<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quantum Yield Measurement of Sodium Salicylate. FL080002, 1 (2008).</li><li>Lakowicz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+Fluorescence+Spectroscopy.">Principles of Fluorescence Spectroscopy.</a> Science and Business Media, LLC. 60, (2006).</li><li>Horigome, J., Wakui, T., Shirasaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Correction+Method+for+Determination+of+Absolute+Fluorescence+Quantum+Yields+of+Solid+Samples+with+a+conventional+Fluorescence+Spectrophotometer.">A Simple Correction Method for Determination of Absolute Fluorescence Quantum Yields of Solid Samples with a conventional Fluorescence Spectrophotometer.</a> Bunseki Kagako. 58 (6), 553-559 (2009).</li></ol>

ルイス·A.·モレノは、この記事で使用されているインストゥルメントを生成します日立ハイテクノロジーズアメリカで採用されています。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>サリチル酸ナトリウムの粉末</td><td>埼玉県和光市</td><td> 191から03142 </td><td>モル。重量160.10 </td></tr></tbody></table>

absolute quantum yield measurement of powder samples

蛍光量子収率の測定は、照明の開発、評価、品質管理や研究の新たなソリューションの検索では重要なツールとなっており、AV機器、有機EL材料、フィルム、フィルター、バイオ産業のための蛍光プローブ。 量子収率は材料によって放出されるフォトンの数に、吸収光子数の比率として計算されます。高い量子収率、蛍光物質の効率良い。 このビデオで紹介さ測定のために、我々は付属品とレポート·ジェネレータ·プログラムを測定する量子収率を搭載した日立F-7000蛍光分光光度計を使用します。提供されるすべての情報は、このシステムに適用されます。 粉末試料における量子収率の測定は、以下の手順に従って実行されます。 <ol><li>励起とemissの計測器の補正係数の生成イオン分光器。これは量子収率の正確な測定のための重要な要件です。それは楽器の全測定範囲のために、事前に行われていると時間の制限により、このビデオには表示されません。 </li><li>球の補正係数を統合するための測定。このステップの目的は、測定に使用する積分球を考慮し反射特性を考慮することである。 </li><li>直接励起と間接励起を使用してリファレンスとサンプル測定。 </li><li>直接および間接励起を用いた量子収率の計算。サンプルは通常の測定セットアップになります直接励起ビームを、直面しているときに直接励起である。しかし、我々は積分球を使用しているため、サンプルの蛍光に起因する放射される光子の一部は、積分球によって反射され、再励起する試料を、我々は考慮間接的な励起を考慮する必要があります。これはアコです。間接的な量子収率を計算し、直接の量子収率の計算を修正し、発光分光器が直面しているポートに配置されたサンプルを測定することにより、mplished。 </li><li>量子収率の計算を修正しました。 </li><li>色度は、レポート·ジェネレータ·プログラムを使用して計算を調整します。 </li></ol>次のように、このアプリケーションの日立F-7000量子収率測定システムの提供の利点： <ul><li>高感度（S / N比800以上RMS）。信号が以下の条件で測定した水のラマンバンドである。例波長350nmのバンドパスExとエムが5nm、レスポンス2秒）、ノイズがラマンピークの最大値で測定されます。高感度でも低量子収率を持つ試料の測定を可能にします。このシステムを用いて、我々は、サリチル酸のサンプルは0.1と低く、マグネシウムタングステンのサンプルは0.8のように高い量子収率を測定した。 </li><li>の6桁のダイナミックレンジと高精度測定大きさは高強度と同様に、低強度の広範な蛍光ピークが同じ条件で、両方のシャープな散乱ピークの測定を可能にします。 </li><li>高い測定スループットとサンプルに縮小露光、最大60,000 nm /分の高いスキャン速度により、自動シャッター機能。 </li><li> 240から800 nmの広い波長範囲にわたって量子収率の測定。 </li><li>正確な量子収率の測定は、測定器のスペクトル応答を収集し、サンプルを測定する前に、球の補正係数を積分した結果です。 </li><li>専用の使いやすいソフトウェアが提供する計算されたパラメータの大きい選択。 </li></ul>このビデオの間に我々は、0.4から0.5の量子収率値を持つことが知られている粉末状のサリチル酸ナトリウムを測定します。

Watch this Scientific Journal Video about Absolute Quantum Yield Measurement of Powder Samples at JoVE.com

Absolute Quantum Yield Measurement of Powder Samples

このビデオでは、絶対量子収率と色度日立F-7000量子収率測定システムを用いた粉末試料に直接座標を測定し、計算のデモンストレーションを行います。

粉末試料の絶対量子収率測定

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

absolute-quantum-yield-measurement-of-powder-samples

Research

JoVE Journal

Biology

27.8K ビュー.  Hitachi High Technologies America. このビデオでは、絶対量子収率と色度日立F-7000量子収率測定システムを用いた粉末試料に直接座標を測定し、計算のデモンストレーションを行います。

ビデオ: Absolute Quantum Yield Measurement of Powder Samples

Large Volume (20L+) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column. Two 20L samples can be filtered simultaneously using a single pump unit equipped with four rotating heads. Filtration is done in the field on extended trips, or immediately upon return for day trips. It is important to record the amount of water passing through each sterivex filter unit. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use. This procedure will take approximately 5 hours plus an additional hour for clean up.

Large Volume 20L+ Filtration of Coastal Seawater Samples

This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column.

沿岸海水サンプルの大容量（20L +）ろ過

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, ...

生物学

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &amp;#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

組織サンプルのγH2AX病巣の定量

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Many types of organic phosphorus (P) molecules exist in environmental samples1. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents2,3. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples4,5. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (31P-NMR) -a method that is expensive and requires specialized technical training6. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system7. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.

This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.

環境試料中の有機Pのハイスループット測定と分類

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

ECIS /タクシーと細胞走化性の測定

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

における寿命の測定<em&gt;キイロショウジョウバエ</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

哺乳動物細胞におけるヒトグランザイムの高収量およびコスト効率の高い発現システム

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

絶対タンパク質定量のための質量分析法を選択反応モニタリング

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

懸濁液中のHEK293細胞の一過性トランスフェクションによる組換えヒトタンパ​​ク質のハイ・イールド式

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification.
A fixed volume (200 &#181;L) of EDTA-plasma was prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50). Total RNA was extracted using commercially available system. Plasma miRNAs were quantified by probe-based RT-qPCR assays which contains miRNA-specific forward/reverse PCR primer and probe. Standard curves for absolute quantification were generated using commercially available synthetic RNA oligonucleotides. A synthetic cel-miR-238 was used as an external control for normalization and quality assessment. The miRNAs that showed quantification cycle (Cq) values above 35 were pre-amplified prior to the qPCR step.
Among the 8 miRNAs examined, miR-122, miR-133a, and miR-192 were detectable without pre-amplification, whereas miR-1, miR-206, and miR-499a required pre-amplification because of their low expression levels. MiR-208a and miR-208b were not detectable even after pre-amplification. Sample processing efficiency was evaluated by the Cq values of the spiked cel-miR-238. In this assay method, technical variation was estimated to be less than 3-fold and the lower limit of quantification (LLOQ) was 102 copy/&#181;L, for most of the examined miRNAs.
This protocol provides a better estimate of the quantity of plasma miRNAs, and allows quality assessment of corresponding data from different studies. Considering the low number of miRNAs in body fluids, pre-amplification is useful to enhance detection of poorly expressed miRNAs.

This report describes a protocol for measuring the absolute levels of plasma miRNA, using quantitative real-time reverse transcription PCR with or without pre-amplification. This protocol affords better understanding of the quantity of plasma miRNAs and allows qualitative assessment of corresponding data from different studies or laboratories.

定量的リアルタイム逆転写 PCR を用いたカニクイザルにおける血中マイクロ Rna の絶対定量

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This ...

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, implantation of transgenic cells and tiny optical sensors. However, intravenous microinjections into small animals, which are mostly used in biological and veterinary laboratories, are very difficult and require trained personnel. Herein, we demonstrate a robust and efficient method for the introduction of microparticles into the circulatory system of adult zebrafish (Danio rerio) by injection into the fish kidney. To visualize the introduced microparticles in the vasculature, we propose a simple intravital imaging technique in fish gills. In vivo monitoring of the zebrafish blood pH was accomplished using an injected microencapsulated fluorescent probe, SNARF-1, to demonstrate one of the possible applications of the described technique. This article provides a detailed description of the encapsulation of pH-sensitive dye and demonstrates the principles of the quick injection and visualization of the obtained microcapsules for in vivo recording of the fluorescent signal. The proposed method of injection is characterized by a low mortality rate (0-20%) and high efficiency (70-90% success), and it is easy to institute using commonly available equipment. All described procedures can be performed on other small fish species, such as guppies and medaka.

This article demonstrates the principles of a quick, minimally invasive injection of fluorescent microparticles into the circulatory system of small fishes and the in vivo visualization of the microparticles in fish blood.

シンプルで効果的な管理と腎臓の注入を使用する小型魚の循環システムの微粒子の可視化

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, ...