著者は彼らの援助と方向にスクリップス研究所の博士盛鼎と北京大学の博士洪鄧に感謝したい。

1。 MEFフィーダープレートの準備<ol><li>培養ES細胞前日、照射E14.5の雪解け1バイアルCF - 1 MEFフィーダー細胞（ES細胞を解凍する方法で私たちの手順を参照）。 </li><li>プレート12ウェルプレートに10,000細胞/ウェルの密度でMEFフィーダー細胞。 37℃、5％CO 2で一晩細胞をインキュベートする。 MEF細胞メディア：DMEM、10％ES - PBS、1Xグルタミンおよび1X非必須アミノ酸を添加した。 </li></ol> 2。マウスES細胞の自己複製の保守<ol><li>トリプシンを用いてES細胞を切り離します。シードSC1を補充/よく増殖培地に予め用意されたMEFフィーダープレート上に50,000個の細胞（15％KSRを添加したノックアウトMEM、4mMのL -グルタミン、0.1mM非必須アミノ酸と1X BME）の密度で細胞所望の濃度での（我々は100nm、300 nm、および1μMを使用）。我々はまた、LIF 1000μ/ mlのを添加した陽性コントロールウェルを追加しました。 37℃、5％CO 2で一晩細胞をインキュベートする。 </li><li>文化37細胞℃、5％、各継代で3〜4日間CO 2。リフレッシュメディア毎に48時間。 </li><li>継代細胞の初期播種密度と同様の密度を維持するためにトリプシンで1:10に1：15。 5継代後、細胞は、免疫細胞化学を用いて典型的な多能性マーカーの発現を調べることができます。 </li></ol> 3。多能性マーカーの免疫細胞化学的検討<ol><li>細胞をPBSで穏やかに3回洗浄する。 </li><li>室温で20分間、固定液500μlで細胞を固定する。 </li><li>細胞をPBSで穏やかに3回洗浄する。 </li><li>室温で1時間500μlのブロッキングバッファーで非特異的結合をブロックする。 </li><li>特定の一次抗体を250μl（：1:500、1:2000、1:500、1:500と我々は以下の希釈でNanogの、レトロウイルスベクター、SSEA1、およびOct4のを使用）で細胞をインキュベート</li><li>細胞をPBSで穏やかに3回洗浄する。 </li><li>室温で2時間二次抗体を250μlで細胞をインキュベートし、光から遠ざけること（我々は、1からのAlexa Fluor ® 488で1:2000とロバ抗ウサギ共役でのAlexa Fluor ® 555と結合ロバ抗マウスを使用：2000）。 </li><li>細胞をPBSで穏やかに3回洗浄する。 </li><li>最後の洗浄のためにDAPIを（最終濃度1μg/ ml）を追加し、核を可視化するために5分間インキュベートする。 </li><li>蛍光顕微鏡下で細胞を分析する。 </li></ol> 4。代表的な結果： 1。形態の結果： マウスES細胞（ESC R1）は未分化状態で自己複製を維持するためにLIFまたはSC1のどちらかの存在下でMEFフィーダー細胞上で増殖させた。第2通路の後、分化がLIFまたはSC1（ネガティブコントロール）することなく細胞内で観察することができたし、5継代後、本質的に未分化コロニーが観察されなかった。 LIF（陽性対照）で処理したコロニーは、5セルの通路を通して未分化状態のまま。 300nm以上は100 nMの濃度でSC1で処理した細胞はまた、しかし、細胞が第四継代後に1μMSC1経験豊富な細胞死で処理、5継代の後に未分化のままであった。 （アプリケーションノートの図2）表1は、形態学的観察結果をまとめたものです。 2。多能性マーカーの発現 5継代維持したマウスES細胞はSSEA1、Oct4の、NanogのとSox2のために固定し、免疫細胞化学によって調べた。マーカーの発現は、LIF内に保持細胞に類似していた。 （アプリケーションノートの図3と4） <img src="/files/ftp_upload/1550/1550fig2a.jpg" alt="図1" /> 図1：LIFまたはSC1に保持されて細胞の形態の比較。形態に差は認められなかった。 <img src="/files/ftp_upload/1550/1550fig2c.jpg" alt="図2および3" /> 図2と図3：Nanogの、SSEA1、レトロウイルスベクター、及びSC1またはLIF（アプリケーションノートの図4）で維持されたES細胞のOct4の染色。 SC1に保持されて細胞はLIFで維持細胞に類似した多能性マーカー発現を示しています。 表1 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> 負の制御 </td><td> LIFポジティブコントロール </td><td> SC1 1uM </td><td> SC1 300nMの </td><td> SC1 100 nMの </td></tr><tr><td> 第一パッセージ </td><td>通常のES細胞の形態</td><td>通常のES細胞の形態</td><td>通常のES細胞の形態</td><td>通常のES細胞の形態</td><td>通常のES細胞の形態</td></tr><tr><td> 第二パッセージ </td><td>いくつかの差別化された形態</td><td>通常のES細胞の形態</td><td>通常のES細胞の形態</td><td>通常のES細胞の形態</td><td>通常のES細胞の形態</td></tr><tr><td> 第三パッセージ </td><td>分化した細胞の大部分</td><td>通常のES細胞の形態</td><td>通常のES細胞の形態</td><td>通常のES細胞の形態</td><td>通常のES細胞の形態</td></tr><tr><td> 第四パッセージ </td><td>いいえESCコロニーなし</td><td>通常のES細胞の形態</td><td>観察された細胞死</td><td>通常のES細胞の形態</td><td>通常のES細胞の形態</td></tr><tr><td> 第五パッセージ </td><td>分化した</td><td>通常のES細胞の形態</td><td>観察された細胞死</td><td>通常のES細胞の形態</td><td>通常のES細胞の形態</td></tr></tbody></table>

<ol>
	<li>Williams, R. L., Hilton, D. J., Pease, S., Willson, T. A., Stewart, C. L., Gearing, D. P., Wagner, E. F., Metcalf, D., Nicola, N. A., Gough, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+leukaemia+inhibitory+factor+maintains+the+developmental+potential+of+embryonic+stem+cells.">Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells.</a> Nature. 336, 684-687 (1988).</li><li>Ying, Q. L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ground+state+of+embryonic+stem+cell+self-renewal.">The ground state of embryonic stem cell self-renewal.</a> Nature. 453, 519-523 (2008).</li><li>Chen, S., Do, J. T., Zhang, Q., Yao, S., Yan, F., Peters, E. C., Scholer, H. R., Schultz, P. G., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+of+embryonic+stem+cells+by+a+small+molecule.">Self-renewal of embryonic stem cells by a small molecule.</a> Proc. Natl. Acat. Sci. USA. 103, 17266-17271 (2006).</li></ol>

この記事の著者は、この記事で使用した試薬と器具を生産するStemgentによって雇用されている。

小分子のSC1は、マウスES細胞の未分化状態で自己複製を維持するために使用することができます。テストされていない細胞株で使用する前に、しかし、それは最適な濃度を決定するために滴定する必要があります。例えば、我々はマウスES細胞株ESC R1の3つの濃度をテストした。 100 nMのと300 nMの濃度は、マウスES細胞を維持することができた、しかし、1μMの濃度では毒性を示した。 SC1は、フィーダーを含まない条件下で使用することができます。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>SC1</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>04-0011</td>
<td>&nbsp;</td>
<tr>
<td>LIF</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>ESG1107</td>
<td>&nbsp;</td>
<tr>
<td>SSEA-1 antibody</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>21702</td>
<td>&nbsp;</td>
<tr>
<td>Nanog antibody</td>
<td>&nbsp;</td>
<td>Abcam</td>
<td>ab21603</td>
<td>&nbsp;</td>
<tr>
<td>Sox2 antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>Ab5603</td>
<td>&nbsp;</td>
<tr>
<td>Oct4 antibody</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>SC-5279</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

the use of sc1 (pluripotin) to support mesc self-renewal in the absence of lif

マウス胚性幹（ES）細胞は、従来、自己複製を維持するために、白血病阻害因子（LIF）で培養する。<sup&gt; 1</sup&gt;しかし、LIFは高価であり、LIF/JAK/STAT3経路の活性化は絶対的に自己複製の状態を維持する必要はありません。<sup&gt; 2</sup&gt; SC1小分子は、LIFへの経済的な代替可能性があります。 SC1のRas - GAPとERK1のデュアル阻害による機能。<sup&gt; 3</supその作用機序の&gt;イラストは自己再生の基本的な分子メカニズムを研究するために有用なツールとなります。ここでは、SC1の存在下で培養マウスES細胞のための手順を示し、それらがLIFの不在下で自己複製を維持することが可能であることを示している。 SC1で培養された細胞は、LIFを維持した細胞に比べ同様の形態を示した。両方は、5継代後、典型的なマウスのESの形態を示した。典型的な多能性マーカーの発現は（Oct4の、レトロウイルスベクター、Nanogの、そしてSSEA1）SC1の存在の5つの継代後に観察された。また、SC1は、マウスES細胞には明白な毒性を生じなかった。

Watch this Scientific Journal Video about The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF at JoVE.com

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1のRas - GAPとERK1のデュアル阻害による機能。我々は、LIFの不在下でマウスES細胞の自己複製をサポートするにSC1の機能をテストし、SC1は、マウスES細胞培養の自己複製を維持することができることを示した。

LIFの不在下で自己再生MESCをサポートするためのSC1（Pluripotin）の使用

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

the-use-sc1-pluripotin-to-support-mesc-self-renewal-absence

Research

JoVE Journal

Biology

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

10.4K ビュー.  Stemgent. SC1のRas - GAPとERK1のデュアル阻害による機能。我々は、LIFの不在下でマウスES細胞の自己複製をサポートするにSC1の機能をテストし、SC1は、マウスES細胞培養の自己複製を維持することができることを示した。

ビデオ: The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

プロテアーゼ活性を測定するためにプロテアーゼ蛍光検出キットの使用

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

生物学

The use of Biofeedback in Clinical Virtual Reality: The INTREPID Project

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life activities. Its high prevalence in general population and the severe limitations it causes, point out the necessity to find new efficient strategies to treat it. Together with the cognitive-behavioral treatments, relaxation represents a useful approach for the treatment of GAD, but it has the limitation that it is hard to be learned. The INTREPID project is aimed to implement a new instrument to treat anxiety-related disorders and to test its clinical efficacy in reducing anxiety-related symptoms. The innovation of this approach is the combination of virtual reality and biofeedback, so that the first one is directly modified by the output of the second one. In this way, the patient is made aware of his or her reactions through the modification of some features of the VR environment in real time. Using mental exercises the patient learns to control these physiological parameters and using the feedback provided by the virtual environment is able to gauge his or her success. The supplemental use of portable devices, such as PDA or smart-phones, allows the patient to perform at home, individually and autonomously, the same exercises experienced in therapist's office. The goal is to anchor the learned protocol in a real life context, so enhancing the patients' ability to deal with their symptoms. The expected result is a better and faster learning of relaxation techniques, and thus an increased effectiveness of the treatment if compared with traditional clinical protocols.

The Project on Virtual Reality Intelligent Multi-sensor System for the Treatment of Anxiety-related Disorders (INTREPID) is aimed at developing a multi-sensor context-aware virtual reality system for the treatment of anxiety-related disorders.

臨床バーチャルリアリティにおけるバイオフィードバックの利用：INTREPIDプロジェクト

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

電離放射線への応答でγH2AX病巣の定量化

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

マウス胚性幹細胞の維持と自己複製を制御する遺伝子を研究するためにOct4GiPレポーターアッセイ

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

微生物システム生物学のケモスタットの使用

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

RNAシーケンシング解析によりEML造血前駆細胞における自己再生および分化を調節する重要な要因の同定

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain slices and neuronal cell cultures. Recently, this technology has been applied to the whole organs. Advances in sensor design have made &#160;possible the measuring of&#160;cell signaling in blood-perfused in vivo kidneys. The present protocols list the steps needed to measure ATP and H2O2 signaling in the rat kidney interstitium. Two separate sensor designs are used for the ex vivo and in vivo protocols. Both types of sensor&#160;are coated with&#160;a thin enzymatic biolayer on top of a permselectivity layer to give fast responding, sensitive and selective biosensors. The permselectivity layer protects the signal from the interferents in biological tissue, and the enzymatic layer utilizes&#160;the sequential catalytic reaction of glycerol kinase and glycerol-3-phosphate oxidase in the presence of ATP to produce H2O2. The set of sensors used for the ex vivo studies further detected analyte by oxidation of H2O2 on a platinum/iridium (Pt-Ir) wire electrode. The sensors for the&#160;in vivo studies are instead based on the reduction of H2O2 on a mediator coated gold electrode designed for blood-perfused tissue. Final concentration changes&#160;are detected by real-time amperometry followed by calibration to known concentrations of analyte. Additionally, the specificity of the amperometric signal can be confirmed by the addition of enzymes such as catalase and apyrase that break down H2O2 and ATP correspondingly. These sensors also rely heavily on accurate calibrations before and after each experiment. The following two protocols establish the study of real-time detection of ATP and H2O2 in kidney tissues, and can be further modified to extend the described method for use in other biological preparations or whole organs.

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors enable real-time measurements of extracellular cell signaling in biologically-relevant concentrations. The following protocols extend the applications of biosensors to the ex vivo and in vivo detection of ATP and H2O2 in the kidney.

酵素バイオセンサの使用は、内因性ATPまたはHを定量します<sub&gt; 2</sub&gt; O<sub&gt; 2</sub&gt;腎臓で

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain ...

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The Drosophila&#160;melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport, due to the powerful genetics of this organism and the accessibility of its renal tubules to physiological study. Here, we describe the use of the Ramsay assay to measure fluid secretion rates from isolated fly renal tubules, with the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid. This assay allows study of transepithelial fluid and ion fluxes of ~20 tubules at a time, without the need to transfer the secreted fluid to a separate apparatus to measure ion concentrations. Genetically distinct tubules can be analyzed to assess the role of specific genes in transport processes. Additionally, the bathing saline can be modified to examine the effects of its chemical characteristics, or drugs or hormones added. In summary, this technique allows the molecular characterization of basic mechanisms of epithelial ion transport in the Drosophila tubule, as well as regulation of these transport mechanisms.

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

This protocol describes the use of the Ramsay assay to measure fluid secretion rates from isolated Malpighian (renal) tubules from Drosophila melanogaster. In addition, the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid, allowing calculation of transepithelial ion flux, is described.

で液分泌とイオンフラックス速度を測定するためのラムゼイアッセイの使用<em&gt;キイロショウジョウバエ</em&gt;マルピーギ管

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

細胞間接触の不在で2つの細胞タイプの挿入共培養システムの開発

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro and in vivo. Fluorescent probes are particularly useful in determining the bio-distribution of administered large molecules, where the addition of a small-molecule fluorescent label is unlikely to affect the kinetics or bio-distribution of the compound. A variety of methods exist to examine bio-distribution that vary significantly in the amount of effort required and whether the resulting measurements are fully quantitative, but using multiple methods in conjunction can provide a rapid and effective system for analyzing bio-distributions.
Ex vivo whole-organ imaging is a method that can be used to quickly compare the relative concentrations of fluorescent molecules within tissues and between multiple types of tissues or treatment groups. Using an imaging platform designed for live-animal or whole-organ imaging, fluorescence within intact tissues can be determined without further processing, saving time and labor while providing an accurate picture of the overall bio-distribution. This process is ideal in experiments attempting to determine the tissue specificity of a compound or for the comparison of multiple different compounds. Quantitative tissue histology on the other hand requires extensive further processing of tissues in order to create a quantitative measure of the labeled compounds. To accurately assess bio-distribution, all tissues of interest must be sliced, scanned, and analyzed relative to standard curves in order to make comparisons between tissues or groups. Quantitative tissue histology is the gold standard for determining absolute compound concentrations within tissues.
Here, we describe how both methods can be used together effectively to assess the ability of different administration methods and compound modifications to target and deliver fluorescently labeled molecules to the central nervous system1.

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Ex vivo whole-organ imaging is a rapid method for determining the relative concentrations of fluorescently labeled compounds within and between tissues or treatment groups. Conversely, quantitative fluorescence histology, while more labor intensive, allows for the quantification of the absolute tissue levels of labeled molecules.

の用法<em&gt; ex vivoで</em&gt;全体の臓器イメージングと定量的組織組織学は、蛍光標識分子のバイオ分布を決定します

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro ...