This work was funded by National Health and Medical Research Project Grant APP1021582 and a Hunter Medical Research Institute grant sponsored by Sparke Helmore/NBN Triathlon and the Estate of the late Leslie Kenneth McFarlane.

このプロトコルのすべての動物の作業はニューカッスル大学の動物倫理委員会に厳密に準拠して行われている手順を承認しました。 楽器、文化メディアと皿の作製<ol><li>プリ暖かいメディア199（TC199）またはダルベッコ改変イーグル培地（DMEM）メディア37へ °C。 95％O 2/5％CO 2でバブリングすることにより事前含酸素媒体。媒体は7.3の最終pHを持っていることを確認してください。 </li><li>各嚢のための2つの5cmのセクションを切断して縫合糸を準備します。ループ閉じられていない結び目に縫合糸。 </li></ol> 2.解剖および消化管の調製<ol><li>安楽死の前に固形食12時間を撤回。必要であれば、この時間の間に栄養ゲルサプリメントに動物を配置します。 </li><li>施設内倫理プロトに従って頸椎脱臼に続いてペントバルビタールナトリウムの過剰摂取（[200ミリグラム/キログラム]、腹腔内注射）によりマウスを安楽死させますCOLSは、腹部と胸部の上に70％エタノールをスプレーし。 </li><li>はさみを使用して、腹部の真ん中に横切開を行い、腹膜を露出させます。 </li><li>分離し、幽門括約筋で胃から小腸上部を切断し、肛門縁で大腸を切断することにより、消化管を除去するために進んでください。優しく腸間膜を削除するために鉗子を使用します。予め温め、酸素媒体中で腸管を置きます。 </li><li> （ 図1）の透過性について評価する腸の部分を特定し、腸管の残りの部分から自由にこのセクションをカット。 <ol><li>動物間の一貫性を維持する胃十二指腸および空腸の相対的なセクションを測定し、盲腸を結腸及び回腸の相対的なセクションを測定するために。 </li><li>組織セグメントを選択する場合、そのようなパイエル板などの粘膜関連リンパ組織の存在に注意してください。これらは小さなうなずきとして同定することができますルーメンの漿膜側のジュール。 </li><li> 1ミリリットルの注射器を使用して、静かに予熱したPBS（37°C）でペトリ皿に腸セグメントの管腔内容をフラッシュします。これらの糞便内容は-80で廃棄または保存することができます 所望のように将来の分析のためにCを°。 </li></ol></li></ol>腸嚢の調製<ol><li>試験化合物または分子の300μlの容量で1ミリリットル注射器を準備します。粘膜の完全性、FITCデキストランM.Wt.の1mg / ml溶液のための4,400を使用してもよいです。 4,400-70,000ダの大きさの範囲のプローブは、増加した感受性のために使用することができます。安全注射器の上に小さな動物の血管カテーテルにフィット。 </li><li>メジャー5腸セグメントの開口部からセンチ、セグメントをネクタイはしっかりとこの時点で、縫合糸ループで閉じます。静かに腸の開口部の周囲に予め結んだ縫合糸ループを配置し、鈍化カテーテルを挿入します。腸のセグメントを確保するために、閉じた縄を引いてそして、すべての溶液が注入されることを確認して、腸にシリンジから300μlのボリュームをリリース。 </li><li>同時に腸嚢の閉鎖を確保​​するために、縫合糸縄を引きながらゆっくりカテーテルを削除します。腸からの緩い腸嚢をカットし、37に予熱酸素培地20mlを充填した50mlコニカルチューブの中に置きます °C。 </li></ol>透水4.測定<ol><li> 37に設定された加熱水浴中で腸の嚢を含む場所コニカルチューブ °C。 0、30、60、90および120分の時点で、各インスタンスで新鮮な培地100μlでボリュームを交換し、円錐管および転送から96ウェルプレートに100μlのサンプルを取ります。 </li><li>最終的なサンプルが採取された後、粘膜表面を露出させ、縫合糸の時点で、セグメントの長さに沿って開いた嚢をカット。 </li><li>各腸のセグメントの長さと幅を測定します。指名打者の場合赤、スナップ-80セグメントと店を凍結 タンパク質または生化学的解析、あるいは、分子アッセイのためのRNA安定化溶液中の店のためのC°。 </li><li> 1から1×10 -6の範囲内のFITCタグ化分子に対する対数希釈の標準曲線を構築します。 </li><li>蛍光プレートリーダー上のFITC用の測定試料および標準、FITCの励起/発光：/ 519 nmの495 nmの。 </li></ol>各個別の腸サックのための見かけ透水5.計算<ol><li>時間単位は秒に変換します。 </li><li>各時点について、累積濃度、Qを計算します Q tの=（C トンの * Vrを）+（Q tの和 * V s）は 、ここで： 時刻tでのQ トン =累積濃度 CのT =時間tにおける濃度 受信機側でのV R =巻 以前のすべてのQ tのQ tの和 =合計 V S =ボリュームをサンプリング </li><li>プロット対時間Q（T）と傾きを計算：δQ/ΔT </li><li>見かけの透過性を計算する（P アプリ ） P アプリ =（δQ/ΔT）/（*株）、場所： 組織のA =エリア C 0 =初期濃度</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。

ここでは、粘膜バリア機能のex vivoで評価するために分離および腸嚢の調製を詳述しています。腸の嚢の調製物は、主に腸を横断候補薬物の吸収を調べ、薬学研究に利用されています。しかし、このアッセイは、腸疾患の研究のためにも同様に適しています。領域と透過性の部位特異的な評価によって大きく異なる場合があります腸の透過性は、消化器疾患における粘膜の整合性の地域...

消化管の腸上皮障壁はヒト成人で400メートル2で推定粘膜表面積です。したがって、常に微生物、摂取薬、栄養素や細菌毒素から挑戦にさらされています。ホストが許容共生細菌と潜在的な病原体を区別しなければならないだけでなく、同​​時に、栄養素の吸収を可能にしながら、上皮関門を通過するから、これらの種とそれらの分泌分子を防止しなければなりません。このように、腸上皮の役割は、管腔の内容物1に対する選択的障壁として作用することです。これは部分的に、構成的および誘導機構2からなる反応性生物系を介して作用する粘膜における先天上皮防御システムによって、達成されます。 上皮バリア機能の喪失は、胃腸疾患の多数の特徴的な病理である。 インビボでの上皮のバリア機能の検査は、トレーサー分子およびその後の血清の分析3の強制経口投与を介して評価することができます。しかし、この技術は、バリア機能障害のサイトへなどの兆候を提供していません。 vitroおよびトランスウェルシステム3を使用し、それぞれのチャンバ4,5をアッシング経上皮抵抗のex vivoでの評価では、一般的に上皮のバリア機能の代理マーカーとして使用するが、動物モデル6の貢献疾患の生理機能を欠いています。このプロトコルでは、レベルの数で粘膜バリア機能を評価するために使用され得る腸の完全性との直接及び局所的な評価を可能にするエクスビボ組織標本のモデルが記載されています。重要なことに、この手法は、疾患の動物モデルに適用することができる、または薬理学的に粘膜障壁機能障害の深さ質問に可能にするように操作することができます。

<table border="0" cellpadding="0" cellspacing="0" width="904">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Dekantel&#160; Non-absorbable Silk suture</td>
			<td style="width:227px;">Braintree Scientific</td>
			<td style="width:227px;">SUT-S 116</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Media 199 (TC199)&#160;</td>
			<td style="width:227px;">Life Technologies</td>
			<td style="width:227px;">11043-023</td>
			<td style="width:227px;">No phenol red as this interferes with fluorescence</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:227px;">Life Technologies</td>
			<td style="width:227px;">21063-045</td>
			<td style="width:227px;">No phenol red as this interferes with fluorescence</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">N-acetylcysteine</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;"></td>
			<td style="width:227px;">Use at 10mM in media</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Small animal vascular cathether: Physiocath</td>
			<td style="width:227px;">Data Sciences International</td>
			<td style="width:227px;">277-1-002</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">FITC-Dextran 4400 MW</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;">FD-4</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">FITC-Dextran 20,000 MW</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;">FD-20</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">FITC-Dextran 70,000 MW</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;">FD-70</td>
			<td style="width:227px;"></td>
		</tr>
	</tbody>
</table>

ex vivo intestinal sacs to assess mucosal permeability in models of gastrointestinal disease

上皮バリアは、胃腸管の最初の先天的防御であり、選択的基盤となる組織区画に内腔からの輸送を調節し、上皮を通過する小さな分子の輸送を制限し、ほぼ完全に上皮高分子輸送を禁止します。この選択性は、親油性分子との両方の頂端受容体および上皮のタイトジャンクションタンパク質複合体の輸送を制限する粘液ゲル層によって決定される。上皮のin vitro細胞培養モデルは便利であるが、モデルとして、それらが欠け微生物、粘液ゲル、上皮および免疫系の間の相互作用の複雑さ。一方、腸管吸収または浸透性のインビボ評価を行うことができるが、これらのアッセイは、部位特異性の兆候と、全体的な消化管吸収を測定する。「腸の嚢」を用いてエクスビボ透過性アッセイ;腸の部位特異性の付加的な利点と、特定の分子の全体的な腸の整合性や比較のトランスポートのいずれかを測定するの迅速かつ高感度な方法です。ここでは、透過性の研究と見かけの透過性（P アプリ ）の計算のための腸嚢の調製を記載 腸管バリアを通過する分子の。この技術は、薬物吸収を評価する方法として使用することができる、または胃腸疾患の動物モデルにおける地域上皮バリア機能障害を検討します。

このプロトコルは、胃腸疾患の動物モデルにおいて腸のバリア機能の地域の変化を調べるために使用することができます。胃腸管7の変化領域に粘膜表面を横切って傍細胞プローブの流量を測定することにより、上皮のタイトジャンクションの完全性を評価することができます。また、大きさ（ 図2）または疎水性（ 図3）、上皮摂動?...

Watch this Scientific Journal Video about Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease at JoVE.com

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

ex vivoで腸嚢は、胃腸疾患のモデルに粘膜透過性を評価するために

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

ex-vivo-intestinal-sacs-to-assess-mucosal-permeability-models

Research

JoVE Journal

Medicine

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

20.9K ビュー.  University of Newcastle. This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

ビデオ: Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

アン<em生体内で&gt;</em血管透過性をテストする&gt;アッセイ

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

医学

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

パーキンソン病における感受性メカニズムのマスク解除を行う遺伝子環境相互作用モデル

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

マウス疾患モデルで嚥下障害を検出し、特徴づけるために、人間のVideofluoroscopicツバメ調査方法を適応させる

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

アン<em&gt;エクスビボ</emヒト乳癌でホルモンの作用を研究するために&gt;モデル

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

生体内および生体外では腹膜脂肪にミルキースポット構造の卵巣がん転移性コロニー形成を研究するためのアプローチで

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

のための多発性嚢胞腎疾患の小齧歯類モデルにおける超高磁場MRIの使用<em&gt;インビボ</em&gt;表現型と薬物モニタリング

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Using an Ingestible Telemetric Temperature Pill to Assess Gastrointestinal Temperature During Exercise

Exercise results in an increase in core body temperature (Tc), which may reduce exercise performance and eventually can lead to the development of heat-related disorders. Therefore, accurate measurement of Tc during exercise is of great importance, especially in athletes who have to perform in challenging ambient conditions. In the current literature a number of methods have been described to measure the Tc (esophageal, external tympanic membrane, mouth or rectum). However, these methods are suboptimal to measure Tc during exercise since they are invasive, have a slow response or are influenced by environmental conditions. Studies described the use of an ingestible telemetric temperature pill as a reliable and valid method to assess gastrointestinal temperature (Tgi), which is a representative measurement of Tc. Therefore, the goal of this study was to provide a detailed description of the measurement of Tgi using an ingestible telemetric temperature pill. This study addresses important methodological factors that must be taken into account for an accurate measurement. It is recommended to read the instructions carefully in order to ensure that the ingestible telemetric temperature pill is a reliable method to assess Tgi at rest and during exercise.

This study describes an accurate, reliable and non-invasive technique to continuously measure gastrointestinal temperature during exercise. The ingestible telemetric temperature pill is suitable to measure gastrointestinal temperature in laboratory settings as well as in field based settings.

運動中の消化管の温度を評価するために摂取可能遠隔測定温度ピルを使用します

Exercise results in an increase in core body temperature (Tc), which may reduce exercise performance and eventually can lead to the development of ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

ヒト腫瘍異種移植片からマウス細胞の枯渇は大幅に標的細胞の下流分析を向上させます

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

糸球体疾患のマウス ・ モデルにおける腎機能の評価

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

生体内で新生児マウスの造血細胞の移動の可能性を評価するためにコロンの細胞の Photolabeling

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

ex vivoで腸嚢は、胃腸疾患のモデルに粘膜透過性を評価するために

要約

さらに動画を探す

この動画の章

アン

パーキンソン病における感受性メカニズムのマスク解除を行う遺伝子環境相互作用モデル

マウス疾患モデルで嚥下障害を検出し、特徴づけるために、人間のVideofluoroscopicツバメ調査方法を適応させる

アン

生体内および生体外では腹膜脂肪にミルキースポット構造の卵巣がん転移性コロニー形成を研究するためのアプローチで

のための多発性嚢胞腎疾患の小齧歯類モデルにおける超高磁場MRIの使用

運動中の消化管の温度を評価するために摂取可能遠隔測定温度ピルを使用します

ヒト腫瘍異種移植片からマウス細胞の枯渇は大幅に標的細胞の下流分析を向上させます

糸球体疾患のマウス・モデルにおける腎機能の評価

生体内で新生児マウスの造血細胞の移動の可能性を評価するためにコロンの細胞の Photolabeling

ex vivoで腸嚢は、胃腸疾患のモデルに粘膜透過性を評価するために

要約

さらに動画を探す

この動画の章

アン

パーキンソン病における感受性メカニズムのマスク解除を行う遺伝子環境相互作用モデル

マウス疾患モデルで嚥下障害を検出し、特徴づけるために、人間のVideofluoroscopicツバメ調査方法を適応させる

アン

生体内および生体外では腹膜脂肪にミルキースポット構造の卵巣がん転移性コロニー形成を研究するためのアプローチで

のための多発性嚢胞腎疾患の小齧歯類モデルにおける超高磁場MRIの使用

運動中の消化管の温度を評価するために摂取可能遠隔測定温度ピルを使用します

ヒト腫瘍異種移植片からマウス細胞の枯渇は大幅に標的細胞の下流分析を向上させます

糸球体疾患のマウス ・ モデルにおける腎機能の評価

生体内で新生児マウスの造血細胞の移動の可能性を評価するためにコロンの細胞の Photolabeling

糸球体疾患のマウス・モデルにおける腎機能の評価