資金はMDに「PROGETTO concorso伊IRCCS病院周辺マッジョーレ総合病院2009年から2010年」、およびAssociazioneバンビーノNefropatico ABN Onlus、ミラノから提供された

全ての動物実験は、科学的な目的のために使用される動物の保護に関する国内および欧州の現行の規制に従って行ったとミラノ大学の倫理委員会によって審査され、承認された。 初代骨芽細胞の1。分離この方法では、わずかな修正を加えて、Dodig ら 14によって記載された手順に従います。 <ol><li> 0.1％コラゲナーゼPおよび0.05％トリプシンHBSS培地に（2.5％トリプシン、10X、無フェノールレッドから開始）（ハンクス平衡塩溶液、無カルシウム、マグネシウム、またはフェノールレッド）を追加することで、メディアを消化準備します。 </li><li> （手順はわずか1などのマウスに適用することができます）3〜4日齢のマウスを使用してください。 </li><li>断頭により犠牲に。 70％エタノールでスプレーヘッド、氷上に保つ。ピンセット及び解剖/外科ハサミで皮膚や筋肉層を除去する。 </li><li>優しく頭頂骨を削除し、2等分に矢状縫合に従うことによって、それらを分離する。骨は縫合糸および任意の接着性組織を含まず、手続きが完了するまでHBSS培地を含むウェルプレート内の個々の骨片を配置していることを確認してください。 </li><li> HBSSを捨て、各ウェルに37℃で15分間消化培地を追加</li><li>上清を捨てる。 </li><li>再び37℃で15分間消化培地を追加</li><li>このとき上清を保存し、α-MEM（最小必須培地）に入れて、10％FBS（ウシ胎児血清）および1％ストレプトマイシン/ペニシリンを補充。 </li><li>繰り返します1.7と1.8倍を繰り返します。 </li><li>プール5分間425グラムで遠心分離して回収した画分3、スピン細胞、上清を捨て、細胞ペレットを再懸濁し、25cm 2のフラスコに転送するために補充したα-MEMの3ミリリットルを追加します。 </li><li>破片および死細胞を除去するために24時間後に培地を変更します。 </li><li>グリコ50μg/ mlのアスコルビン酸（AA）および3mMの追加cerol 2 - リン酸二ナトリウム塩水和物（GP）α-MEM培地である。 </li><li>その後、一日おきに（α-MEMプラスAA / GP）培地を変更してください。 </li></ol> 2。ATRAプロトコル<ol><li>不活性化を防止する暗い部屋でATRA（オールトランス型レチノイン酸）ですべての実験を行っています。 </li><li> HBSS培地中の1Xトリプシン溶液を調製し、使用するまで37℃で維持する。 </li><li>フルエント細胞から培地を除去し、慎重にHBSSでそれらを洗ってください。 </li><li> 1ミリリットルのトリプシンを添加し、穏やかにすべての細胞は、トリプシンによって覆われることを可能にするために、フラスコを回転させ、37℃で3分間インキュベート</li><li>倒立顕微鏡下で細胞の実際の剥離を確認してください。 </li><li>トリプシンを不活性化するα-MEM培地の3ミリリットルを追加します。 </li><li>細胞を回収し、425グラムで15分間遠心する。 </li><li>新鮮な培地中で細胞ペレットを再懸濁する。 </li><li>血球計数器の計数室の低下を置き、数分のために解決することを可能にし、CEを数えるLL番号。 </li><li> 10,000個の細胞のα-MEMプラスAA / GPと/ cm 2の密度で25cm 2のフラスコ内で場所細胞。細胞密度が細胞突起および細胞間の接点の最適な開発に関連していることに注意してください。出発密度より高く、10,000細胞/ cm 2の細胞プロセスの適切な発達を阻害するが、より低い細胞数は、細胞間の連絡先が存在しないため、時期尚早の細胞死をもたらす。 </li><li> 24時間毎に培地から5μmのATRAを追加します。 </li><li>形態を評価するために日々のセルを監視：形態学的変化は、一般的に48時間後に観察される。場合は、最初の24〜48時間後に、細胞は、依然として上記の密度でreplate、増殖している。成熟した骨細胞の均質な集団は、4〜5日後に存在している。必要に応じて、12〜15日まで、任意の時点で細胞を使用する。 </li><li>細胞は、（このようなカバースリップまたはウェルプレートのような）他の支持体上にメッキされている場合、上記の濃度が維持されていることを確認してくださいと使用するまで、ATRAごとに24時間を追加していく。実験の前に少なくとも24時間のための新しいサポートに付着する細胞を残す。 </li></ol> 3。免疫染色<ol><li>免疫染色のために、カバースリップ上の細胞をプレート（例えばバリー15を参照）確立された方法に従ってください。 </li><li>例えば、間接的免疫蛍光のために、室温で30分間、次いで暗所で二次蛍光標識抗体と、加湿チャンバー内で室温で1時間一次抗体で順次インキュベートし、4％パラホルムアルデヒドまたは冷アセトン中で固定し、加湿チャンバー内。 </li><li>核対比のために、DAPIまたは類似体を使用し、退色防止媒体にマウントします。 </li></ol> 4。アリザリンレッド染色と抽出<ol><li>カバースリップ上の細胞をプレート。 </li><li> 10分間、70％エタノールでカバースリップを浸漬することによって細胞を固定。 </li><li>アリザリンレッド染色溶液（1 mg / ml、pH5.5）に数滴を有する細胞をカバー30分間。 </li><li>水道水でカバースリップを洗ってください。マウントスライドガラス上グリセロールの降下と直立10Xと20X顕微鏡で倍率を用いて画像を取る。 </li><li>上記の手順の後、水道水でスライドを浸漬することにより、カバースリップを回復する。 </li><li>ウェルプレートの各カバースリップを置く。 </li><li> 60％過塩素酸（量は細胞を完全にカバーするのに十分でなければなら）を追加し、穏やかに撹拌しながら4℃でO / Nのままにしておきます。 </li><li> 450 nmの波長で分光光度法により上清の光学濃度を読み取る。 </li><li>細胞数の値を正規化するために、10分間、細胞調製物を覆うようにヤヌスグリーン（PBS中の0.2％溶液）を加える。 </li><li>超純水で丁寧に洗ってください。 </li><li> 0.5NのHClを追加する（数量はすべてのセルをカバーするのに十分でなければなら）、数秒間、手でそっと振る。 </li><li>分光光度計で615 nmの吸光度を読んでください。 </li></ol>

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著者らは、開示することは何もありません。

最後の年で骨細胞は、骨の中の最も中心的な細胞として浮上している。研究の進歩は、徐々に骨様々な疾患のための新規およびより良い治療を設計するために多大な価値が以前に実証されていない又は疑われていない骨細胞の特性の数を明らかにしている。しかしながら、骨細胞生物学の調査は骨細胞様細胞株MLO-Y4が利用可能になる前に骨芽細胞が長期にわたってサロゲート細胞として使用?...

骨細胞、最も豊富な骨細胞型は、深く骨格内に位置する高度に分岐した細胞は最終分化している。成熟した骨細胞の細胞体は、骨小腔に含まれており、多様な形状を有する;丸みを帯びた骨細胞は、骨梁1に、より頻繁であるのに対し、細長い細胞体と骨細胞は、皮質骨で発見されています。分枝樹状突起は、他の骨ではなく、他の骨の細胞型、骨髄、血管及び関連する周皮細胞を有するだけでなく、複数の連絡先を行う複雑なウェブを形成する、細管と呼ばれる小さなチャネルにおいて細胞体から延びて存在する。小腔と細管間質液中に含まれる介して、骨細胞はまた、最終的に循環系に接続されているので、それらはローカルでなく、イベントだけでなく、全身に影響を与えることができ、その逆もその挙動は、局所および全身の両方の変化によって調節することができる2。 ザ·骨細胞の研究は、最近、このような組織および細胞特異的なトランスジェニック動物の生成、強力な顕微鏡技術のハイスループット分子スクリーニング3,4の使用などのいくつかの技術的な進歩のおかげで勢いを得ている。しかしながら、これらの細胞の知識は、主に、in vitroモデルにおいて適切な相対的希少性のために、依然として不完全である。実際に、骨細胞が原因の深い位置の取得と文化の中で維持するためには、常に困難であった、そしてそのような分化した細胞の種類を特徴づける低レベルの増殖している。 それらは一般的に低い細胞収量を生産し、さらにいくつかの混入線維芽細胞は急速に骨08過増殖というリスクを運ぶのに年に沿って、多数の方法は、一次骨5-7を単離するために開発されてきた。この理由のために、 インビトロの実験作業の中で最も、十分に確立された骨細胞の細胞リットルでこれまで行われてきたINE MLO-Y4 9。 追加のin vitroでの方法論は、これらの細胞を研究する可能性を高めることができるし、骨細胞生物学と病態生理の解析を向上させることができます。主に採用される、そのような方法は、再現することが容易であるべきであり、細胞成熟に達するために、特別な器具類又は非常に長い時間を必要としない。初代細胞に該当する場合重要なことに、彼らはそれが可能なトランスジェニック動物を利用することであろう。我々は最近、レチノイン酸による骨芽細胞株MC3T3-E1のと初代骨芽細胞の治療は骨10の形態学的および分子的特徴をもつ分岐した細胞の均質な集団の開発につながる迅速な表現型変化を誘導することを説明した。 オールトランスレチノイン酸（ATRA）は、核レチノイン酸受容体（RARは）を結合することにより遺伝子転写を調節するビタミンAの活性代謝産物である。 RARは最終的に、レチノイン酸（RA）応答性標的遺伝子の11の変調をもたらす、レチノイドX受容体（RXRの）とヘテロダイマーとしてDNAに結合する。 ATRAは、例えば、神経細胞12および有足13のような他の分岐した細胞は、それらのうち、いくつかの細胞型の分化および成熟を ​​調節することが示されている。 ここで説明する方法は、一次骨芽細胞に対するATRAの加算に基づいています。効果的にするために、ATRAは、定義された密度で播種した細胞上の正確な成熟段階で添加されなければならない;我々の経験では、これらの条件は、骨芽細胞から骨細胞への表現型のスイッチを得るために重要である。

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			<td height="20" style="height:20px;width:283px;">Name</td>
			<td style="width:239px;">Company</td>
			<td style="width:219px;">Cat #</td>
			<td style="width:183px;">comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase P</td>
			<td>Roche Applied Science</td>
			<td>11213857001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Gibco, Life Technologies</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco, Life Technologies</td>
			<td>14175129</td>
			<td>Pre-warm at 37&#176;C before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alpha-MEM</td>
			<td>Invitrogen, Life Technologies</td>
			<td>22571038</td>
			<td>Pre-warm at 37&#176;C before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptomycin/Penicillin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4403</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glycerol 2-phosphate disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ATRA</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
			<td>Protect ATRA from light</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Dissolve in PBS and filter before use. Work always under a chemical hood.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>32670</td>
			<td>Can be added to the secondary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alizarin red</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Janus Green</td>
			<td>Sigma-Aldrich</td>
			<td>201677</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Perchloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>176745</td>
			<td>Use with caution (skin and eye protection are recommended)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HCl</td>
			<td>Sigma-Aldrich</td>
			<td>320331</td>
			<td>Use with caution (skin and eye protection are recommended)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fluorsave</td>
			<td>Calbiochem - Merck</td>
			<td>345789</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Economy Tweezers #7, 0.40 x 0.5mm tips</td>
			<td>World Precision Instruments</td>
			<td>501981</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Economy Tweezers #4, 0.40 x 0.45mm tips</td>
			<td>World Precision Instruments</td>
			<td>501978</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting Scissors, straight,10cm curved</td>
			<td>World Precision Instruments</td>
			<td>14394</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Scissors, 14 cm, straight, S/S</td>
			<td>World Precision Instruments</td>
			<td>501218</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Culture flasks</td>
			<td>Corning</td>
			<td>430168</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Well-plates</td>
			<td>Corning</td>
			<td>3335</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermanox coverslips</td>
			<td>Thermo Scientific Nunc</td>
			<td>12-565-27</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microscope</td>
			<td>Zeiss Apotome</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">spectrometer</td>
			<td>Safas Xenius</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

application of retinoic acid to obtain osteocytes cultures from primary mouse osteoblasts

<html>骨細胞培養の必要性は、骨の研究者のコミュニティによく知られている。一次骨細胞の単離は困難であり、低細胞数を生成する。したがって、最も広く使用される細胞系は、骨細胞様MLO-Y4細胞株である。 ここで説明する方法は、形態学的および分子骨細胞の機能と分岐した細胞の均質な集団を生成するために、レチノイン酸の使用を意味する。 マウス頭蓋冠骨芽細胞からの単離後、全トランスレチノイン酸（ATRA）を細胞培地に添加され、セル監視を倒立顕微鏡下で毎日行われる。最初の形態学的変化は、治療の2日後に検出可能であり、差別化は、一般的に骨芽細胞マーカーのレギュレーションダウンとアップレギュレーション骨細胞特異的分子の細胞外マトリックスを産生する能力が失われ、樹状突起の漸進的発展に伴い、5日間で完了する。 コンテンツ &quot;&gt;毎日のセル監視が原因初代細胞の固有の変動性に必要とされており、プロトコルは、異なるマウス系統から得た細胞への最小限の変化に適応し、トランスジェニックモデルに適用することができる。 この方法は、実行が容易であり、特別な器具類を必要とせず、それは非常に再現性があり、かつ急速に無制限の生物学的用途のためのこれらの細胞の使用を可能にする、細胞外マトリックスの完全な非存在下で成熟した骨細胞集団を生成する。

結果は5〜10の独立した実験から得られた。 細胞形態学 AA / GPは初代細胞は主に玉石のような機能、成熟骨芽細胞の特性を持って処理した。散在枝状細胞は、（ 図1Aの赤い矢印で示すように）可能性がいくつかの骨を表す、見出すことができる。 ATRA処理によって、細胞が急速に一般的に2日後に観察される波及効果を、表?...

Watch this Scientific Journal Video about Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts at JoVE.com

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

レチノイン酸によるマウスの主骨芽細胞の治療は、骨細胞の形態学的および分子的特徴をもつ分岐した細胞の均質な集団を生成します。この方法は、培養中の一次骨細胞を取得し、維持することの難しさを克服し、トランスジェニックモデル由来の細胞を研究することが有利である。

初代マウス骨芽細胞から骨細胞培養物を入手するレチノイン酸のアプリケーション

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

application-retinoic-acid-to-obtain-osteocytes-cultures-from-primary

Research

JoVE Journal

Biology

13.3K ビュー.  Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico. レチノイン酸によるマウスの主骨芽細胞の治療は、骨細胞の形態学的および分子的特徴をもつ分岐した細胞の均質な集団を生成します。この方法は、培養中の一次骨細胞を取得し、維持することの難しさを克服し、トランスジェニックモデル由来の細胞を研究することが有利である。 

ビデオ: Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

後期ステージショウジョウバエ蛹の脳からの初代神経培養

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

生物学

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

げっ歯類の新生児から一次分離した中脳ドーパミン細胞培養

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

げっ歯類からプライマリ大人の線維芽細胞培養の確立

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

から初代細胞培養ショウジョウバエ原腸胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

マウスの心臓から初代メラノサイト様細胞を単離する

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

初代ヒト線維芽細胞培養からナイーブ成人幹細胞の分離

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

マウスおよびヒト結腸組織からの一次筋線維芽細胞の分離

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

基礎脊椎動物の系統からの一次筋原前駆細胞/筋芽細胞培養液の調製

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...