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1。カルボキシ- H 2 DCFDAを使用してROSの検出カルボキシ- H 2 DCFDAは非蛍光性であるが、ROSの存在下では、この試薬 ​​が酸化されるとき、それは蛍光緑色になります。 <ol><li>使用直前に、無菌ジメチルスルホキシド（DMSO）または100％エタノールでカルボキシ- H 2 DCFDAの新鮮なストック溶液を調製。あなたの染料の複数の融解/凍結サイクルを避けてください。 </li><li> HEPESで細胞を洗浄しても、元の媒体のトレースを削除するには、塩溶液（HBSS）またはリン酸緩衝生理食塩水（PBS）を。 </li><li>染料（と制御色素、CFのノートセクション）で細胞をロードする。このアッセイでは、Jurkat細胞、ヒト白血病細胞株を使用。低血清との定期的な培地（2％）に1μMの最終濃度でカルボキシ- H 2 DCFDAを使用してください。 </li><li>従来のインキュベーター（37℃、5％CO2）で、暗所で30分間培養をインキュベートする。すべての未使用の色素溶液を捨てます。 </li><li>カルボキシを削除します。- H 2 DCFDAは、培地を含有し、HBSSまたはPBSで2回洗浄する。前進このステップから、光から細胞を保護する。 </li><li>カルボキシ- H 2 DCFDA -ロードされた細胞に選択のあなたの薬剤を含有する新鮮培地を追加し、必要に応じて培養する。この例では、1時間、H 2 O 2（0.03％）を使用してください。 </li><li>直ちにFL1チャンネル（緑色蛍光）を使用してフローサイトメトリーによって、または蛍光プレートリーダーによって、または蛍光顕微鏡であなたの細胞を解析することによってROSを評価する。蛍光は、緑色の蛍光に適した励起および発光波長を用いて検出することができます。 </li></ol>注：コントロールは、カルボキシ- H 2 DCFDA -ロードされた未処理細胞と未染色の未処理の細胞が含まれている必要があります。カルボキシ- H 2 DCFDAは、過酸化物を検出することが知られていますが、他のROSにより酸化することができます。この試薬はまた、5のように、他の手段によって酸化を区別しないコントロールの染料を変更することができます - （および-6） - カルボキシ-2&#39;、7&#39; - dichlorofluoresceinジアセテート（カルボキシ- DCFDAが）したがって、試験に含まれるべきです。 2。一酸化窒素（NO）産生の測定あなたは、スルファニルアミドとN - 1 - napthylethylenediamine二塩酸塩（NED）ソリューション、および亜硝酸の標準が必要になります。このアッセイは、グリースアッセイと呼ばれています。 NED液：5％リン酸で希釈したスルファニルアミドの1％溶液を作る：滅菌water.Sulfanilamide液で希釈したN - 1 - napthylethylenediamine二塩酸塩の0.1％溶液を作る。亜硝酸標準：滅菌培地に100μMに0.1Mの標準ストック亜硝酸ナトリウムを希釈し、同じ培地で連続希釈を行う。 保管条件：として、室温で製造業者の指示ストアの化学物質。再構成する場合は、NEDとスルファニルアミド溶液は直ちに4℃、暗所で、3ヶ月、最長使用後に格納されています。 <ol><li> 96ウェルプレートの培養細胞、使用するには、各条件のために三連、および実験に応じて適切なコントロールが含まれています。 </li><li> NO産生を誘導しないため、細胞を扱います。私たちの実験では我々の細胞を治療するためにリポポリサッカライド（LPS）（100 ng / ml）および組換えIL - 4を使用してください。このプロトコルでは、我々はRAW 264.7、マウスのマクロファージ細胞株（マウス白血病単球マクロファージ細胞株）を使用。 </li><li>検定の日に、室温に両方の試薬をもたらす。 </li><li>お皿をスピン、細胞上清を収集し、新しい96ウェルプレートに50μlのを転送する。標準曲線を作るためにあなたの標準原液の限界希釈井戸を準備します。 </li><li>各サンプルと同様の制御にスルファニルアミド溶液の50μLを加え、よく混ぜる。 </li><li>暗所で10分間室温でインキュベートする。 </li><li>各サンプルと同様の制御にN - 1 - napthylethylenediamine二塩酸塩溶液を50μlずつを加え、よく混ぜる。 </li><li>暗所で10分間室温でインキュベートする。 </li><li> absorbaを測定すぐに520nmと550nmの間の波長のフィルターとプレートリーダーを使用してNCE。 </li></ol>あなたが別のプレート/ディッシュのサイズを利用する場合、各ソリューションおよびサンプルの上清について1/1/1ボリュームを使用してください。 紫の色は、陽性ウェルに表示されます。標準で得られた結果は、あなたのソリューションの安定性を確認するのに役立ちます。 3。 ROS作用の検出：酸化タンパク質 ROSの産生を識別するために、別の方法は、タンパク質の酸化を検出することにより、最後の結果を見ることです。確かに、ROS変更グルタチオン、ほとんどの細胞で発現している抗酸化物質は活性酸素に対する保護的な役割を果たしている。 ROSによって以下の酸化、ジスルフィド架橋を介して第グルタチオンにリンクされて、そのシステインのスルフヒドリル（チオール）基の還元型グルタチオン（GSH）の結果の変更。これは、（酸化タンパク質のGSSG）二量体の蛋白質の形成につながる。 GSHは酵素グルタチオンの還元酵素によりGSSGの変更で復元することができます。 GSSG / GSH比の増加は、酸化ストレスを反映している。以下のアッセイは、これらの酸化タンパク質の検出および定量に基づいています。このメソッドは、特定のROSに対して選択的ではないのではなく、NOの影響を検出する、H 2 O 2、O ​​2 -およびその他のROS。ここでは、生物発光シグナルを用いて酸化（GSSG）および還元（GSH）グルタチオンの総量を測定する。 <ol><li>いつものように96ウェルプレートにシードあなたの細胞（透明/白、フラットボトム）。私たちの実験では、我々は、接着RAW 264.7及びA549細胞とサスペンションのJurkat細胞のために、ウェル当たり1 × 10 4細胞を使用していました。あなたの細胞の大きさに応じて、これらの数値を調整することができます。テストは、それらがプレートにアタッチできるようにする前に我々は、プレート接着細胞の日にお勧めします。十分な井戸は、酸化還元タンパク質の検出のためだけでなく、&quot;メディアのみ&quot;のための準備としてくださいコントロールとして未処理の細胞。すべての条件のための三連を使用してください。 </li><li>必要な時間については、薬剤を使用して、細胞を扱います。ここでは、H 2 O 2（5mmと2.5mmのそれぞれにおいて）とJurkat細胞と1HのためのA549細胞を処理した。 RAW 264.7細胞は16時間LPSの200ng/mlで処理したこのインキュベーション時間中に、室温（RT）にすべての試薬をもたらす。 30分以上のテストを実行する前にすべての試薬を加えます。以下の表は、ウェルあたりの試薬の量を示しています。可能な場合、それはテストを続行する前に、還元剤を含有する培地を除去することが重要である。 </li></ol><table border="1"><tbody><tr><td rowspan="2"></td><td colspan="2"> 付着細胞 </td><td colspan="2"> 浮遊細胞 </td></tr><tr><td> 合計Gluの溶解 </td><td> 酸化Gluの溶解 </td><td> 合計Gluの溶解 </td><td> 酸化Gluの溶解 </strong&gt; </td></tr><tr><td> NEM、25mMの</td><td>なし</td><td> 0.5μl </td><td>なし</td><td> 0.5μl </td></tr><tr><td>ルシフェリン- NT </td><td> 1μL </td><td> 1μL </td><td> 1μL </td><td> 1μL </td></tr><tr><td>受動的溶解バッファー、5X </td><td> 10μlの</td><td> 10μlの</td><td> 10μlの</td><td> 10μlの</td></tr><tr><td>蒸留水</td><td> 39.0μl </td><td> 38.5μl </td><td> 14μl </td><td> 13.5μl </td></tr><tr><td> ウェルあたりの最終容量</td><td> 50μlの</td><td> 50μlの</td><td> 25μlの</td><td> 25μlの</td></tr></tbody></table><ol start="3"><li>接着細胞のウェルから培地を取り除く。浮遊細胞のウェルの培地を除去しないでください。 </li><li>対応するウェルに還元型グルタチオン溶解試薬または酸化型グルタチオンの溶解試薬を追加し、室温で5分間振盪する。 </li><li>ルシフェリンの世代の試薬とi​​ncubatを追加します。室温で30分間のe。 </li><li>ルシフェリン検出試薬を追加し、室温で15分間反応させます。 </li><li> 0.25から1秒あたりのウェルプレートリーダーのルミノメーターを使用しての統合時に生物発光シグナルを読む。 </li></ol><table border="1"><tbody><tr><td></td><td> 付着細胞 </td><td> 浮遊細胞 </td></tr><tr><td>ウェルあたりの細胞数</td><td> 1 × 4 </td><td> 1 × 4 </td></tr><tr><td>ウェルあたりの細胞懸濁液+薬</td><td> 3.4前に削除するに100μlの、 </td><td> 25μlの、削除されない</td></tr><tr><td>還元型グルタチオンの溶解試薬</td><td> 50μlの</td><td> 25μlの</td></tr><tr><td>酸化型グルタチオン溶解試薬</td><td> 50μlの</td><td> 25μlの</td></tr><tr><td>ルシフェリン生成試薬</td><td> 50μlの</td><td> 50μlの</td></tr><tr><td>ルシフェリン検出試薬</td><td> 100μlの</td><td> 100μlの</td></tr></tbody></table> 4。代表的な結果： <img alt="図1" src="/files/ftp_upload/3357/3357fig1.jpg" /> カルボキシ- H2DCFDA色素を用いてROSの図1検出 。 H 2 O 2で処理したJurkat細胞（ヒト白血病細胞株）の非処理細胞と比較した。 ROSは、H 2 O 2処理した細胞は、酸化を区別しない染料と非で染色対照のピーク（と比較してO 2処理した細胞は、シフトフローサイトメトリーによって検出される緑色蛍光を発するカルボキシ- H 2 DCFDAの変更、H 2の蛍光ピークを誘導カルボキシ- H 2 DCFDAで染色処理した細胞）。結果は、処理した細胞ではROSの存在を確認する。 <img alt="図2" src="/files/ftp_upload/3357/3357fig2.jpg" /> 図2検出OF NOグリース試薬を使用して。 RAW 264.7細胞（マウスマクロファージ）はLPSとIL - 4で処理した。重要なNO産生増加は、未処理の細胞をコントロールと比較して処理した細胞で検出された。 <table border="1"><tbody><tr><td> 細胞株 </td><td> 無処理 </td><td> 治療 </td></tr><tr><td> RAW 264.7 </td><td> 13.0 </td><td> 8.3 </td></tr><tr><td> A549 </td><td> 21.6 </td><td> 10.5 </td></tr><tr><td> Jurkat細胞</td><td> 5.2 </td><td> 2.8 </td></tr></tbody></table> ROSの表1検出は、タンパク質の酸化を仲介 。 RAW 264.7細胞はLPS、Jurkat細胞及びA549（ヒト肺癌）で処理した細胞は、H 2 O 2で処理した。結果は、比減少（GSH）/酸化（GSSG）グルタチオンのように表される。グルタチオン（GSH）/（GSSG）の低い比率は、に比べて処理した細胞で検出された未処理の細胞を制御し、タンパク質がより多くの処理した細胞で酸化されたことを明らかにする。

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このような炎症性疾患、がん、虚血/再灌流を行い、そして、放射線や化学療法などの治療法として、いくつかの病的状態は（すなわちシスプラチン）ROSの過剰産生を誘導する。従って、ROSレベルを検出および測定することは、多くの基本的な、前臨床および臨床研究において重要である。しかし、ROSは非常に短い半減期を持っていると検出するために複雑にすることができます。ここで、我...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td> 5 - （および-6） - カルボキシ-2&#39;、7&#39; -ジクロロフルオレセインジアセテート（カルボキシ- DCFDA） </td><td>分子プローブ</td><td> C369 </td><td>の制御</td></tr><tr><td>カルボキシ- H 2 DCFDA </td><td>分子プローブ</td><td> C400 </td><td></td></tr><tr><td>ファニルアミド</td><td>シグマ</td><td> S9251 - 100G </td><td></td></tr><tr><td> N - 1 - napthylethylenediamine二塩酸塩</td><td>シグマ</td><td> N9125 - 10G </td><td></td></tr><tr><td>亜硝酸塩の標準</td><td>シグマ</td><td> 237213 - 100G </td><td></td></tr><tr><td> GSH / GSSG -グロアッセイ</td><td>プロメガ</td><td> V6612 </td><td>定量化、酸化減少または酸化/還元型グルタチオンに</td></tr></tbody>
</table>

production and detection of reactive oxygen species (ros) in cancers

活性酸素種は分子に損傷を与えるDNAとRNAの数が含まれており、タンパク質や脂質（脂質のperoxydation）を酸化する。 （OH - ）、hydrochlorous酸（HOCl）、およびヒドロキシルラジカルこれらの反応性分子は、酸素が ​​含まれており、H 2 O 2（過酸化水素）、NO（一酸化窒素）、O 2が含まれます- - （酸化物アニオン）を、ペルオキシナイトライト（尾さん） 。 酸化種のみならず病理学的状況（癌、/虚血再灌流、神経学的および心血管病変、感染症、炎症性疾患1、自己免疫疾患2、等...）の下だけでなく、細胞の代謝3のような生理的（非病理学的）な状況の間に生産されています、4。確かに、ROSは（増殖、細胞の活性化5、6、マイグレーション7等。）多くの細胞内シグナル伝達経路において重要な役割を果たしている。 ROSは有害です（それはその後と呼ばれています&quot;酸化およびニトロソ化ストレス&quot;）一般的に保護するスーパーオキシドジスムターゼ（SOD）とカタラーゼ（CAT）、グルタチオンペルオキシダーゼ（GPX）とグルタチオン（GSH）と酸化防止剤をアップレギュレートすることによってROSへの応答の細胞内区画と細胞内に多量に生産するときに無害な分子（すなわち水）に危険なフリーラジカルを変換することによって、それら。ビタミンCとEは、ROSのスカベンジャー（抗酸化物質）として記載されている。 フリーラジカルは、低量3で有益です。マクロファージと好中球性免疫応答は、ウイルス、病原体や腫瘍増殖8を阻害NOの産生および放出を伴います。 NOはまた、他のROSと反応していないので、またデトックス（ROSのスカベンジャー）としての役割を持っています。長時間運動9、10〜筋肉の適応のために重要である血流を調節するために船で最後にNOを作用しません。いくつかの出版物はまた、ROSはインシュリンのSENSに関与していることが示されているitivity 11、12。 ROS産生を評価するための多数のメソッドが使用可能です。この記事では、いくつかの、シンプルで速く、そして手頃なアッセイを提案する、これらのアッセイは、多くの出版物によって検証され、日常的に哺乳動物細胞でROSやその効果を検出するために使用されます。これらのアッセイのいくつかは複数のROSを検出しながら、他は単一のROSを検出する。

Watch this Scientific Journal Video about Production and Detection of Reactive Oxygen Species ROS in Cancers at JoVE.com

Production and Detection of Reactive Oxygen Species ROS in Cancers

ここでは、細胞内の活性酸素種の存在をテストし、評価するための簡単​​な方法を提案する。

がんの生産と活性酸素種の検出（ROS）

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

production-and-detection-of-reactive-oxygen-species-ros-in-cancers

Research

JoVE Journal

Medicine

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

69.9K ビュー.  Baylor College of Medicine. ここでは、細胞内の活性酸素種の存在をテストし、評価するための簡単​​な方法を提案する。

ビデオ: Production and Detection of Reactive Oxygen Species ROS in Cancers

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

肝微小循環における血流と酸素消費量の可視化と分析：急性肝炎モデルへの応用

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

医学

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

がん治療のモニタリングのためのチェレンコフ発光イメージング（CLI）

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

バイオアッセイとしてウシ腸間膜動脈と静脈の切片を分離して使用すると、小腸で血管作用をテストするために

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

と伝達性海綿状脳症種の障壁を評価する<em&gt;インビトロ</em&gt;プリオンタンパク質変換アッセイ

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

人間A53T変異αシヌクレインを過剰発現するトランスジェニックマウスの脳における強化ELISAによる疾患関連αシヌクレインの検出

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

反応性神経膠症のトリガ<em&gt;インビボ</em&gt;前脳刺し損傷によって

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In VitroとIn Vivo検出、ひと細胞、線虫、マウス Mitophagy の

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

VASP のリン酸化を測定することによって血液中の一酸化窒素の血小板ベースの検出

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

造影剤としてのプロトン磁気共鳴イメージングと酸素を用いたヒト肺における特定の換気の定量的マッピング

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When deoxygenated, HbS can polymerize, forming rigid rods of hemoglobin, resulting in the sickling of red blood cells (RBCs). These sickled RBCs have significantly reduced deformability, causing vaso-occlusion, which leads to numerous SCD-related clinical complications, including pain, stroke, and organ damage. RBC deformability is also reduced by RBC dehydration, resulting in dense red blood cells that are more likely to sickle. To date, there is not a single widely available, rapid, and reproducible laboratory assay capable of predicting the disease severity or directly monitoring the treatment effects for novel, non-fetal hemoglobin inducing therapies. In this study, we describe a protocol to measure RBC deformability as a function of pO2 that allows for the quantitation of sickling behavior in SCD patients. Oxygen gradient ektacytometry measures RBC deformability, expressed as the elongation index (EI), as a function of pO2. RBCs are exposed to a fixed shear stress of 30 Pa during one round of deoxygenation and reoxygenation. Six readout parameters are produced. Of these, the point of sickling (PoS), defined as the pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient&#8217;s pO2 at which sickling starts and the minimal deformability of a patient&#8217;s red blood cells, respectively. PoS is associated with an individual patient&#8217;s hemoglobin affinity for oxygen, whereas EImin shows a strong correlation with fetal hemoglobin levels. We conclude that oxygen gradient ektacytometry is a promising technique to monitor the treatment of patients with SCD, as a biomarker for anti-sickling agents in clinical and preclinical trials, and an important tool to study sickling behavior of RBCs from individuals with SCD and sickle cell traits.

Here, we present oxygen gradient ektacytometry, a rapid and reproducible method to measure red blood cell deformability in samples from patients with sickle cell disease under controlled deoxygenation and reoxygenation. This technique provides a way to study red blood cell sickling and to monitor sickle cell disease treatment efficacy.

酸素勾配エクタキトメトリーを用いた制御自動脱酸素時の鎌の特性評価

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When ...