著者らは、このビデオを制作し、ミズーリ大学学術支援センターでクリエイティブサービスチームに感謝したいと思います。この作品は、国立衛生研究所の母子保健と人間開発のユニスケネディシュライバー国立研究所、助成番号HD055231によってサポートされていました

1。マウス胎盤の解剖<ol><li> マウスの交配のペアを設定します。 </li><li>次の日のそれぞれで、膣栓の有無をチェックする。プラグインが検出された日は0.5日後に性交（DPC）7とみなされます。 </li><li>胎盤は、d 10.5成熟胎盤の発達と胎盤可能初めのクリーン郭清を、妊娠の所望の段階で収集することができる。ここでは、14.5 DPCを実証している。指定されたボリュームで、10.5 DPCに40から60胎盤、および14.5 DPCに10から20胎盤がはっきり見える栄養膜細胞層が得られます。 </li><li>妊娠マウスを安楽死させると、子宮を取り出し、子宮頸部で、かつ子宮間膜に沿って切断する。 </li><li>各受胎産物（ 図1A）との間で切断鋭いはさみを使用して。 </li><li>鉗子の2ペアを使用して、カットエッジで子宮壁をつかみ、引き離し、（ 図1B）子宮筋層を除去<html> 。ワンペア鋸歯、（湾曲またはストレート）適度に細かい鉗子プラス1対の非常に細かい鉗子（＃5、例えば）を推奨します。 </li><li>胎盤向かい、胎盤を露出して、鉗子またはハサミで脱落を開きます。 1鉗子ペアで子宮組織を把持すると、分離するために胎盤（ 図1C）の下に別の鉗子をスライドさせます。胎盤から、必要に応じて独立した胎児と胎膜場合。また、胎盤組織を失うことなく、切開によってすべて脱落膜組織を削除することはできませんが、追加の脱落膜組織は、（青白い）そっとピンセットで剥離することにより、胎盤の表面から除去することができる。我々は、さらに精製するためのパーコール勾配に依存するようになります。 </li><li>すべての解剖が完了するまで、50 mlコニカルチューブに25 mlの氷冷解離バッファ内の各解剖胎盤を置きます。後の段階で、粗く解離バッファに転送する前にハサミやカミソリの刃で胎盤組織をミンチ。 </li></ol>栄養膜細胞のTLE &quot;&gt; 2。アイソ<ol><li> 45〜60分間37℃の水浴中で緩くキャップコニカルチューブおよび場所。約10分毎にピペッティングすることにより積極的に解離液を混ぜる。合計消化時間は密接に監視されるべきであると可能性が高いいくつかの試行錯誤が最適な細胞の回復を達成するために必要になります。組織は、10mlのピペットを通過することができるべきであるが、組織の目に見える部分が残ったままになります。過剰消化が悪く、細胞の生存やめっき効率になります。少数の細胞の塊が存在するときに最適な細胞の生存および平板効率はむしろ完全な解離が達成されたときよりも、発生します。逆に、underdigestionはパーコール分離後純度減少につながる。適切な消化のポイントに達した場合には、コラゲナーゼ活性を停止するためにチューブを氷上に置きます。 </li><li>消化されていない物質を除去するために細胞ストレーナーを通してソリューションを渡すと、5分間@ 500×gで遠心する。 </li><li>洗浄するには、削除10mlの洗浄溶液の上清と細胞を懸濁。 5分間@ 500×gで遠心します。 </li><li>遠心分離の間に、9.6ミリリットルパーコール、13.4ミリリットルの洗浄液と高速遠心分離に適した50mlチューブで1.1ミリリットル10倍培地199を混合することにより、パーコール溶液を調製します。 </li><li> 2 mLの洗浄液に上澄みを再懸濁し、細胞ペレットを取り外します。 </li><li> 4℃で30分間溶液と遠心@3万XGをパーコールにセルを追加℃に</li><li>勾配を乱すことなく、遠心分離器からチューブを取り外します。明るい光源の前に勾配、場所の管を可視化する。トロホブラストバンド（ 図2）を見つけて、下の赤血球バンドを動かさないように注意しながら、ピペットで吸引します。栄養バンドの音量は5-8 mlとなります。 </li><li> 50mlコニカルチューブに栄養膜細胞を転送します。 45ミリリットルの全体積に対するソリューションを洗う追加。 </li><li> 5分間@ 500×gで遠心します。上清を除去し、培養メディに再懸濁し、（NCTC-135）ええと。細胞を37℃、5％CO 2で薄層マトリゲルまたは組織培養プラスチック上で培養することができます。 10.5日で、各胎盤は80〜90％が肯定しているケラチンの約50,000生きた細胞を、もたらします。 </li></ol> 3。栄養膜浸潤アッセイ<ol><li> 37℃、5％30から90分間、CO 2で各マトリゲル室とインキュベートに1-2 mlの培地（NCTC-135）を追加します。 </li><li>培地を除去し、上部チャンバーに2ミリリットル中に1.5ミリリットル×10 5新たに単離した細胞を追加します。低いメンブレン表面を覆うのに、下部チャンバーに1 mlの培地を追加します。 </li><li> 18〜24時間用のインサートで培養細胞。 </li><li>スポンジ綿棒を使ってインサートからの細胞とマトリゲルをこすり取る。 </li><li> 2mlのPBSでチャンバの上部と下部をすすぐ。 </li><li> PBS中で1ミリリットルを4％パラホルムアルデヒドで5分間固定してください。 </li><li> 400μlのDAPI（300nm以下）と下部チャンバーを充填することによって染色し、30分間インキュベートします。 </li><li> PBS中で簡潔に3回洗浄する。 </li><li&gt;は鋭いメスの刃のインサートからの膜を取り除きます。 </li><li>スライドさせて取り付け培地（PBS中50％グリセロール）10μlのドロップを追加します。ピンセットを使って、ドロップ上の膜に横たわっていた。媒体の別の10μlのドロップを追加してから、慎重にカバースリップ。 </li><li>写真とカウント細胞。使用されている顕微鏡の適切東洋メンブレン（ダウンセルサイドアップまたはセル·サイド）に確認してください。画像Jソフトウェア（国立衛生研究所）、Adobe Photoshopまたは手動のカウンターができるように、この目的のために使用することができる手動計数機能を備えています。 </li></ol> 4。代表的な結果<img src="/files/ftp_upload/3202/3202fig1.jpg" alt="図1" /> 図1マウスの胎盤の解剖。 （a）は、子宮はmesometrial表面に沿って、子宮頸部（下）で、卵管（黄色）に沿って切断することにより除去される。個々の受胎は、次に示すように、ハサミで切断することによって分離することができる。 （b）の子宮<html>壁はカット子宮の表面に2鉗子を置き、胎児近く離れて胎盤から、抗mesometrial側面に沿って引き裂くことによって除去される。 （c）の胎盤は、その後、鉗子で把持し、胎盤と子宮の組織​​との間に別のものをスライドさせて分離されています。臍帯および他の胚体外膜は、その後胎盤から分離することができる。 <img src="/files/ftp_upload/3202/3202fig2.jpg" alt="図2" /> 図2：栄養膜細胞のパーコール分離。左の漫画に示すように、遠心分離した後、三大バンドが見えるようになりますと、右の写真。上部のバンドは、細胞破片および繊維芽細胞が含まれており、最も明らかになるであろう。赤血球を含む下部付近タイト（赤）バンドはちょうどびまん性栄養膜バンドの下に表示されます。ちょうど写真の矢印の左側に示すように2のように、細胞凝集塊は、おそらく、栄養膜帯で見られるでしょう。 ove_content &quot;&gt; <img src="/files/ftp_upload/3202/3202fig3.jpg" alt="図3" /> 図3侵襲栄養膜細胞（白矢印）は、膜の底面に現れる、マトリゲル層と膜孔を通過することになる。毛穴（黒矢印）も表示されます。全体の膜表面または代表試料領域のセルの数をカウントすることができる。蛍光DAPIで核染色のグレースケール画像は、細胞の可視化を向上させるために反転されました。 <img src="/files/ftp_upload/3202/3202fig4.jpg" alt="図4" /> 図4：栄養膜細胞が同定され、単離された集団の純度はサイトケラチン7（センター、マゼンタ）の免疫染色により、評価することができる。細胞はDAPI（上、青）で対比されています。マージされた画像、下部。

<ol>
	<li>Thordarson, G., Folger, P., Talamantes, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+placental+cell+culture+system+for+studying+the+control+of+mouse+placental+lactogen+II+secretion.">Development of a placental cell culture system for studying the control of mouse placental lactogen II secretion.</a> Placenta. 8, 573-585 (1987).</li><li>Petroff, M. G., Phillips, T. A., Ka, H., Pace, J. L., Hunt, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+term+human+trophoblast+cells.">Isolation and culture of term human trophoblast cells.</a> Methods. Mol. Med. 121, 203-217 (2006).</li><li>Kliman, H. J., Nestler, J. E., Sermasi, E., Sanger, J. M., Strauss, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+characterization,+and+in+vitro+differentiation+of+cytotrophoblasts+from+human+term+placentae.">Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae.</a> Endocrinology. 118, 1567-1582 (1986).</li><li>Blaschitz, A., Weiss, U., Dohr, G., Desoye, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+reaction+patterns+in+first+trimester+placenta:+implications+for+trophoblast+isolation+and+purity+screening.">Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening.</a> Placenta. 21, 733-741 (2000).</li><li>Librach, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=92-kD+type+IV+collagenase+mediates+invasion+of+human+cytotrophoblasts.">92-kD type IV collagenase mediates invasion of human cytotrophoblasts.</a> J. Cell. Biol. 113, 437-449 (1991).</li><li>Schulz, L. C., Widmaier, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+leptin+on+mouse+trophoblast+cell+invasion.">The effect of leptin on mouse trophoblast cell invasion.</a> Biol. Reprod. 71, 1963-1967 (2004).</li><li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> Science. 282, 2072-2075 (1998).</li></ol>

我々は、開示することは何もない。

このビデオでは、マウスの胎盤から栄養膜細胞の単離を示しています。次に、栄養膜侵入のためのin vitroアッセイを示しています。この手順を用いて、主にではなく、完全に、栄養膜細胞の純粋な集団を得ることができる。さらに純度が要求されている場合は、一次ヒト栄養膜細胞のために記載されているように、磁性ビーズ分離またはフローサイトメトリーは、実行することができる。コ?...

Specific reagents and equipment:
Wash Solution (filter sterilize after mixing)
<table border='1'>
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Amount</td>
 <td>Final concentration</td>
 </tr>
 <tr>
 <td>10x Medium 199</td>
 <td>50ml</td>
 <td>1X</td>
 </tr>
 <tr>
 <td>Hepes</td>
 <td>2.383 g</td>
 <td>0.02M</td>
 </tr>
 <tr>
 <td>sodium bicarbonate</td>
 <td>0.42 g</td>
 <td>0.01M</td>
 </tr>
 <tr>
 <td>Penicllin-Streptomycin</td>
 <td>5 mL</td>
 <td>100 U- 100 &mu;g /ml</td>
 </tr>
 <tr>
 <td>Sterile dH20</td>
 <td>to 500 mL</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

Dissociation solution (make fresh and filter sterilize)
<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Amount</td>
 <td>Final concentration</td>
 </tr>
 <tr>
 <td>Wash Solution</td>
 <td>100 mL</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DNase</td>
 <td>1 vial (2000 U)</td>
 <td>20 U/mL</td>
 </tr>
 <tr>
 <td>Collagenase</td>
 <td>100 mg</td>
 <td>125 digestion units/mL</td>
 </tr>
 </tbody>
</table>


<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Collagenase</td>
 <td>Sigma</td>
 <td>C9891</td>
 <td>This is Clostridium collagenase.
Bovine pancreatic collagenase would likely work as well</td>
 </tr>
 <tr>
 <td>NCTC-135</td>
 <td>Sigma</td>
 <td>D4263</td>
 <td>Add FBS to 10%</td>
 </tr>
 <tr>
 <td>Matrigel invasion chambers</td>
 <td>BD Biosciences</td>
 <td>354481</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10x Medium 199</td>
 <td>Sigma</td>
 <td>M9163</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Invitrogen</td>
 <td>15140</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DNase</td>
 <td>Sigma</td>
 <td>D4263</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>100 &mu;m Cell strainer</td>
 <td>Fisher</td>
 <td>22363549</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Antibody to cytokeratin 7</td>
 <td>DAKO</td>
 <td>M7018</td>
 <td>Used at 1:100 dilution</td>
 </tr>
 </tbody>
</table>

Equipment Standard cell culture equipment including a CO2 incubator is required. In addition, a centrifuge rotor capable of spinning 50 ml tubes at 30,000 x g is needed. Note that this exceeds the maximum speed of most conical centrifuge tubes and will likely require an Oak Ridge style tube.

isolation of primary mouse trophoblast cells and trophoblast invasion assay

胎盤は、胎児への栄養、ガスや成長因子の輸送だけでなく、廃棄物の除去を担当しています。したがって、胎盤発育における欠陥は胎児と母親のための重要な結果を持っており、胚性致死の主な原因です。胎盤の胎児部分の主要な細胞型は栄養です。初代マウス胎盤栄養膜細胞は、正常および異常な胎盤の発達を研究するための有用なツールであり、細胞株とは異なり、単離され、妊娠の特定の段階で栄養を研究するために使用することができる。また、トランスジェニックマウスからの栄養膜細胞の初代培養を胎盤細胞中の特定の遺伝子の役割を研究するために用いることができる。ここで紹介するプロトコルはThordarson らの説明に基づいています。パーコール勾配が単離したマウスの胎盤から比較的純粋な栄養膜細胞集団を得るために使用されている1。それは、より広く使用するために類似しています人間の栄養膜細胞単離2-3 dの方法。純度は、サイトケラチン7 4の単離された細胞の免疫細胞化学的染色によって評価することができる。ここでは、単離された細胞は、その後、 体外 5-6 に栄養膜侵襲性を評価するために、マトリゲル浸潤アッセイを用いて分析する。浸潤細胞をカウントするための免疫細胞化学及び染色によって分析される。

Watch this Scientific Journal Video about Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay at JoVE.com

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

このプロトコルでは、パーコール勾配を用いた栄養膜細胞の分離に続いて妊娠d10.5、上のマウスから胎盤の解剖について説明します。次に、マトリゲル浸潤アッセイで単離された細胞の使用例を示します。

マウスの主栄養膜細胞と絨毛浸潤アッセイの分離

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

isolation-primary-mouse-trophoblast-cells-trophoblast-invasion

Research

JoVE Journal

Biology

23.4K ビュー.  University of Missouri. このプロトコルでは、パーコール勾配を用いた栄養膜細胞の分離に続いて妊娠d10.5、上のマウスから胎盤の解剖について説明します。次に、マトリゲル浸潤アッセイで単離された細胞の使用例を示します。

ビデオ: Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

胚盤胞からマウス栄養膜幹細胞の導出

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

生物学

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

ラット肝細胞の単離及び初代培養

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

原発腫瘍から免疫細胞の分離

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

マウスプライマリ膵臓腺房細胞の単離と培養

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

直接標識プローブを用いた堅牢な3DのDNA FISH

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

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 ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

マウス大動脈内皮細胞の単離および初代培養

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

新生児および成人マウス皮膚由来の初代マウスケラチノサイトの単離および培養

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

マウスの主な腎尿細管上皮細胞の分離と性質、高スループットの細胞フラックスの解析

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

ミニチュアブタからの原発性大動脈内皮細胞の単離と培養

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...