td style="width:253px;">Sigma Aldrich, St Louis, MO</td>
			<td style="width:128px;">C5138</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:169px;">1X HBSS with Ca2+/Mg2+</td>
			<td style="width:253px;">Mediatech/Cellgro, Manassas, VA</td>
			<td>MT21020CV</td>
			<td style="width:219px;">If HBSS wihout Ca2+/Mg2+ is obtained, CaCl2 and MgSO4 can be added to 1.26 and 0.5 mM, respectively.&#160;</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:169px;">RPMI 1640 w/o glucose</td>
			<td style="width:253px;">Thermo Fisher Scientific/Life Technologies, Waltham, MA</td>
			<td>11879-020</td>
			<td style="width:219px;">Glucose needs to be added to specific levels to not interfere with seusequent islet usage.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Glucose</td>
			<td style="width:253px;">Sigma Aldrich, St Louis, MO</td>
			<td>G-7021&#160;</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:169px;">polysucrose and sodium diatrizoate solution</td>
			<td style="width:253px;">Sigma Aldrich, St Louis, MO</td>
			<td>Histopaque-10771</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Stereoscope</td>
			<td style="width:253px;">Carl-Zeiss, Oberkochen, Germany</td>
			<td>Stemi2000</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Stereoscope</td>
			<td style="width:253px;">Leica, Wetzlar, Germany</td>
			<td>Leica M165</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Microcentrifuge</td>
			<td style="width:253px;">Eppendorf, Hauppauge, NY</td>
			<td>Centrifuge 5417C</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Centrfuge</td>
			<td style="width:253px;">Eppendorf, Hauppauge, NY</td>
			<td>Centrifuge 5810R</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">15-ml centrfuge tubes&#160;</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>89039-666</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">50-ml centrfuge tubes&#160;</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>89039-658</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Precision balance</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>VWR-225AC</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Microfuge tubes</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>87003-294</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Pipetman P1000</td>
			<td style="width:253px;">Fisher Scientific,&#160; Waltham, MA</td>
			<td>F123602</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Pipetman P20</td>
			<td style="width:253px;">Fisher Scientific,&#160; Waltham, MA</td>
			<td>F123600</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">100X15 millimeter dish</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>25384-088</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">60X15 millimeter dish</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>25384-168</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:169px;">12-well plates</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>665-180</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:169px;">Scissor</td>
			<td style="width:253px;">Fine Scientific Tools, Foster City, CA</td>
			<td>14080-11</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Tweezers</td>
			<td style="width:253px;">Fine Scientific Tools, Foster City, CA</td>
			<td>5708-5</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:169px;">CD1 mice</td>
			<td style="width:253px;">Charles River Laboratories, Wilminton, MA&#160;</td>
			<td>CD-1</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:169px;">C57BL/6J</td>
			<td style="width:253px;">The Jackson laboratory, Farmington, CT&#160;</td>
			<td>C57BL/6J</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">B6CBAF1/J</td>
			<td style="width:253px;">The Jackson laboratory, Farmington, CT&#160;</td>
			<td>B6CBAF1/J</td>
			<td style="width:219px;"></td>
		</tr>
	</tbody>
</table>

effective isolation of functional islets from neonatal mouse pancreas

大型哺乳動物の膵臓からの灌流ベースの膵島分離プロトコルは十分に確立されています。このようなプロトコルは容易に準備ができて、コラゲナーゼ注入とその後の組織潅流を可能にする膵管の大きなサイズのため、多くの研究室で行われています。灌流は小さな膵臓で容易に達成可能ではないので、コントラスト、小さな膵臓から膵島単離では、新生児マウスのような、挑戦的です。ここでは、視覚的な支援を受けて、新たに生まれたマウスからの膵島の相当数を回復する詳細な簡単な手順について説明します。新たに切開全膵臓を微量遠心管中、37℃で、ハンクス平衡塩溶液（HBSS）中に溶解し0.5ミリグラム/ mLのコラゲナーゼIVで消化しました。チューブは、組織の分散を助けるために、定期的にタップされました。組織の大部分を1mmの周りの小さいクラスタに分散させたときに、溶解物を、10％ウシ胎児血清（FBS）で培養培地で3〜4回洗浄しました。アイレットクラスター、認識腺房組織を欠く、その後ステレオスコープを解剖下で回収することができます。新たに生まれたマウスの膵臓あたりの大型小島に80小 - この方法は、20を回収します。これらの島は、インスリン分泌、遺伝子発現、および文化を含むほとんどの考えられる下流のアッセイに適しています。インスリン分泌アッセイの例は、単離プロセスを検証するために提示されています。遺伝的背景と消化の程度が収量を決定する最大の要因です。それは内分泌外分泌分離に役立つように、高い活性を有する新たに作られたコラゲナーゼ溶液が好ましいです。洗浄中のすべてのソリューションやウシ胎児血清中の陽イオンの存在[カルシウム（Ca 2+）とマグネシウム（Mg 2+）] /ピッキングメディアは、適切な整合性を持つ島の良好な収率のために必要です。良好なコントラストおよび倍率の解剖スコープにも役立ちます。

各小マウスの膵臓から80小島 - 最適な条件の下では、提示された方法は、20を得ることができます。この数は、マウスの遺伝的背景、年齢に依存し、島の大きさは、回収されます。一般的に使用され、CD1の中で繁殖させたアウトとC57BL / 6J純血マウスCD1とC57BL / 6の間のハイブリッドまたは商業B6CBAF1 / Jマウスが何よりも小さいサイズの小さい島を生産します。一般的にピ...

Watch this Scientific Journal Video about Effective Isolation of Functional Islets from Neonatal Mouse Pancreas at JoVE.com

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

我々は、新生児マウスから無傷の膵島を単離するために、本明細書のプロトコルを記述します。膵臓は、部分的に洗浄し、ハンドピッキング続いて、コラゲナーゼで消化しました。 20から80膵島クラスタは、いくつかの島の研究に適しており、新たに生まれたマウスから膵臓ごとに取得することができます。

新生児マウスの膵臓からの機能性膵島の効果的な分離

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

effective-isolation-of-functional-islets-from-neonatal-mouse-pancreas

Research

JoVE Journal

Developmental Biology

10.0K ビュー.  Vanderbilt University Medical School. 我々は、新生児マウスから無傷の膵島を単離するために、本明細書のプロトコルを記述します。膵臓は、部分的に洗浄し、ハンドピッキング続いて、コラゲナーゼで消化しました。 20から80膵島クラスタは、いくつかの島の研究に適しており、新たに生まれたマウスから膵臓ごとに取得することができます。 

ビデオ: Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

新生児ラット心筋細胞の単離と凍結保存

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

発生生物学

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

ゼブラフィッシュ胚からの単一細胞の単離とキャラクタリゼーション

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

インビトロコロニーアッセイでトライ強力な前駆細胞を特徴付けるための大人のマウスの膵臓から分離されました

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

マウス胚性造血内皮細胞の単離

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

マウス骨格筋からのI型およびII型ペリクルの単離

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

心血管イメージング (30/45 MHZ) の高周波超音波を用いた胎児マウス

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

効率的な世代の膵臓・十二指腸ホメオ蛋白質 1+後部の前腸・膵前駆細胞接着培養における hPSCs から

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological conditions. Post-mitotic neurons represent a major cell type affected by aging. Although multiple mammalian models of neuronal aging exist, they are challenging and expensive to establish. The roundworm Caenorhabditis elegans is a powerful model to study neuronal aging, as these animals have short lifespan, an available robust genetic toolbox, and well-cataloged nervous system. The method presented herein allows for seamless isolation of specific cells based on the expression of a transgenic green fluorescent protein (GFP). Transgenic animal lines expressing GFP under distinct, cell type-specific promoters are digested to remove the outer cuticle and gently mechanically disrupted to produce slurry containing various cell types. The cells of interest are then separated from non-target cells through fluorescence-activated cell sorting, or by anti-GFP-coupled magnetic beads. The isolated cells can then be cultured for a limited time or immediately used for cell-specific ex vivo analyses such as transcriptional analysis by real time quantitative PCR. Thus, this protocol allows for rapid and robust analysis of cell-specific responses within different neuronal populations in C. elegans.

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

回虫カエモルハブ炎エレガンスからの特定ニューロン集団の分離

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

マウス骨格筋移植からの原発性筋芽生の単離と分化

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

ゼブラフィッシュ胚の簡易かつ効果的な移植装置

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...