FMは、PNPグラント、研究所Universitaire·ド·フランスなどソルボンヌパリシテ（ANR-10-LABX-0023とANRでLabex UniverEarthプログラムを通じてchaireドール優秀IDEXソルボンヌパリシテ、INSUを通じてANRからの資金調達を認めます-11-IDEX-0005から02）。また、欧州共同体のH2020フレームワークプログラム/ ERC助成契約＃637503（プリスティン）の下で欧州研究評議会からの資金に感謝します。

注：動物を含む手順は、大学のパリディドロにおける施設内動物管理使用委員会（IACUC）によって承認されています。 材料の作製<ol><li>サブ沸騰不純物からそれらを精製するために酸（HNO 3、HBrを）の1 Lを蒸留します。 </li><li>きれいに熱い（〜100℃）でビーカーと先端アダプタは、少なくとも2日間HNO 3酸浴を濃縮しました。 </li><li>数日間の冷3 N HNO 3浴中でピペットチップを洗浄し、脱イオン水で個別に3回すすいでください。 </li></ol> 2.サンプル調製<ol><li>ケタミンとキシラジンの腹腔内注射によりマウスを麻酔。つま先のピンチ方式により麻酔を評価します。 </li><li> 1.5ミリリットルチューブ内のヘパリンの存在下での心臓穿刺により血液を収集します。 </li><li>遠心分離（10分、1,500×gで）により血液細胞から血漿を分離し、ポリプロピレンcにプラズマを転送しますポリプロピレンのヒントを使用してryogenicバイアル。 </li><li>肝静脈を切断し、心臓にDPBSを注入することにより器官から残りの血液を除去。頚椎脱臼によりマウスの死を評価します。 </li><li>滅菌ステンレス鋼の機器との臓器を収穫、もしあれば脂肪周囲のそれらを解放し、スナップ凍結してポリプロピレンの低温バイアル中。 </li></ol> 3.化学精製<ol><li>まず、濃（30％）〜1ミリリットルH 2 O 2、濃縮（15〜M）HNO 3の約1 mlと混合物中にサンプルを溶解します。ヒュームフード内のすべての手順を実行してください。 <ol><li> 15ミリリットルのテフロンビーカーに関心のある器官全体を配置します。その後、ビーカー5にH 2 O 2 / HNO 3を追加。有機物の酸化反応およびCO 2の放出に起因する飛沫を避けるために、数分間開放ビーカーにしてください。 </li><li>最後に、ABOのホットプレート上でビーカーを置きます時間のカップルのための、または溶液が完全に透明になるまで100℃でユタ。 </li></ol></li><li>ビーカーを開き、約100℃のホットプレート上に溶液を乾燥。 </li><li>サンプルが乾燥すると、サンプルに1.5 NのHBrの1ミリリットルを追加します。ビーカーを閉じて、それが時間のカップルのため、100℃のホットプレート上で溶解させます。 </li><li>一方、500μlのカラムを準備します。 <ol><li>列にAG1X8 200〜400メッシュの樹脂の500μlを添加して、その下のゴミ箱のビーカーでカラムラックの上に置きました。 5ミリリットル18.2のMΩ⋅センチ水、0.5 N 5mlのHNO 3、5mlの水、0.5 N 5mlのHNO 3、および水の後、5ミリリットルを：交互に樹脂を洗浄。 1.5 NのHBr 5mlで樹脂を調整。 </li></ol></li><li>ホットプレートからビーカーを取り出して、約30分間超音波浴に入れ、その後、ビーカーを室温まで冷却させます。 </li><li>ビーカーを冷却し、樹脂を洗浄した後、ビーカーを開きます。 tに先端アダプタを入れて彼は、注射器、ピペットチップを追加します。サンプルの1ミリリットルをピペット（樹脂を攪拌させないために、非常にゆっくりと）樹脂上にロードします。 </li><li>すべての液体がカラムを通過した後、1.5 NのHBrの5ミリリットルを追加します。 </li><li> 1.5 NのHBr 5mlをカラムを通過すると、きれいな15ミリリットルビーカーでゴミのビーカーを交換してください。 </li><li>一度に0.5 NのHNO 3 2.5ミリリットルの5ミリリットルを追加します。この段階で、Znが樹脂から溶出されます。 </li><li> HNO 3 5mlをカラムを通過したら、ビーカーを除去し、乾燥するまで、100℃のホットプレート上に置きます。 </li><li>カラムホルダーから列を削除します。樹脂をゴミ箱（各サンプルのための新しい樹脂を使用します）。 </li><li>サンプルが乾燥したら、小さい列に酸（100μl）を同容量のプロトコルを繰り返した後、乾燥するまで、ホットプレートの上に置きます。サンプルは、質量分析のための準備ができました。 </li></ol> 4.質量分析測定<ol><li>亜鉛同位体コンポを分析マルチコレクタ - 誘導結合プラズマ質量分析計（MC-ICP-MS）にジション。 <ol><li> 表1にまとめた機械パラメータを使用してください。 </li></ol></li><li> 62のNi、63銅、64亜鉛、銅65、66のZn、67 Znおよび68 Znを質量（M / Z）で収集するために、ファラデーカップを置きます。 </li><li>同位体分析のために、0.1M HNO 3で500 ppbでのZnを含む溶液を調製します。 </li><li> 100μL/分テフロンネブライザーと組み合わせたスプレーチャンバを使用して、亜鉛の500 ppbの液を分析。各サンプルについて、各スキャンの積分時間は8.389秒である30のスキャン（30サイクルの1ブロック）を測定します。 </li><li>ブランク溶液（サンプルを再溶解するために使用される、0.1M HNO 3溶液）からのピークゼロ強度を差し引くことにより、バックグラウンドを修正してください。 </li><li> 62 Niのピークの強度を測定することによって制御し、正しい可能性64 Niの同重体干渉。64のNi / 62 Ni比が（0.2548）自然で、楽器のマスバイアスから、この値を修正し、などのマス64の64のNiを削除することを想定します。 64 Znの実 = 64 Znを測定 - 64のNi = 64のZnを測定 - （64のNi / 62 Ni）の自然のx 62 Niを測定しました 。 </li><li> JMCリヨンのZn標準の500 ppbの標準溶液（またはIRMM-3702のような別の利用可能な標準）での各サンプルをブラケットにより楽器マスバイアスを修正してください。 （ 式1を参照）は、2つの標準サンプルマイナス1前後で測定し、千を乗じの66のZn / 64のZn比の平均による試料の66のZn / 64 Znの比を割ることにより、標準のブラケットを実行します。 JMCリヨンのZn標準の一般的な外部精度は0.05パーミル/ AMU（2標準偏差、2 SD）です。 </li></ol>

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著者らは、開示することは何もありません。

測定の再現性は、異なる分析セッション中に行うと同じサンプルの複製された分析によって評価されます。例6のために、私たちは同じ地上岩を7回繰り返しており、我々は表2に報告された結果を得ました。 （15〜17 例えば、隕石11-13、植物3-5、深海堆積物14、動物）、これまで任意の太陽系材料で測定同位体分?...

高精度（より良い100ppm以下/原子質量単位）の測定は、亜鉛安定同位体組成は約15年間、マルチコレクタプラズマソース質量分析計の開発のおかげで可能になった、それ以来、主に地球に適用されています惑星科学。医療分野への応用は、新規であり、亜鉛（ 例えば、アルツハイマー病）の代謝を変更する疾患のバイオマーカーとしての大きな可能性を持っています。本稿では、種々のマウス臓器で高精度に亜鉛の自然な安定同位体比を測定する方法を報告します。同じことが、ヒトサンプルに適用可能です。この方法は、臓器、原子の残りの部分からの亜鉛の化学的精製、その後、質量分析計での同位体比の分析の溶解で構成されています。 亜鉛同位体測定の質は、化学的精製（亜鉛の純度、低ブランクCOMPの品質に依存していますサンプル中のZnの存在量、手順の高い化学収率）にし、楽器のバイアスの制御にared。最終的な亜鉛画分の純度は、マトリックス効果を作成両方同重体干渉と非同重体干渉を除去するために必要とされます。同重体核種が直接干渉（ 例えば、64のNi）を作成します。非同重体干渉は、いわゆる「マトリックス」効果を生成し、サンプルが1と比較される純亜鉛の標準に比べてイオン化の条件を変更することにより、測定値の分析精度を変化させます。低ブランク（&lt;10 ngの）そのバイアス測定同位体組成を、外付けのZnによる試料のコンタミネーションがないことを示しています。亜鉛同位体は、イオン交換クロマトグラフィーの間に2分画できるように、すべてのZn原子の集合は、化学的手順は、完全な収率を有していなければならないことを意味する、いかなる同位体分別が起こらないことを保証します。最後に、質量分析測定中の楽器の同位体分別の補正は、「標準ブラケット &quot;メソッドを介して行われます。 そのため、正確な測定値を得るための主な困難は、他の原子または分子のクリーンで完全降伏化学的精製を製造、および質量分析計で楽器同位体分別の補正、外部汚染（ すなわち、低ブランク）を制御しています。本稿では、マウスの臓器と同様に質量分析の測定値からのZnを分離するために私たちの分析プロトコルを記述します。 抽出は、陰イオン交換樹脂のマイクロカラム（0.5μLおよび0.1μL）で希釈した酸（HBrを/ HNO 3メディア）の少量を使用して行われます。これは、完全な収率を有しており、測定値が66のZn / 64のZn比で50ppmのよりも優れた外部再現性を持っています。メタの別の利点ODが、それは非常に高速であるということです。この方法は、極めてよくつこれらの分析法が開発された地球科学に比べて多数のサンプルを分析する必要のある、医学に適合されます。

<table><tbody><tr><td>Multi-collection inductively-coupled-plasma mass-spectromter</td><td>Thermo-Fisher</td><td></td><td></td></tr><tr><td>Anion-exchange resin AG1 X8 200-400</td><td>Bio-Rad</td><td>140-1443-MSDS</td><td></td></tr><tr><td>Teflon beakers</td><td>Savillex&amp;nbsp;</td><td>200-015-12</td><td></td></tr><tr><td>In-house-made teflon colunms made with shrinkable teflon</td><td></td><td></td><td></td></tr></tbody></table>

high precision zinc isotopic measurements applied to mouse organs

我々は、マウスの臓器における高精度の亜鉛同位体比で測定するための手順を提示します。亜鉛は、マウスの臓器の間に自然に分画される5安定同位体（64 Znを、66のZn、67のZn、68 Znおよび70 Zn系）で構成されています。まず、Zn原子を解放するために、異なる臓器を溶解する方法を示しています。このステップは、HNO 3とH 2 O 2の混合物によって実現されます。それから、希のHBr / HNO 3媒体に陰イオン交換クロマトグラフィーによる同重体干渉（ 例えば 、Ni）の、より具体的には、他のすべての要素からの亜鉛原子を精製します。これらの最初の2つのステップは、高純度化学薬品を使用して、きれいな実験室で行われています。最後に、同位体比は、低解像度のマルチコレクタ誘導結合プラズマ質量分析計を用いて測定されます。サンプルはcorrecあるスプレーチャンバーと質量分析計により誘導される同位体分別を用いて注入されています標準（標準ブラケット技術）の比にサンプルの比率を比較することにより、テッド。この完全な典型的な手順は、50 ppmの（2 SD）再現性の同位体比を生成します。

1.5 NのHBrでは、主亜鉛種（ZnBr3-）陰イオン交換樹脂でフォーム非常に強い複合体は、他のほとんどの要素は、樹脂と相互作用しないながら。亜鉛は、その後、樹脂6,7から放出される2+ Znに亜鉛の分化を変化させる、希釈HNO 3に培地を変更することによって回収されます。 同位体比は、典型的には、標準に比べて千の偏差当たりの部として表現さ​?...

Watch this Scientific Journal Video about High Precision Zinc Isotopic Measurements Applied to Mouse Organs at JoVE.com

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

We present the technique to measure with high precision zinc isotope ratios in mouse organs.

マウス臓器に印加される高精度の亜鉛同位体測定

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn ...

high-precision-zinc-isotopic-measurements-applied-to-mouse-organs

Universidad de Cartagena UDEC

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JoVE Journal

Chemistry

7.4K ビュー.  Université Paris Diderot. We present the technique to measure with high precision zinc isotope ratios in mouse organs.

ビデオ: High Precision Zinc Isotopic Measurements Applied to Mouse Organs

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

評価するための総合的な手順<em&gt;インビボ</em&gt;がんNanomedicinesのパフォーマンス

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

がん研究

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

の調製と評価<sup&gt; 99メートル</sup&gt; Tc標識三座キレートバイオ直交化学反応を用いて事前ターゲティングするため

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

化学

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

哺乳類細胞内の細胞内亜鉛プールを測定するための原子吸光度分光法

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

生化学

Shotgun Lipidomics of Rodent Tissues

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Shotgun mass spectrometry-based lipidomics delivers a sensitive quantitative snapshot of a broad spectrum of lipid classes simultaneously in a single measurement from various rodent tissues.

げっ歯類組織のショットガンリピドミクス

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have ...

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

マウスの複数の臓器からのジカウイルスの単離と定量

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

免疫・感染学

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

身体組成と高脂肪給電マウスにおける代謝ケージ分析

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

医学

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

平滑筋細胞系譜トレース マウスの高度な動脈硬化病変における細胞構成の定量的解析

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

マウスにおける肝細胞核プロイドの推定に対するSitu法におけるハイスループット

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects.
This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches.

陽電子放射断層撮影法を用いた実験的自己免疫性脳脊髄炎マウスモデルにおけるCD19+ B細胞のイメージング

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological ...

マウス臓器に印加される高精度の亜鉛同位体測定

要約

さらに動画を探す

この動画の章

評価するための総合的な手順

の調製と評価

哺乳類細胞内の細胞内亜鉛プールを測定するための原子吸光度分光法

げっ歯類組織のショットガンリピドミクス

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of ⁶⁸Ga-Labelled Nano Molecules

マウスの複数の臓器からのジカウイルスの単離と定量

身体組成と高脂肪給電マウスにおける代謝ケージ分析

平滑筋細胞系譜トレースマウスの高度な動脈硬化病変における細胞構成の定量的解析

マウスにおける肝細胞核プロイドの推定に対するSitu法におけるハイスループット

陽電子放射断層撮影法を用いた実験的自己免疫性脳脊髄炎マウスモデルにおけるCD19⁺ B細胞のイメージング

マウス臓器に印加される高精度の亜鉛同位体測定

要約

さらに動画を探す

この動画の章

評価するための総合的な手順

の調製と評価

哺乳類細胞内の細胞内亜鉛プールを測定するための原子吸光度分光法

げっ歯類組織のショットガンリピドミクス

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

マウスの複数の臓器からのジカウイルスの単離と定量

身体組成と高脂肪給電マウスにおける代謝ケージ分析

平滑筋細胞系譜トレース マウスの高度な動脈硬化病変における細胞構成の定量的解析

マウスにおける肝細胞核プロイドの推定に対するSitu法におけるハイスループット

陽電子放射断層撮影法を用いた実験的自己免疫性脳脊髄炎マウスモデルにおけるCD19+ B細胞のイメージング

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of ⁶⁸Ga-Labelled Nano Molecules

平滑筋細胞系譜トレースマウスの高度な動脈硬化病変における細胞構成の定量的解析

陽電子放射断層撮影法を用いた実験的自己免疫性脳脊髄炎マウスモデルにおけるCD19⁺ B細胞のイメージング