この研究は、江蘇省高等教育機関の優先学術プログラム開発(PAPD)および中国薬科大学の教育改革研究プロジェクト(2019XJYB18)の支援を受けました。

1.半固体培地の調製
<ol><li>従来の半固体寒天
	<ol><li>基本成分11を使用して、細菌運動性試験培地レシピに従って伝統的な半固体寒天を調製する。トリプトース10 g、NaCl15 g、寒天4 gを十分な蒸留水に溶解し、pHを7.2±0.2に調整し、最終容量を1,000 mLにします。</li>
		<li>寒天を121°Cで20分間オートクレーブし、高さ3cmの半固体培地として10mLの試験管に分注します。</li>
	</ol></li>
	<li>TTCを含む従来の半固体寒天
	<ol><li>従来の半固体培地をオートクレーブした後、50°Cに冷却し、100mLの培地に5mLの滅菌1%TTC溶液を加えて混合し、10mLの試験管に分注して高さ3cmの半固体培地を形成します。</li>
	</ol></li>
</ol>2.細菌株
注:水生環境から80株を分離し、大腸菌、緑膿菌、サルモネラ属菌、ビブリオ属菌、肺炎桿菌、およびアエロモナス親水性菌を含む自動細菌識別装置(資料表を参照)を使用して同定しました(表1)。黄色ブドウ球菌(材料の表を参照)は、負の非運動性対照として使用されました。大腸菌、緑膿菌、およびネズミチフス菌(材料表参照)をポジティブコントロール株として使用しました。
<ol><li>運動性分析に使用する試験細菌株を特定します。</li>
	<li>負の非運動性対照および運動性陽性対照株を含める。</li>
</ol>3. TTC増強細菌運動性観察
<ol><li>寒天プレートから被験菌の単一コロニーを選び、接種針を用いた穿刺により、上記の2つの半固体培地(ステップ1.1.2および1.2.1)に接種します。</li>
	<li>チューブをインキュベーター内で37°Cで24〜48時間培養し、結果を観察した。</li>
	<li>成長状態を観察する:穿刺線だけが赤の場合は、細菌を非運動性(-)として特徴付けます。赤色が穿刺線12に沿ってわずかに外側に広がる場合、細菌を運動性(+)として特徴付ける。</li>
</ol>4. 寒天濃度の違いが細菌の運動に及ぼす影響
<ol><li>0.3%、0.5%、0.8%の寒天を含む半固体培地を調製し、上記のように穿刺して接種する。インキュベーションの24-48時間後に結果を観察してください。</li>
</ol>

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著者は開示する利益相反を持っていません。

半固体培地法による細菌の運動性の検出は、多くの要因の影響を受けます13,14。酸素(寒天表面での好気性、半固体培地を含むチューブの底部での非好気性)、pH、温度などの細菌増殖条件は、細菌のべん毛の生存率に影響を与える可能性があり、運動性の低下または運動性の喪失につながる可能性があります15。さらに、それらの運...

細菌の運動性は、細菌の病原性、バイオフィルム形成、および薬剤耐性において重要な役割を果たします1。細菌の運動性は病原性と密接に関連しており、宿主細胞の早期感染中の細菌のコロニー形成に必要です2。バイオフィルムの形成は細菌の運動性と密接に関連しており、細菌は運動性を介して固体培地の表面に付着します。細菌の運動性は、バイオフィルム形成と正の相関があると長い間考えられてきました。バイオフィルムによる高度の細菌薬剤耐性は、人間の健康を脅かす持続感染につながる可能性があります3,4,5。したがって、細菌の運動性を検出することが重要です。細菌の運動性試験は、主に生きている状態にあるさまざまな形態の細菌の運動性を調べるために使用され、べん毛の有無を間接的に決定できるため、細菌の同定に重要な役割を果たします。
細菌の運動性を検出するための直接的および間接的な方法があります6。べん毛を有する細菌は運動性を示すため、べん毛の有無を検出することで間接的に細菌が運動性であるかどうかを検出することができる。例えば、細菌が運動性であることを示すために電子顕微鏡観察およびべん毛染色によって間接的に運動性を検出することができる。また、懸濁液落下法や半固体穿刺法などの直接法による検出も可能です。 
細菌の運動性を検出するために学部微生物学研究室で一般的に使用される半固体穿刺法は、細菌の増殖の方向に応じて、0.4〜0.8%寒天を含む半固体寒天培地の穿刺に細菌を接種します。細菌が穿刺線に沿って増殖して周囲に広がると、雲のような(ブラシのような)成長痕跡が現れ、べん毛の存在、したがって運動性を示します。穿刺線の成長痕跡がない場合、細菌は鞭毛も運動性もありません。 
しかしながら、この方法はその欠点を有する:細菌は無色透明であり、べん毛活性は生きている細菌の生理学的特性および他の要因、ならびに寒天の濃度および試験管の小径によって影響を受ける。さらに、好気性細菌は寒天表面での増殖にのみ適しており、細菌の運動性の観察に影響を与えます。したがって、この実験を改善するために、2,3,5-トリフェニルテトラゾリウムクロリド(TTC)(無色)を培地に添加して、TTC 7,8,9,10の赤色生成物の形成を触媒する細胞内デヒドロゲナーゼを使用する現在の直接穿刺法よりも、細菌の運動性を決定するより信頼性が高く直感的な方法を確立しました。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bacto Agar</td>
			<td>Difco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli</td>
			<td>ATCC</td>
			<td>ATCC25922</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Pseudomonas aeruginosa</td>
			<td>ATCC</td>
			<td>ATCC27853</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Salmonella typhimurium</td>
			<td>ATCC</td>
			<td>ATCC14028</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus</td>
			<td>ATCC</td>
			<td>ATCC25923</td>
			<td>Negative nonmotile control</td>
		</tr>
		<tr>
			<td>Tryptose</td>
			<td>&#160;OXOID</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TTC</td>
			<td>Sigma</td>
			<td>298-96-4</td>
			<td></td>
		</tr>
		<tr>
			<td>VITEK 2 automated microbial identification system</td>
			<td>Bio M&#233;rieux</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing bacterial motility based on a color reaction

細菌の運動性は、細菌の病原性、バイオフィルム形成、および薬剤耐性にとって非常に重要です。細菌の運動性は、多くの病原性種の侵入および/または拡散にとって非常に重要です。したがって、細菌の運動性を検出することが重要です。酸素、pH、温度などの細菌の増殖条件は、細菌の増殖と細菌べん毛の発現に影響を与える可能性があります。これは、運動性の低下または運動性の喪失につながり、細菌の運動性の不正確な評価をもたらす可能性があります。生細菌の細胞内デヒドロゲナーゼによる2,3,5-トリフェニルテトラゾリウムクロリド(TTC)の呈色反応に基づいて、TTCを細菌の運動性検出のために従来の半固体寒天に添加しました。その結果、このTTC半固体寒天法は、細菌の運動性を検出するための簡便で操作が容易であり、大型で高価な機器を必要としないことが示されました。結果はまた、0.3%寒天で調製された半固体培地で最も高い運動が観察されることを示した。従来の半固体培地と比較して、結果は評価が容易で正確です。

運動性検出のために標準株と単離株の両方を比較し、結果を表1に示した。べん毛がないため、黄色ブドウ球菌と肺炎桿菌は、従来の培地とTTC半固体培地の両方で接種された線に沿ってのみ成長しました。一方,緑膿菌,大腸菌,ネズミチフス菌はTTC半固体培地で24時間培養した後,接種ライン付近で全方向に増殖を示した。これは、48時間培養後にさ?...

Watch this Scientific Journal Video about Visualizing Bacterial Motility Based on a Color Reaction at JoVE.com

Visualizing Bacterial Motility Based on a Color Reaction

ここでは、呈色反応に基づいて細菌の運動性を検出するためのプロトコルを提示します。この方法の主な利点は、評価が容易で精度が高く、特殊な機器を必要としないことです。

呈色反応に基づく細菌の運動性の可視化

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or ...

visualizing-bacterial-motility-based-on-a-color-reaction

Research

JoVE Journal

Biology

3.8K ビュー.  China Pharmaceutical University. ここでは、呈色反応に基づいて細菌の運動性を検出するためのプロトコルを提示します。この方法の主な利点は、評価が容易で精度が高く、特殊な機器を必要としないことです。

ビデオ: Visualizing Bacterial Motility Based on a Color Reaction

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

アガロースゲルベースの​​マイクロ流体デバイスを用いた神経幹細胞運動の評価

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

生物学

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor.  While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents.  Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection.  Mononuclear cells are purified from peripheral blood by Ficoll underlay.  CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads.  Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2.  The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25.  After labeling, the cells can be directly analyzed by flow cytometry.  The tetramer positive cells typically form a distinct population among the expanded CD4+ cells.  Tetramer positive cells are usually CD25+ and often CD4 high.  Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer.  Multiple variations of this basic assay are possible.  Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays.  Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies.  

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

抗原特異的CD4 + T細胞はMHCクラスIIテトラマーを用いての可視化

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

蛍光顕微鏡による可視化単分子のDNAの複製

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人間のDNA修復タンパク質の遺伝的研究は、モデルシステムとして酵母を使用

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

可視化RNAの局在にアフリカツメガエル卵母細胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Gastrointestinal Motility Monitor (GIMM)

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility in isolated segments of guinea pig distal colon. The complete system consists of a computer, video camera, illuminated organ bath, peristaltic and heated water bath circulating pumps, and custom GIMM software to record and analyze data. Compared with traditional methods of monitoring colonic peristalsis, the GIMM system allows for continuous, quantitative evaluation of motility. The guinea pig distal colon is bathed in warmed, oxygenated Krebs solution, and fecal pellets inserted in the oral end are propelled along the segment of colon at a rate of about 2 mm/sec. Movies of the fecal pellet proceeding along the segment are captured, and the GIMM software can be used track the progress of the fecal pellet. Rates of propulsive motility can be obtained for the entire segment or for any particular region of interest. In addition to analysis of bolus-induced motility patterns, spatiotemporal maps can be constructed from captured video segments to assess spontaneous motor activity patterns. Applications of this system include pharmacological evaluation of the effects of receptor agonists and antagonists on propulsive motility, as well as assessment of changes that result from pathophysiological conditions, such as inflammation or stress. The guinea pig distal colon propulsive motility assay, using the GIMM system, is straightforward and simple to learn, and it provides a reliable and reproducible method of assessing propulsive motility.

Gastrointestinal Motility Monitor GIMM

Evaluation of colonic motility in the guinea pig distal colon with the Gastrointestinal Motility Monitor (GIMM) is a straightforward and simple to learn approach to quantitatively evaluate propulsive motility in the gastrointestinal tract.

消化管運動のモニター（GIMM）

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

準備、イメージング、および細菌表面運動性アッセイの定量化

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

使用して小胞体カルシウムの恒常性の監視<em&gt;ガウ</em&gt;ルシフェラーゼSERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

外酸生産の測定と解析は、解糖速度を決定するために

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

セノ治療薬同定のためのSA-β-ガラクトーシダーゼベーススクリーニングアッセイ

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

呈色反応に基づく細菌の運動性の可視化

要約

さらに動画を探す

この動画の章

アガロースゲルベースのマイクロ流体デバイスを用いた神経幹細胞運動の評価

抗原特異的CD4 + T細胞はMHCクラスIIテトラマーを用いての可視化

蛍光顕微鏡による可視化単分子のDNAの複製

人間のDNA修復タンパク質の遺伝的研究は、モデルシステムとして酵母を使用

可視化RNAの局在にアフリカツメガエル卵母細胞

消化管運動のモニター（GIMM）

準備、イメージング、および細菌表面運動性アッセイの定量化

使用して小胞体カルシウムの恒常性の監視

外酸生産の測定と解析は、解糖速度を決定するために

セノ治療薬同定のためのSA-β-ガラクトーシダーゼベーススクリーニングアッセイ

呈色反応に基づく細菌の運動性の可視化

要約

さらに動画を探す

この動画の章

アガロースゲルベースの​​マイクロ流体デバイスを用いた神経幹細胞運動の評価

抗原特異的CD4 + T細胞はMHCクラスIIテトラマーを用いての可視化

蛍光顕微鏡による可視化単分子のDNAの複製

人間のDNA修復タンパク質の遺伝的研究は、モデルシステムとして酵母を使用

可視化RNAの局在にアフリカツメガエル卵母細胞

消化管運動のモニター（GIMM）

準備、イメージング、および細菌表面運動性アッセイの定量化

使用して小胞体カルシウムの恒常性の監視

外酸生産の測定と解析は、解糖速度を決定するために

セノ治療薬同定のためのSA-β-ガラクトーシダーゼベーススクリーニングアッセイ

アガロースゲルベースのマイクロ流体デバイスを用いた神経幹細胞運動の評価