Os autores gostariam de agradecer ao Dr. Sheng Ding do The Scripps Research Institute e Dr. Deng Hongkui da Universidade de Pequim por sua assistência e direção.

1. Preparação das placas de alimentação MEF <ol><li> Um dia antes de as células ES de cultura, frasco de descongelar um dos irradiados E14.5 CF-1 células alimentadoras MEF (veja nosso procedimento para descongelar células ES). </li><li> A placa de células alimentadoras MEF a uma densidade de 10.000 células / poço em uma placa de 12 poços. Incubar as células durante a noite a 37 ° C e 5% CO 2. MEF célula mídia: DMEM suplementado com 10% ES-PBS, 1X e 1X glutamina não-aminoácidos essenciais. </li></ol> 2. Manutenção da auto-renovação das células ES rato <ol><li> Separar as células ES usando tripsina. Semente as células a uma densidade de 50.000 células / poço sobre as placas MEF previamente preparado alimentador em meios de crescimento (MEM suplementado com Knockout KSR 15%, 4 mM L-glutamina, 0,1 mM não-aminoácidos essenciais e 1X BME) suplementado com SC1 na concentração desejada (usamos 100 nM, 300 nM e 1 mM). Nós também adicionamos um controle positivo bem que foi complementado com 1000 μ / ml de LIF. Incubar as células durante a noite a 37 ° C e 5% CO 2. </li><li> Cultura das células a 37 ° C e 5% CO 2 por 3-4 dias em cada passagem. Atualizar media a cada 48 horas. </li><li> Células passagem 1:10 - 01:15 com tripsina para manter uma densidade semelhante como a densidade de semeadura inicial. Depois de cinco passagens, as células podem ser examinados para a expressão de marcadores de pluripotência típico usando imunocitoquímica. </li></ol> 3. Exame imunocitoquímico de marcadores pluripotência <ol><li> Lave suavemente as células três vezes com PBS. </li><li> Fixar as células com 500 mL de fixador por 20 minutos em temperatura ambiente. </li><li> Lave suavemente as células três vezes com PBS. </li><li> Bloco de ligação não específica com 500 mL de buffer de bloqueio para uma hora em temperatura ambiente. </li><li> Incubar as células com 250 mL do anticorpo primário específico (usamos Nanog, Sox2, SSEA1 e Oct4 nas seguintes diluições: 1:500, 1:2000, 1:500 e 1:500) </li><li> Lave suavemente as células três vezes com PBS. </li><li> Incubar as células com 250 mL de anticorpo secundário por duas horas à temperatura ambiente, mantendo-se longe da luz (usamos burro anti-rato conjugada com Alexa Fluor ® 555 a 1:2000 e anti-burro coelho conjugados com Alexa Fluor ® 488 em 1 : 2000). </li><li> Lave suavemente as células três vezes com PBS. </li><li> Adicionar DAPI (concentração final de 1 mg / ml) para a última lavagem e incubar 5 minutos para visualizar núcleos. </li><li> Analisar as células em um microscópio fluorescente. </li></ol> 4. Resultados representativos: 1. Morfologia resultados: Células de camundongo ES (ESC R1) foram cultivadas em células alimentadoras MEF na presença de qualquer LIF ou SC1 para sustentar a auto-renovação em um estado indiferenciado. Após a segunda passagem, a diferenciação pode ser observada em células sem LIF ou SC1 (controle negativo) e depois de 5 passagens essencialmente nenhuma colônias indiferenciado foram observados. LIF (controle positivo) colónias tratadas permaneceu em um estado indiferenciado, durante o 5 passagens celular. Células tratadas com SC1 em concentrações de 300 nm ou 100 nM também permaneceu indiferenciado após 5 passagens, no entanto, as células tratadas com 1 mM morte celular SC1 experimentado após a quarta passagem. (Fig. 2 de nota app) A Tabela 1 resume as observações morfológicas. 2. Expressão de marcadores pluripotência Células-tronco embrionárias de rato mantidos por 5 passagens foram fixados e analisados ​​por imunocitoquímica para SSEA1, Oct4, Sox2 e Nanog. Expressão do marcador foi semelhante às células mantidas em LIF. (Fig. 3 e 4 de nota app) <img src="/files/ftp_upload/1550/1550fig2a.jpg" alt="Figura 1" /> Figura 1: comparação Morfologia de células mantidas em LIF ou SC1. Nenhuma diferença na morfologia foi observada. <img src="/files/ftp_upload/1550/1550fig2c.jpg" alt="Figura 2 e 3" /> Figura 2 e Figura 3: Nanog, SSEA1, Sox2 e Oct4 coloração de células ES mantido com SC1 ou LIF (fig. 4 de nota app). Células mantidas em SC1 mostram expressão do marcador semelhante pluripotência das células mantidas em LIF. Tabela 1 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> Controle negativo </td><td> LIF Controle Positivo </td><td> SC1 1um </td><td> SC1 300 nM </td><td> SC1 100 nM </td></tr><tr><td> 1 Passagem </td><td> Morfologia das células normais ES </td><td> Morfologia das células normais ES </td><td> Morfologia das células normais ES </td><td> Morfologia das células normais ES </td><td> Morfologia das células normais ES </td></tr><tr><td> Passagem 2 </td><td> Alguns morfologia diferenciada </td><td> E normaisS morfologia celular </td><td> Morfologia das células normais ES </td><td> Morfologia das células normais ES </td><td> Morfologia das células normais ES </td></tr><tr><td> Passage 3 </td><td> Maioria das células diferenciadas </td><td> Morfologia das células normais ES </td><td> Morfologia das células normais ES </td><td> Morfologia das células normais ES </td><td> Morfologia das células normais ES </td></tr><tr><td> Passagem 4 </td><td> Não colônias ESC </td><td> Morfologia das células normais ES </td><td> Morte celular observado </td><td> Morfologia das células normais ES </td><td> Morfologia das células normais ES </td></tr><tr><td> Passagem 5 </td><td> diferenciado </td><td> Morfologia das células normais ES </td><td> Morte celular observado </td><td> Morfologia das células normais ES </td><td> Morfologia das células normais ES </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>

Os autores deste artigo são empregados por Stemgent que produz reagentes e instrumentos utilizados neste artigo.

O SC1 pequena molécula pode ser usada para manter a auto-renovação, com um estado indiferenciado em células ES mouse. Antes de usar em uma linhagem de células não testados, no entanto, deve ser titulada para determinar a concentração ideal. Por exemplo, testamos três concentrações no ES célula do rato linha ESC R1. 100 nM e 300 nM concentrações foram capazes de sustentar as células ES mouse, no entanto, uma concentração de 1 mM era tóxico. SC1 também pode ser usado sob alimentação livre de condições.

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<td>SC1</td>
<td>&nbsp;</td>
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<td>04-0011</td>
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<td>LIF</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>ESG1107</td>
<td>&nbsp;</td>
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<td>SSEA-1 antibody</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>21702</td>
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<td>Nanog antibody</td>
<td>&nbsp;</td>
<td>Abcam</td>
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<td>Sox2 antibody</td>
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the use of sc1 (pluripotin) to support mesc self-renewal in the absence of lif

Rato-tronco embrionárias (ES), as células são convencionalmente cultivados com fator inibitório de leucemia (LIF) para manter a auto-renovação.<sup&gt; 1</sup&gt; No entanto, LIF é caro e ativação da via LIF/JAK/STAT3 não é absolutamente necessário para manter o estado auto-renovação.<sup&gt; 2</sup&gt; A molécula SC1 pequena pode ser uma alternativa econômica para LIF. SC1 funções através da inibição dual de Ras-GAP e ERK1.<sup&gt; 3</sup&gt; Ilustração de seu mecanismo de ação faz com que seja uma ferramenta útil para estudar o mecanismo molecular fundamental de auto-renovação. Aqui nós demonstrar o procedimento para a cultura de células ES mouse na presença de SC1 e mostrar que eles são capazes de manter a auto-renovação, na ausência de LIF. Células cultivadas com SC1 apresentaram morfologia similar ao mantido com células LIF. Ambos exibiram morfologia ES típico do mouse depois de cinco passagens. Expressão de marcadores de pluripotência típico (Oct4, Sox2, Nanog e SSEA1) foi observada após cinco passagens na presença de SC1. Além disso, SC1 não causou toxicidade evidente nas células ES mouse.

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 funções através da inibição dual de Ras-GAP e ERK1. Testamos a função de SC1 no apoio célula do rato ES auto-renovação, na ausência de LIF e mostrou que SC1 é capaz de manter a auto-renovação de culturas de células de rato ES.

O uso de SC1 (Pluripotin) para Suporte MESC auto-renovação na ausência de LIF

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

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

10.4K visualizações.  Stemgent. SC1 funções através da inibição dual de Ras-GAP e ERK1. Testamos a função de SC1 no apoio célula do rato ES auto-renovação, na ausência de LIF e mostrou que SC1 é capaz de manter a auto-renovação de culturas de células de rato ES.

Vídeo: 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.

Uso do Kit de Detecção de Protease Fluorescente para determinar a atividade de protease

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

Biologia

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.

O uso de Biofeedback na Clínica de Realidade Virtual: O Projeto INTREPID

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.

Quantificação de γH2AX Focos em resposta à radiação ionizante

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.

Oct4GiP Ensaio Reporter para o Estudo de genes que regulam o mouse Manutenção células-tronco embrionárias e auto-renovação

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.

O Uso de quimiostatos em Microbial Biologia de Sistemas

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.

Identificação dos fatores chave que regula a auto-renovação e diferenciação em células precursoras hematopoéticas EML por Análise RNA-seqüenciamento

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.

Uso de biossensores para quantificar enzimática endógena ATP ou H<sub&gt; 2</sub&gt; Ó<sub&gt; 2</sub&gt; No rim

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.

A utilização do Ramsay ensaio para medir secreção de fluidos e Ion Flux Preços no<em&gt; Drosophila melanogaster</em&gt; Tubule Malpighian

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.

Desenvolvimento de um Sistema Insert Co-cultura de dois tipos celulares na ausência de célula-célula 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 ...

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.

O uso de<em&gt; Ex Vivo</em&gt; Imagem de órgãos inteiros e Histologia Tissue quantitativa para determinar o Bio-distribuição das moléculas fluorescente etiquetado

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