我々は、この作品[R33 CA174616-01A1]を資金調達NIHから国立癌研究所のIMATプログラムに感謝します。

1.ターゲット細胞の調製<ol><li> 37°Cの時に（10％の熱不活性化ウシ胎児血清（FBS）、4nmのL-グルタミン、500 IU / mlペニシリン/ストレプトマイシンを、RPMI 1640培地）増殖培地中の標的細胞（ 例えば 、Raji細胞リンパ腫細胞）を維持5％CO 2インキュベーター中。 </li><li>遠心分離機15ミリリットルチューブに標的細胞をペレット化するためのサンプル。遠心分離時間は、異なる細胞型によって変化する。 Raji細胞のための468×gで3分間遠心する。 </li><li>最初の上清の表面に形成された気泡を吸引し、全上清を除去。 </li><li>チューブに10mlのPBSを追加します。再懸濁細胞塊を分割するには、上下に数回ソリューションをピペッティングすることにより細胞を。 </li><li>遠心分離機468×gで3分間のサンプル。 </li><li>上清の表面に形成された気泡を吸引し、全上清を除去。 </li></ol>細胞マイクロアレイの作製<ol><li> Obtain細胞付着の96ウェルプレートキットまたは単一セルアレイ8ウェルチャンバースライド（材料/試薬の表を参照）。 </li><li>キットからのDNA試薬にアクティベーターの1.2μlのを適用します。バイアルにキャップを、それを反転し、優しく溶液を混合する振る。 </li><li>ソリューションは、RTで20分間反応させる。 </li><li> （15mlチューブ中で）標的細胞ペレットを混合した溶液を塗布する。 </li><li> 500 nMの最終濃度緑色蛍光細胞質ゾル染料を追加します。 </li><li>オービタルシェーカー上でRTで30分間混合した溶液および色素で標的細胞をインキュベートする。 </li><li> 5mLのPBSで細胞を2回洗浄し、10mlの増殖培地中で細胞を再懸濁する。 </li><li>各ウェルにステップ2.7から溶液100μlを適用します。血球計算盤を用いて細胞数を数える。細胞数はウェル当たり1×10 6の細胞に5×10 4の範囲であることを確認してください。 </li><li> RTでインキュベーションの10〜15分後、静かに200から30で二回のサンプルを洗う未結合の細胞を除去する0μlの増殖培地。 注：約5-10μlの増殖培地は、洗浄後に各ウェルに残っています。 </li></ol> 3.全血細胞毒性アッセイ<ol><li>各サンプルウェルにヒト全血180μlのを追加します。 </li><li>ミリリットル/ 5mgのに抗CD20抗体溶液を得、そして作るために連続希釈を行い50、20、10、5、2,1、1.5 mlチューブ中の抗CD20抗体溶液を0.5μg/ mlであった。よく三重を作る各サンプルに抗CD20抗体の各濃度の20μlのを追加します。 </li><li>各ウェルに500 nMでの最終濃度でヨウ化プロピジウムを追加する。 </li><li> 16時間、5％CO 2、37℃で、得られたプレートをインキュベート。 </li><li>画像スライドまたは8X倍率に自動撮像システム96ウェルプレート上の細胞。 </li><li>結果を定量化するために自動撮像システムの細胞計数プログラムを使用します。 NOTE：自動撮像システムのソフトウェアは、単純な提供細胞計数のためのステップバイステップの操作。このソフトウェアを使用するプロトコルは、プロバイダのウェブサイトで見つけることができます。 </li><li>ソフトウェアのメインページから[レビュー結果]を選択します。 </li><li> [サンプルのために保存したファイル]を選択します。 </li><li>最初のゲートは、FITCその後ゲートが強度&gt; 20を意味TxRed、&gt; 50強さを意味します。 </li><li> [プレートの概要]を選択します。 </li><li> [エクスポートテーブル]を選択します。 注：プログラムのパーセンテージ細胞殺傷式を以下に示す： ％の標的細胞の死滅=（緑と赤の両方の染色された細胞の数/細胞の＃は緑色に染色）* 100％ </li></ol>

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著者は、競合する経済的利益を持っていません。

WCAは、理想的にはそのようなCDCおよびADCCアッセイ9-11、などと前臨床動物試験の前に、伝統的なターゲットの上映の後に利用され、単一細胞の解像度12-16、を用いたin vitro抗がんスクリーニングツールにおいて重要である。現在、CDCまたはADCCアッセイの一次標的スクリーニングアッセイは、すべての単純化されたメディアまたは緩衝液系中で実施される。?...

製薬業界における最近の進歩は、腫瘍細胞抗体パーソナライズ癌治療の特定の識別を実現する上で関心が高まってきた。しかし、いくつかの障害は、プロセスで遭遇している。前臨床開発中の抗がん活性を有する薬剤のわずか5％がフェーズII-III試験の1,2で十分な有効性を示した後にライセンスされています。多くの例では抗腫瘍薬3-5主としてそれらの異なる血液成分にヒトおよび実験動物では異なる挙動を示すことが示されているように（in vitroおよびin vivoの両方で ）前臨床戦略は最適以下である。 抗腫瘍薬物スクリーニングプラットフォームの必要性に対処するために費用のかかる動物実験および臨床試験前のチェックポイントを提供するために、ヒト全血の細胞毒性アッセイ（WCA）は、より関連性の生物学的環境において抗腫瘍薬の有効性を評価するために提案されている。ザ·全血の細胞毒性アッセイは、抗体およびヒト全血の他の薬物候補への個々の細胞の応答を評価するために用いることができる。 WCAは、ハイスループットおよびハイコンテントイメージング6でハイスループット細胞測位技術を組み込むことによって現像される。自動化されたイメージングシステムを利用することにより、生きている細胞および死細胞の両方の数を高精度に判定することができる。による標的細胞は、同一の焦点面上に固定されているという事実のため、WCAは、赤血球を除去することなく、リアルタイムでの定量的細胞毒性分析を提供することができる。また、自動イメージングシステムは、その指定した基準に達した場合にのみ、標的細胞（ 例えば 、蛍光標識された細胞、および細胞形態）などのいくつかの利点ゲートされ、処理さを提供する。また、一日あたり144のプレートを製造することができる。したがって、この撮像能力とスループットが高いcの実行を可能にするontentハイスループット実験を同時に。 WCAおよび自動画像化システムを組み合わせることにより、高スループット定量的細胞毒性分析は、複数の生物学的関連性環境内で達成することができる。

<table border="0" cellpadding="0" cellspacing="0" width="761">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Name of Material/ Equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:135px;">Name/Discription</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:336px;">Cell attachment 96 well plate kits&#160;</td>
			<td style="width:172px;">Adheren</td>
			<td style="width:119px;">AP9601</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:336px;">Suspension Single cell array 8 well chamber slide</td>
			<td style="width:172px;">Adheren</td>
			<td style="width:119px;">SS0801</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:336px;">Adherent Single cell array 8 well chamber slide</td>
			<td style="width:172px;">Adheren</td>
			<td style="width:119px;">SS0802</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Lymphoma cell line CD20+</td>
			<td style="width:172px;">ATCC</td>
			<td style="width:119px;">CCL-86</td>
			<td style="width:135px;">Raji cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Lymphoma cell line CD20-</td>
			<td style="width:172px;">ATCC</td>
			<td style="width:119px;">CRL-8083</td>
			<td style="width:135px;">MC/CAR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">RPMI 1640 with L-glutamine</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">11875-119</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Fetal Bovine Serum</td>
			<td style="width:172px;">Thermo</td>
			<td style="width:119px;">SH30070.01HI</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Peni/Strep</td>
			<td style="width:172px;">Life Technologies</td>
			<td align="right" style="width:119px;">15070063</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Cytosolic dye</td>
			<td style="width:172px;">Life Technologies</td>
			<td style="width:119px;">C7025</td>
			<td style="width:135px;">Cell Tracker Green</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Rituxan (Biosimilar)&#160;</td>
			<td style="width:172px;">Eureka Therapeutics</td>
			<td style="width:119px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Human whole blood</td>
			<td style="width:172px;">Allcells</td>
			<td style="width:119px;">WB001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Propidium Iodide</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:119px;">P4170-10MG</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Automatic imaging system</td>
			<td style="width:172px;">Molecular Devices</td>
			<td style="width:119px;">Contact Vendor</td>
			<td style="width:135px;">Cell Reporter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Cell counting program</td>
			<td style="width:172px;">Molecular Devices</td>
			<td style="width:119px;">Contact Vendor</td>
			<td style="width:135px;">Cell Reporter</td>
		</tr>
	</tbody>
</table>

real-time cytotoxicity assays in human whole blood

全体的な細胞毒性の時間情報へのアクセスを可能にする生細胞ベースの全血の細胞毒性アッセイ（WCA）は、ハイスループット細胞測位技術で開発されている。標的腫瘍細胞集団は、最初の配列形式に固定化予めプログラム、および緑色蛍光の細胞質ゾル色素で標識されている。セルアレイを形成した後、抗体医薬品は、ヒト全血との組合せで添加される。ヨウ化プロピジウム（PI）は、細胞死を評価するために添加される。セル配列は、自動画像化システムを用いて分析される。細胞質ゾルの色素が標的腫瘍細胞集団にラベルを付けながら、PIは死んだ腫瘍細胞集団にラベルを付けます。従って、標的癌細胞の殺傷の割合は死んだ標的細胞の数の標的細胞の生存数を計算することによって定量することができる。この方法では、研究者は、時間依存性および用量依存性の細胞毒性の情報にアクセスすることができる。驚くべきことに、有害ラジオません化学物質が使用されている。 WCAは、ここで提示リンパ腫、白血病、および固形腫瘍細胞株を用いて試験された。したがって、WCAは研究者が、関連性の高いex vivoの状態で薬効を評価することができます。

抗CD20抗体およびリンパ腫細胞（Raji細胞およびMC / CAR）は、全血の細胞毒性アッセイ（WCA）7,8を実証するためのモデル系として選択した。 MC / CAR細胞がその膜上のCD20の低コピー数を有していたRaji細胞では、細胞表面上のCD20の高いコピー数を有していた。標的細胞は、最初に緑色蛍光細胞質色素で緑色に染色し、96ウェルプレート上に配列した。万 - 5万標的リンパ腫細胞を、各ウェル?...

Watch this Scientific Journal Video about Real-time Cytotoxicity Assays in Human Whole Blood at JoVE.com

Real-time Cytotoxicity Assays in Human Whole Blood

全血細胞毒性アッセイ（WCA）は、蛍光顕微鏡および自動化された画像処理をハイスループット細胞測位技術を組み込むことによって開発された細胞毒性アッセイである。ここでは、抗CD20抗体で処置したリンパ腫細胞の定量的な細胞傷害性分析を提供するために、ヒト全血中でリアルタイムに分析することができる方法について説明します。

ヒト全血でのリアルタイム細胞毒性アッセイ

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

real-time-cytotoxicity-assays-in-human-whole-blood

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JoVE Journal

Medicine

12.1K ビュー.  Adheren, Inc. 全血細胞毒性アッセイ（WCA）は、蛍光顕微鏡および自動化された画像処理をハイスループット細胞測位技術を組み込むことによって開発された細胞毒性アッセイである。ここでは、抗CD20抗体で処置したリンパ腫細胞の定量的な細胞傷害性分析を提供するために、ヒト全血中でリアルタイムに分析することができる方法について説明します。 

ビデオ: Real-time Cytotoxicity Assays in Human Whole Blood

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

生体内イメージングを使ってリアルタイムで腫瘍におけるナノ粒子の取り込みの評価

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

医学

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

全血におけるエノキサパリン抗凝固効果の迅速なポイントオブケアアッセイ

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

定量的リアルタイムPCR（定量PCR）による前立腺腫瘍におけるマイクロRNAの検出

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

パフォーマンスのリアルタイムのフィードバックを使用して再教育運動

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

定量リアルタイムPCRを用いて脳脊髄液マイクロRNAプロファイリング

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

化学療法誘発性血管毒性 - リアルタイム<em&gt;インビボ</em船舶減損の&gt;イメージング

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

リアルタイムで中皮細胞の卵巣癌スフェロイドアタッチメントおよび浸潤の評価

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

定量的リアルタイムPCRによるマウス乳腺腫瘍モデルにおける肺転移の評価

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via a liver laceration that results in consistent blood loss, hemodynamic alterations, and survival.
Mice undergo a standardized resection of the left-middle lobe of the liver. They are allowed to bleed without mechanical intervention. Hemostatic agents can be administered as pre-treatment or rescue therapy depending on the interest of the investigator. During the time of hemorrhage, real-time hemodynamic monitoring via a left femoral arterial line is performed. Mice are then sacrificed, blood loss is quantified, blood is collected for further analysis, and organs are harvested for analysis of injury. Experimental design is described to allow for simultaneous testing of multiple animals.
Liver hemorrhage as a model of uncontrolled hemorrhage exists in the literature, primarily in rat and porcine models. Some of these models utilize hemodynamic monitoring or quantify blood loss but lack consistency. The present model incorporates quantification of blood loss, real-time hemodynamic monitoring in a murine model that offers the advantage of using transgenic lines and a high-throughput mechanism to further investigate the pathophysiologic mechanisms in uncontrolled hemorrhage.

Uncontrolled hemorrhage, an important cause of mortality among trauma patients, can be modeled using a standard liver laceration in a murine model. This model results in consistent blood loss, survival, and allows for testing hemostatic agents. This article provides the step-by-step process to perform this valuable model.

リアルタイム血行動態モニタリングを用いたマウスの肝梗塞モデルによる制御不能出血性ショック

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

血液脳関門メッシュ様血管形成および崩壊を評価するためのインビトロアッセイ

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...