試薬の準備<ol><li> BSAを含むPBS（0.5％）、および2mM EDTAを含むバッファーを加えます。気泡は、MACS分離カラムをブロックすることができるため、バッファーは2〜8℃使用前に脱気し、格納する必要があります。 </li><li>我々は、5％のマウス血清を含むRPMI 1640培地を使用してください。培地は、これらの化合物が細胞刺激の特異性を変えてしまうように、BSAまたはFCSを含めてはいけません。 </li><li>マウスIL - 17 Secretion Assayは - ミルテニーバイオテク-から細胞分離キット。 IL - 17キャッチ試薬、IL - 17検出抗体（ビオチン）、抗ビオチン- PE、およびAnti - PE MicroBeadsは：キットには、次のコンポーネントが含まれています。 </li></ol>脾細胞を刺激する<ol><li>このプロトコルは、このような非刺激脾細胞とT細胞のための対比として、陰性と陽性対照の存在下で行われる。このプロトコルは、無菌操作を用いて行われる。 </li><li> gentleMACS™のDissociatorを用いて分離されたマウスの脾細胞の単一の細胞懸濁液を準備します。細胞の濃度は、細胞計数を介して所定の必要があります。室温で10分間、200 × gでペレットは、細胞。 </li><li>遠心分離に続いて、ピペットを用いてペレットから上清を吸引除去する。ペレットの損失を避けるためにチューブをデカントしてください。 </li><li>今、培地中の細胞を再懸濁し、その後ウェルに加える。 mLのごとに10万セルと平方センチメートルあたり5万個の細胞の濃度に対して十分なメディアを追加。 </li><li>私たちの再懸濁した細胞の免疫応答を刺激するために、我々は、サンプルにイオノマイシン（1μg/ mLの）とPMA（10 ng / mL）を加え、穏やかにピペッティングして混合液を。その後、井戸はそれに応じてラベルが付いています。 </li><li>今、我々は37℃で3時間、私たちの細胞をインキュベートする予定° C刺激期間を開始するためにないミキシングを持つ。刺激の開始からのIL - 17の分析3時間に進みますので、それに応じて計画しています。 </li><li>分泌を停止するには、細胞を氷の上に置き、我々は静かに冷緩衝液をピペッティングによって刺激された細胞を収集している。次いで、細胞をウェルからのチューブに移していると二回目を洗浄しています。 </li><li>すべてのセルが収集されることを保証するために、それは顕微鏡下でお皿をチェックすることをお勧めします。細胞がまだ接続されている残っている場合には、冷緩衝液で皿を洗浄することにより、残りの細胞を収集することができます。あなたの細胞懸濁液内の任意のセルの塊は、事前に分離フィルタを使用して削除できます。 </li></ol>キャッチ試薬で細胞をラベリング<ol><li>それは、IL - 17分泌細胞の2％未満が存在する場合、このアッセイは最適に動作することを、注意することは重要です。 </li><li> IL - 17分泌細胞の濃度はそれに応じてボリュームを調整する2％を超えると予想される場合。 <img src="/files/ftp_upload/old/1037_2008_12_8_6/figure8.png" alt="図8" width="500" height="162" /></li><li>この手順の1つの潜在的な落とし穴は、それによって偽陽性を生成する、隣接するリンパ球から分泌される。二重特異性抗体は、非分泌性T細胞とトラップに結合するIL - 17を標識する際に発生する可能性がキャッチ試薬のクロスコンタミネーション、です。 </li><li>この問題を回避するためには、それはダウン前の冷緩衝液でラベリングや仕事へのIL - 17の拡散を遅くし、このクロスコンタミネーションを避けるために、細胞を冷却することが重要です。細胞は冷たい保つことに加え、それらは定義されている濃度に保たれている必要があります。 </li><li>ラベリングの手順を開始するには、我々は、15ミリリットル閉鎖可能なチューブに10万セルを使用してください。高い細胞数を使用するがある場合は、単にそれに応じてすべてのボリュームをスケールアップ。最適な細胞濃度が取得された後、冷緩衝液10mlを添加することにより細胞を洗浄。 </li><li>冷却遠心機（2〜8℃）で10分間、300 × gで細胞をスピンダウン。 </li><li>遠心分離に続いて、完全にピペットを用いて上清を吸引除去する。これは、セルの損失や不正確なボリュームにつながるとして、上清をデカントしてください。コー​​ルドバッファ、遠心分離、そして吸引10mlのを追加することで構成され、洗浄ステップを繰り返します。 </li><li>今我々は、所望の純度のペレットを持っていることを、冷たい培地80μLに細胞を懸濁します。それらにラベルを付けるには、我々は今マウスIL - 17キャッチ試薬20μLを追加します。 </li><li>氷上で5分間細胞をインキュベート。 </li><li>氷上で5分間のインキュベーション期間の後、チューブを取り外し、37℃の暖かい培地10 mlに細胞を希釈する。その後MACSmixチューブローテーター上にチューブを固定し、37チューブをインキュベート° Cを連続的に動きの下で45分間。温度を37増加させると、° Cは、サイトカインの分泌を再起動します。 </li></ol> IL - 17検出抗体（ビオチン）とAnti - Biotin - PEと細胞のラベリング<ol><li> 37℃で45分間の分泌期間の後、氷上ですぐにチューブを置きます。これは、サイトカインの分泌を停止します。ここからその上に氷の上で細胞を維持することが重要です。コー​​ルドバッファを操作するキャッチ試薬のクロスコンタミネーションを避けることができます。 </li><li> 2〜8℃で300xgで冷緩衝液および遠心機でチューブを埋める℃で10分間。完全に上清を吸引除去する。洗浄工程の1つのより多くの時間を繰り返します。 </li><li>細胞ペレットを冷緩衝液80μLに再懸濁され、チューブを氷上に配置されます。 </li><li>抗体の非特異的な結合を避けるために、我々は、5分間氷上で10μlをのFcRブロッキング試薬とインキュベートの追加をお勧めします。その後、我々は、氷上で10分間、ビオチンとインキュベートする共役であるマウスIL - 17検出抗体（ビオチン）の20μLを加える。 </li><li>我々は2〜8℃で300xgでコールドバッファと遠心10mLを加えることによって前に行っているので、再度、細胞を洗浄℃で10分間。 </li><li>今すぐ上清を吸引し、冷バッファー80μlで細胞ペレットを再懸濁します。 </li><li>私たちの二次抗体、抗ビオチン- PEの20μLを追加。 </li><li>細胞および二次抗体溶液を混合し、再び氷上で10分間チューブをインキュベートする。 </li><li>この2回目のインキュベーションが完了したら、10 mLの冷緩衝液および遠心で細胞を洗浄する。 </li><li>ペレットは現在、冷緩衝液500μlで再懸することができます。 </li><li>細胞はさらに下流の分析のために分離する必要がある場合、それらは磁気抗PE - MicroBeadsで標識し、MACSカラムとMACSのセパレーターを使用して手動または自動分離することができます。 </li><li>我々は現在、脾細胞のラベリングを完了し、分析のために準備が整いました。 </li></ol> FACS解析<ol><li>我々の分析では、フローサイトメトリーにより刺激と刺激脾細胞を比較します。 </li><li>フローサイトメトリー分析の前に、細胞が死細胞外ゲートに0.5μg/ mlで、ヨウ化プロピジウムで染色されています。 MACSQuant™アナライザーは、サンプルごとに20万セルにロードされており、我々は現在、サンプル中の相対的な抗体の反応性の散布図を見に行くされています。希少細胞の解析のための2つの重要な考慮事項 - IL - 17陽性細胞のような - フローサイトメトリーでは、次のとおりです。 <ul><li>前方散乱対側の散布図でリンパ球にゲートを設定</li><li>と死細胞（ヨウ化プロピジウムで染色）およびB -細胞（非特異的バックグラウンド染色を引き起こす可能性があります）をゲーティングは、さらに検出感度を高めるために</li></ul></li><li>我々はT細胞とB細胞を検出するためにCD45R/B220-PerCP抗体を検出するためにCD4 - APC抗体を使用。我々は、試料の前方および側方散乱の特性を参照してくださいとリンパ球集団にゲートを適用します。 </li><li>秒のゲートは、染色した死細胞と染色したB細胞（Y軸）を参照に適用した。これらの細胞は、T細胞の解析から除外されます。 </li><li>約0.003％（図4） - この例では、基本的に私たちのネガティブコントロールである、我々は非常に少数のIL - 17刺激を受けていない試料の分泌CD4陽性T細胞があることがわかります。 0.367％程度（図5A）分離前とMACS技術（図5B）と磁気分離後の60.76パーセント - 刺激T細胞集団を見ると、我々はその分泌IL - 17 CD4陽性T細胞のかなりの数を見ることができます。 </li></ol>

すべてのプロトコルとデータシートはwww.miltenyibiotec.comでご利用いただけます。 警告 試薬はアジ化ナトリウムが含まれています。酸性条件下でアジ化ナトリウムは極めて毒性がアジ化水素酸を、もたらします。アジド化合物は、廃棄する前に流水で希釈してください。これらの予防措置は、爆発の危険が生じる可能性のある配管内の付着物を避けることをお勧めします。

Th17細胞は適応免疫と炎症反応において重要な役割を果たし、自己免疫炎症を推進する上で重要なコンポーネントです。 細胞分離キット（PE） - 国内でのみ有効のマウスIL - 17 Secretion Assayは使用方法をこのビデオドキュメント。この手順を行うときには、出発細胞調製物におけるIL - 17分泌細胞の頻度の推定をすることが重要です。ラベリングおよびサイトカイン分泌の期間中に適切な細胞濃度は懸濁状態で他の細胞によるクロスコンタミネーションを防ぎ、信頼性の高い結果を保証します。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Mouse IL-17 Secretion Assay Cell Enrichment and Detection Kit (PE) </td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-094-213</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_98_1005_Mouse_IL_17_Secretion_Assay_Cell_Enrichment_and_Detection_Kit.aspx">http://www.miltenyibiotec.com/en/PG_98_1005_Mouse_IL_17_Secretion_Assay_Cell_Enrichment_and_Detection_Kit.aspx</a>
</td>
</tr>
<tr>
<td>RPMI 1640</td>
<td>Medium</td>
<td>Miltenyi Biotec</td>
<td>130-091-440</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_243_293_RPMI_1640.aspx">http://www.miltenyibiotec.com/en/PG_243_293_RPMI_1640.aspx</a></td>
</tr>
<tr>
<td>CD4-APC</td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-091-611</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_599_327_CD4.aspx">http://www.miltenyibiotec.com/en/PG_599_327_CD4.aspx</a></td>
</tr>
<tr>
<td>CD8a-FITC</td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-091-605</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_599_328_CD8a.aspx">http://www.miltenyibiotec.com/en/PG_599_328_CD8a.aspx</a></td>
</tr>
<tr>
<td>Propidium Iodide Solution</td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-093-233</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx">http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx</a></td>
</tr>
<tr>
<td>FcR Blocking reagent, mouse </td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-092-575</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_601_435_FcR_Blocking_Reagent_mouse_.aspx">http://www.miltenyibiotec.com/en/PG_601_435_FcR_Blocking_Reagent_mouse_.aspx</a></td>
</tr>
<tr>
<td>Dead Cell Removal Kit </td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-090-101</td>
<td>&nbsp;</td>
<tr>
<td>MACSmix Tube Rotator</td>
<td>Instrument</td>
<td>Miltenyi Biotec</td>
<td>130-090-753</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx">http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx</a></td>
</tr>
<tr>
<td>gentleMACS&#x2122; Dissociator</td>
<td>Instrument</td>
<td>Miltenyi Biotec</td>
<td>130-093-235</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx">http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx</a></td>
</tr>
<tr>
<td>autoMACS Pro Starting Kit</td>
<td>Instrument</td>
<td>Miltenyi Biotec </td>
<td>130-092-545</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_651_679_autoMACS_Pro_Starting_Kit.aspx">http://www.miltenyibiotec.com/en/PG_651_679_autoMACS_Pro_Starting_Kit.aspx</a></td>
</tr>
<tr>
<td>MACS Columns</td>
<td>Tool</td>
<td>Miltenyi Biotec</td>
<td>&nbsp;</td>
<td><a href="http://www.miltenyibiotec.com/en/NN_115_Columns_at_a_glance.aspx">http://www.miltenyibiotec.com/en/NN_115_Columns_at_a_glance.aspx</a></td>
</tr>
<tr>
<td>MACS Separator</td>
<td>Tool</td>
<td>Miltenyi Biotec</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

detection and isolation of viable mouse il-17-secreting t cells

MACSサイトカイン分泌アッセイ技術は、単一細胞レベルと実行可能なサイトカイン分泌細胞の高感度分離に分泌されたサイトカインを検出することができます。 IL - 17分泌細胞を標識するためには、マウス脾細胞の単一細胞の懸濁液を37に調製し、刺激°回転PMA /イオノマイシンとCは、サイトカイン分泌を誘導する。停止するには分泌細胞は、氷の上に配置され、IL - 17キャッチ試薬には、細胞表面の近くに分泌され、捕捉されたとして、白血球の細胞表面上のCD45に特異的に結合し、IL - 17に二重特異性抗体を公開されています。分泌は、その後、温度を37増加させることによって再起動され° CとIL - 17がキャッチ試薬によってトラップされる。分泌は、その後、氷上で細胞を配置することにより、再び停止されます。トラップされたIL - 17を検出するために、細胞はビオチンに結合した第二のIL - 17特異的抗体とAnti - Biotin - PE抗体とインキュベートされています。セルが、直接、フローサイトメトリーによって分析またはAnti - PEコンジュゲートマイクロビーズと、その後の標識によって分離と濃縮のために調製することができる。

Watch this Scientific Journal Video about Detection and Isolation of Viable Mouse IL-17-Secreting T Cells at JoVE.com

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

この手順では、刺激により活発に分泌するIL - 17マウスTh17細胞の白血球の検出と分離を説明します。

生存マウスIL - 17分泌T細胞の検出と分離

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable ...

<a class="grey_button" href="/video/1044">
ChineseVersion
</a>


<a class="grey_button" href="/video/1233">
JapaneseVersion
</a>

detection-and-isolation-of-viable-mouse-il-17-secreting-t-cells

Research

JoVE Journal

Biology

11.1K ビュー.  Miltenyi Biotec,GmbH.  ChineseVersion JapaneseVersion 

ビデオ: Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

CD4 +ミルテニーMACS精製を使用してマウスのリンパ節からT細胞の分離

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

生物学

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

マウスES胚細胞の単離と導出

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

弁内皮細胞の単離

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

マウス唾液腺幹細胞の単離

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

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 in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

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 of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

マウス気管から基底細胞および粘膜下腺管細胞の単離

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

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 ...

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

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 of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

マウスの冠動脈内皮細胞の単離

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...