国立癌研究所の助成金1R03CA172923によって部分的にサポートされています。

倫理文：研究におけるマウスの使用は、それが基本的な生物学のまたは疾患の最終的な治療に向けた人間の理解を進める可能性があるインスタンスでのみ許容されます。 1.注文<ol><li>購入雌のC57BL / 6マウス（6歳 - 8週間）、群当たり5匹のマウス。マウスのための数日間は、調整することができます。 </li></ol> B16-BL6細胞の調製<ol><li>無菌フード内で37度のインキュベーターと場所から細胞培養プレートを取り外します。プレートは、B16-BL6マウス黒色腫細胞と約70％コンフルエントであるべきです。大きなコンフルエント細胞に転移能を変化させることができます。 </li><li> 1分間放置し、プレートあたり1ミリリットル0.05％のトリプシン-EDTAを追加し、トリプシン培地溶液を吸引除去します。 </li><li>プレート当たりトリプシンの1ミリリットルを加え、10分間インキュベーターにプレートを返します。 </li><li>無菌フードにインキュベーターからプレートを移動させた後、無血清ローズウェルパーク記念研究所Mediuの4ミリリットルを追加各プレートにメートル（RPMI）。 </li><li>滅菌、電動式ピペットを用いて細胞を収集します。細胞を破壊するために、非常にわずかな角度で押したチップを用いて、プレートの底から細胞を取り出します。そうでなければ排出針を詰まらせる可能性があり、細胞塊の十分な分離を確実にするために数回繰り返します。思い出すと50mlコニカルチューブに移します。 </li><li>細胞密度を決定するために、血球計数器を使用してください。一定のB16-BL6細胞の総数を維持し、無血清RPMIの量を決定すること0、0.065、0.015、0.25、0.5万個の細胞/ mlの最終濃度に到達するために必要。 </li><li>同様に15ミリリットルコニカルチューブに50ミリリットルチューブ内のメディアを分取。 5分間、2250×gでコニカルチューブを遠心。メディアをデカント。無血清RPMIの適切な量を追加し、血球計数器を経由して所望の細胞密度を確認します。追加のRPMIを追加するか、回転し、それに応じて小さくVOLUME-に再懸濁することによりdensities-調整します。 </li><li>アリコート500μlの氷の上で標識された1.8ミリリットルチューブにそれぞれの細胞懸濁液。 </li></ol> 3.尾静脈注射<ol><li>尾が保持しているマウスを、取り、マウスレストレーナーにリア最初にそれを導きます。制止のメインチャンバー内の胴体により、外側尾で、拘束の終わりに向かってマウスを引っ張ります。マウスが安全になるまでプッシュし続け、拘束具、プランジャーを挿入します。 </li><li>外側尾静脈のいずれかが上を向いていることを90度、マウスを回転させます。アルコールパッドを使用して、積極的に尾静脈が最も表示されている場合は特に、マウスの尾の側面を清掃してください。 注：良い照明はB57BL / 6Jマウスでの効果的な尾静脈の可視化のために不可欠です。加熱ランプまたは加熱手術パッドは、必要ではないが、また、尾静脈の容積を増加させることによって助けます。 </li><li>無菌培養フードで2万個の細胞/ mlのチューブからの細胞培養培地を300μlを収集します。秒をフリック、針を反転IDEとシリンジから気泡を排出するために、プランジャーを押します。 250μlの最終容量をもたらすことが文化-RPMIを取り出します。 </li><li>非利き手でマウスの尾を拡張します。非利き手の人差し指と親指で押し下げ、尾の先端部に上昇尾の近位端部と尾を持ちます。 注：このように、尾静脈を横位置に保持され、尾部のより遠位部分の可能な一致するエントリと、拘束面に平行です。 </li><li>そもそも分下向きの角度で、遠位端に向かって、尾静脈に針を挿入し、さらに挿入時に尾静脈の角度に合わせて、針を調整（針に注射器の相対的なプランジャー端を下げます） 。 </li><li>尾静脈に針約1cmを挿入します。挿入後、着実に針を保持し、プランジャーをプッシュし始めます。 注針が尾静脈内にあり、針の端部がNである場合にotのは、メディアが静脈に妨げられることなく流れるべき遮ら。効果的な注入は、静脈からの血液クリアランスによって特徴付けられます。そこに抵抗がある、またはバブルは、注射中の部位で形成し始める場合は、針を除去し、尾静脈に沿ってより近位の場所で再試行してください。 </li><li>静脈から針を外し、それを捨てます。任意の出血を止めるために進入部位に対するガーゼを保持します。 </li><li>拘束からプランジャーを取り外し、受信者のケージにマウスを置きます。 </li><li>他のすべての濃度について繰り返します。 注：肺病巣確立は約2取る-細胞株と焦点の所望の大きさに応じて変動して、3週間を暫定で9に 、マウスは、過度のあえぎのように、早期の安楽死を保証可能性が症状を監視する必要があります。 </li></ol> 4.肺病巣を数えます<ol><li> CO 2を CO 2チャンバー内に配置し、100％に曝露することによって、マウスを安楽死させます</sUB&gt;は5分間分あたりチャンバ容積の10から30までパーセントで導入しました。頚椎脱臼で従ってください。 </li><li>発泡スチロールの上でマウスをスプレイ。手術用はさみを使用して、胸骨に沿って外科的切開を作成します。 </li><li>胸骨切開の上からと胸郭を露出させ、マウスの肩に向かって分岐の両方、二つの追加の切開を行います。 </li><li>肺と心臓を露出し、胴体から胸郭を削除するには、2カット、胸骨の側面に沿って各してください。 </li><li>手術用ピンセットで心を持ち、マウスから心臓や肺を解放し、食道と気管をカット。より良い視覚化のために、10倍の倍率で、解剖顕微鏡の下に置きます。 </li><li>肺から心臓を解剖し、胸腺を除去し、2つだけ肺を残します。 </li><li>解剖範囲の下の肺では、肺の病巣をカウントし始めます。各病巣は、肺の表面上に円形、茶色の出しゃばりとして表示されます。 <ol><li>ためにマウスとの間の整合性は、葉の表面に病巣の数をカウントし、その葉を反転します。 4ローブの各操作を行うと、個別に簡単にカウントすることができます。 </li></ol></li><li>遺体を捨て、そうでない場合は、保存して、IHCを行った場合、肺を修正。 </li></ol>

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著者は、彼らが競合する金融利害関係を持たないことを宣言します。

尾静脈注射からの循環腫瘍細胞の一次成長のサイトを回避し、時折遠隔地の毛細血管床で自分自身を確立し、血流やリンパ系のような経路を通って移動するの転移を表す。上記の14のプロトコルは二次のモデルとして機能します転移。注入されたB16-BL6細胞の最適な数では、実験者は、癌細胞の転移に投与された薬剤または免疫細胞集団、ポスト中和の相対的な効果を決定することができます。 このプロトコルには限界があります。循環腫瘍細胞が転移する能力のために選択され、従って、非免疫原性である、主要組織適合性複合体クラスの下位レベルの1分子を有している。12さらに、転移の多数の突然のクラスタ化導入は、患者における一般的なシナリオとは異なっていますここで、転移性腫瘍細胞の少数原発腫瘍の場所から長期間にわたって放散します。また、静脈内導入した腫瘍細胞は、組織培養物からではなく、原発腫瘍の起源です。したがって、これらの細胞は、細胞接着、遊走、免疫認識および受信者の臓器の確立の変化に起因する二次転移とは違っている。15,16 代替プロトコルは、原発腫瘍の発生や得られた「自発的な」肺転移の分析のためのB16 / BL6の皮下注射であろう。これらの「自発的」転移がより密接に人間の患者のものと一致する、彼らの &quot;実験&quot;の対応よりも多くの不均一性を含んでいることが分かってきました。プロトコルは、転移の実験化合物の影響を決定する能力に制限されます。外側尾静脈注射で、血流中に注入黒色腫細胞の数は、hを介して近似することができます。emocytometer。 「自発的」転移と、しかし、肺病巣を生じたの数に追加の変数を追加し、腫瘍の間に大きな不均一性がある。16 結論として、尾静脈B16-BL6マウス黒色腫モデルは、転移の治療または免疫療法の影響を決定するための単純なモデルを表します。静脈循環腫瘍細胞を注射することによって、研究者は、患者が後転移を、ある程度まで、複製することができます。癌患者における転移性腫瘍細胞の破壊的な影響を考慮すると、このモデルは、転移の多段階プロセスを理解するための強力なツールです。

転移は悪性腫瘍の患者の死亡の主要な原因です。癌細胞の転移性播種の過程はよくわかっていないが、隣接組織への浸潤を含む、いくつかの段階、リンパ管および血管系への血管内、循環系内の生存と転座、転移部位の血管から血管外漏出が関与すると表示され、適応新しい新しいサイトの微小環境、および二次性腫瘍の増殖および形成することにより、新しいサイトでの植民地に。1、これらの生物学的事象のそれぞれは、転移性腫瘍細胞の形質転換、普及および生存を伴います。例えば、細胞外マトリックスとの正常な相互作用と相まって間葉表現型への腫瘍細胞の上皮表現型の変換は、隣接する組織に侵入し、身体の他の部位に転移することを可能に。2,3さらに、これらの転移細胞循環血流内で生き残るためには、ホストからの免疫監視を回避する必要があります。本体内に離れた部位に移植された場合4,5最後に、腫瘍細胞が増殖し、二次腫瘍を形成するために、それらの微小環境に適応しなければならない。6,7ので転移は、がん患者の間で共通の現象であるが、転移性腫瘍細胞は、治療的介入の影響を受けやすい脆弱性の複数の領域を持っています。 メラノーマの免疫原性の性質、および免疫療法の最近の関心を考えると、黒色腫のためのモデルがますます便利です。8米国だけでも、メラノーマは年間推定9000人の死亡の原因である。黒色腫転移の9一般的な場所は、骨、脳であります、肝臓、および肺。遠隔部位への転移の大部分は、血流を介して起こります。血液中の循環腫瘍細胞は、免疫クリアランスを回避遠位臓器の毛細血管床に到達しなければならないし、正常に自分自身を確立するために、血管の内皮細胞を介して侵入する。4-7,10、11 転移の一般的および病原性の現象を模倣するために、マウスB16-BL6細胞株を作成しました。親のC57BL / 6メラノーマ細胞株B16-F0の被相続人は、B16-BL6は、膀胱膜浸透のための6連続した選択に続いて（B16-F10の結果）は、静脈内注射からの肺転移のための10の連続した​​選択範囲の最終産物です。 12このように、静脈内注射、特にとき、マウスにおける転移巣の確立のための信頼性の黒色腫細胞株である。13 B16 / BL6細胞の移植および増殖を可能にするために、二つ以上の週間続くB57BL / 6マウスの尾静脈へのB16-BL6細胞の十分な量の注射後、転移病巣は、肺に形成します。解剖顕微鏡による安楽死や検査の際、数個々の病巣の存在を定量することができます。注入されたB16-BL6細胞の数は、肺の表面に形成された病巣の数と相関し、これは、今度は、用量転移効果を確立するために使用することができます。このモデルは、既知の転移性細胞は、調査の肺、肝臓、または他の器官の迅速かつ予測可能な広がりとの確立を容易に、血流に直接導入される「実験的転移」として知られています。これは、しばしば、皮下、腫瘍細胞が移植されている「自然転移」とは対照的であり、転移は有機癌細胞を流し由来する。14,15 マウスの尾静脈にB16 / BL6細胞の適切な量を注入することが重要です。あまりにも多く、および肺は、転移で覆われると、隣接病巣が互いに区別できないであろう。少なすぎると、治療薬の影響が原因で本来のバリに識別できないだろう注射の間ations。 C57BL / 6マウスの肺の病巣の最適な数を得るためには、アカウントに個別の識別性をながら、肺の表面上に確立された転移巣の量を注入した細胞の数を相関させる必要があります焦点。このプロトコルは、C57BL / 6マウスに対する転移性マウス黒色腫モデルのデモンストレーションを行います。

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			<td height="21" style="height:21px;width:377px;">Trypsin</td>
			<td style="width:275px;">Corning</td>
			<td style="width:288px;">MT25052CV</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium</td>
			<td>Corning</td>
			<td>MT15040CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phase Contraast Hemacytometer</td>
			<td>Hausser</td>
			<td>02-671-54</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-Fine IV Insulin Syringes</td>
			<td>BD</td>
			<td>14-829-1D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Alcohol Prep Pads</td>
			<td>Fisherbrand</td>
			<td>22-363-750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Restrainer</td>
			<td>Braintree Scientific</td>
			<td>NC9999969</td>
			<td>Restrainer choice depends on age/size of mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating Pad</td>
			<td>Harry Schein</td>
			<td>NC0012697</td>
			<td>Optional</td>
		</tr>
	</tbody>
</table>

injection of syngeneic murine melanoma cells to determine their metastatic potential in the lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

目視検査の際に、表面の病巣の変化は明らかです。解剖顕微鏡下で腫瘍の数を計数すること注入腫瘍細胞の数と得られると数えられる病巣の数の間の直線関係を生じるはずです。注射用細胞の最適な数を使用して、彼らは、図1Aの500,000個の細胞/ mlであるように、目標は、肺が完全に覆われていないものであり、彼らは62,500細胞/ mlと125,000個の細胞であるとしてあまりにもまばら/、カバーされていませんミリリットル。これはまた、線形相関（ 図1B）によって定量可能です。 <img alt="図1" src="/files/ftp_upload/54039/54039fig1.jpg" /> 尾静脈注射したB16 / BL6細胞から得られた図1肺病巣。 （A）肺病巣は、茶色の円形の塊（矢印の下で代表病巣）としてC57BL / 6マウスからの肺の表面に可視化することができます。洞穴のように（各画像上）注射し、細胞培養の性が増えるので、（各画像の下）、肺病巣を生じた対応する数がありません。注入した細胞の密度が最も高いマウスからの肺は、肺病巣の最大の視覚的なカバレッジを持っています。スケールバーは20mmでを表す。細胞培養密度に対して肺病巣の相対的な（B）列挙。血球計によって決定されるように125,000細胞/ ml以上のほぼ直線的な関係が存在する。 <a href="https://www.jove.com/files/ftp_upload/54039/54039fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs at JoVE.com

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

同系マウスメラノーマ細胞の注入は、肺におけるそれらの転移能を決定するために、

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

injection-syngeneic-murine-melanoma-cells-to-determine-their

Research

JoVE Journal

Medicine

17.5K ビュー.  Beth Israel Deaconess Medical Center & Harvard Medical School. Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

ビデオ: Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

アンインビトロ</em転移性増殖に腫瘍の休眠とスイッチを研究する>システム

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

医学

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

弱視における抑制の測定と治療

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

競争力のあるマウス骨髄移植モデルにおけるドナー細胞の生着を​​決定するために、定量的リアルタイムPCRを用いて

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

豚における心筋梗塞および機能的結果の評価

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

変更された<em&gt;インビトロ</em媒介する癌細胞浸潤におけるホルモン、サイトカインおよび/または成長因子の潜在的な役割を決定するために&gt;浸潤アッセイ

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

フォーカス形成：細胞ベースのアッセイ遺伝子の発癌性を決定するために、

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

マウス肺における圧力 - 容積曲線の測定

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

新しい治療戦略を開発するための膵臓癌幹細胞特性を研究

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

肝細胞癌転移の「患者様」同所同系マウスモデル

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

健康なボランティアからの内皮前駆細胞の単離および心臓手術後の血清試料の影響を受け彼らの渡り鳥の可能性

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...