Gostaríamos de agradecer DA Coulter (CHOP) por nos permitir usar seu microscópio dois-fotão para adquirir as imagens 2-fótons mostrada no início do vídeo, e H. Takano por compartilhar conosco seus conhecimentos técnicos.

<ol>
	<li>Costa, M. a. r. c. e. l. l. o., Furness, J. o. h. n. . B., Llewellyn-Smith, I. J., Johnson, L. e. o. n. a. r. d. . R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> in Physiology of the Gastrointestinal Tract, edited by Leonard R. , 1-1 (1987).</li><li>Obaid, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+patterns+of+activity+in+an+intact+mammalian+network+with+single-cell+resolution:+optical+studies+of+nicotinic+activity+in+an+enteric+plexus.">Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus.</a> J Neurosci. 19 (8), 3073-3073 (1999).</li><li>Obaid, A. L., Loew, L. M., Wuskell, J. P., Salzberg, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+naphthylstyryl-pyridinium+potentiometric+dyes+offer+advantages+for+neural+network+analysis.">Novel naphthylstyryl-pyridinium potentiometric dyes offer advantages for neural network analysis.</a> J. Neurosci. Methods. 134 (2), 179-179 (2004).</li><li>Hirst, G. D., McKirdy, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+potentials+recorded+from+neurones+of+the+submucous+plexus+of+guinea-pig+small+intestine.">Synaptic potentials recorded from neurones of the submucous plexus of guinea-pig small intestine.</a> J Physiol (Lond). 249 (2), 369-369 (1975).</li><li>Salzberg, B. M., Barker, J., McKelvy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Methods+in+Cellular+Neurobiology.">Current Methods in Cellular Neurobiology.</a> , 139-139 (1983).</li><li>Salzberg, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultra-stable+non-coherent+light+source+for+optical+measurements+in+neuroscience+and+cell+physiology.">An ultra-stable non-coherent light source for optical measurements in neuroscience and cell physiology.</a> Journal of Neuroscience Methods. 141, 165-16 (2005).</li><li>Obaid, A. L., Nelson, M. E., Lindstrom, J., Salzberg, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+studies+of+nicotinic+acetylcholine+receptor+subtypes+in+the+guinea-pig+enteric+nervous+system.">Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system.</a> J Exp Biol. 208 (Pt 15), 2981-2981 (2005).</li><li>Obaid, A. L., Zou, D. J., Rohr, S., Salzberg, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+recording+with+single+cell+resolution+from+a+simple+mammalian+nervous+system:+Electrical+activity+in+ganglia+from+the+submucous+plexus+of+the+guinea-pig+ileum.">Optical recording with single cell resolution from a simple mammalian nervous system: Electrical activity in ganglia from the submucous plexus of the guinea-pig ileum.</a> Biol. Bull. 183, 344-344 (1992).</li><li>Salzberg, B. M., Davila, H. V., Cohen, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+recording+of+impulses+in+individual+neurones+of+an+invertebrate+central+nervous+system.">Optical recording of impulses in individual neurones of an invertebrate central nervous system.</a> Nature. 246 (5434), 508-508 (1973).</li></ol>

Este protocolo foi escrito com dois objetivos em mente. O primeiro é convencer outros investigadores que, graças aos avanços tecnológicos muitas da década passada, a gravação ótica da atividade elétrica utilizando corantes sensíveis à tensão tornou-se um dos mais poderosos, metodologias confiáveis ​​e acessíveis para o estudo de redes neuronais intacta, na verdade , poderia ser facilmente implementado, mesmo em um laboratório com recursos modestos. O segundo objetivo é promover o sistema nervoso ent?...

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		<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>
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<tr>
<td>Medium 199</td>
<td>&nbsp;</td>
<td>Sigma, St. Louis, MO</td>
<td>M3769</td>
<td>With Earle&#x2019;s salts Without L-Glutamine, Phenol Red and Sodium Bicarbonate</td>
</tr>
<tr>
<td>Sylgard</td>
<td>&nbsp;</td>
<td>Dow Corning Corp., Midland, MI</td>
<td>184 silicone elastomer kit</td>
<td>&nbsp;</td>
<tr>
<td>Insect pins used for dissection</td>
<td>&nbsp;</td>
<td>F.S.T., Foster City, CA</td>
<td>26001-30</td>
<td>&nbsp;</td>
<tr>
<td>Dumont forceps</td>
<td>&nbsp;</td>
<td>F.S.T., Foster City, CA</td>
<td>11203-23 or 11200-33</td>
<td>&nbsp;</td>
<tr>
<td>Moria MC19/B Pascheff-Wolff Spring Scissors</td>
<td>&nbsp;</td>
<td>F.S.T., Foster City, CA</td>
<td>15371-92</td>
<td>&nbsp;</td>
<tr>
<td>Pins to hold the tissue during the experiment</td>
<td>&nbsp;</td>
<td>Seirin Corp., Shizuoka, Japan</td>
<td>Seirin Spinex</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase VII</td>
<td>&nbsp;</td>
<td>Sigma, St. Louis, MO</td>
<td>C0773</td>
<td>&nbsp;</td>
<tr>
<td>Protease IX</td>
<td>&nbsp;</td>
<td>Sigma, St. Louis, MO</td>
<td>P6141</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen, Carlsbad, CA</td>
<td>10439-016</td>
<td>&nbsp;</td>
<tr>
<td>PenStrep</td>
<td>&nbsp;</td>
<td>GIBCO, BRL</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine 100X</td>
<td>&nbsp;</td>
<td>GIBCO, BRL</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Nifedipine</td>
<td>&nbsp;</td>
<td>Sigma, St. Louis, MO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Di-4-ANEPPDHQ</td>
<td>&nbsp;</td>
<td>Molecular Probes, Eugene, OR</td>
<td>D36802</td>
<td>&nbsp;</td>
<tr>
<td>4-Channel Stimulus Generator</td>
<td>&nbsp;</td>
<td>Multi Channel System MCS GmbH, Reutlingen, Germany</td>
<td>2000 Series</td>
<td>&nbsp;</td>
<tr>
<td>Peristaltic pump</td>
<td>&nbsp;</td>
<td>Rainin, Woburn, MA</td>
<td>2-channel Dinamax RP-1</td>
<td>&nbsp;</td>
<tr>
<td>High-speed camera</td>
<td>&nbsp;</td>
<td>RedShirtImaging, LLC Decatur, GA</td>
<td>NeuroCCD-SM</td>
<td>&nbsp;</td>
<tr>
<td>1:10 demagnifier / relay lens</td>
<td>&nbsp;</td>
<td>Optem, Qioptiq Linos, Inc., Rochester, NY</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Upright microscope</td>
<td>&nbsp;</td>
<td>Olympus Optical Co., Tokyo, Japan</td>
<td>BX61TRF</td>
<td>&nbsp;</td>
<tr>
<td>Gibraltar fixed stage with motorized X-Y translator</td>
<td>&nbsp;</td>
<td>Gibraltar, Burleigh Instruments, EXFO, Quebec, Canada</td>
<td>PSSG--BX2</td>
<td>&nbsp;</td>
<tr>
<td>Linear matrix electrodes</td>
<td>&nbsp;</td>
<td>FHC, Inc., Bowdoin, ME</td>
<td>Custom made</td>
<td>&nbsp;</td>
<tr>
<td>Active vibration-isolation table</td>
<td>&nbsp;</td>
<td>Halcyonics,Inc., Menlo Park, CA</td>
<td>Micro 60</td>
<td>&nbsp;</td>
<tr>
<td>Ultra-low-ripple feedback stabilized power supply</td>
<td>&nbsp;</td>
<td>Kepco, Flushing, NY</td>
<td>ATE 75-15M</td>
<td>&nbsp;</td>
<tr>
<td>Heat filter</td>
<td>&nbsp;</td>
<td>Schott Optical Glass, Duryea, PA</td>
<td>KG-1</td>
<td>&nbsp;</td>
<tr>
<td>High-Q interference filter</td>
<td>&nbsp;</td>
<td>Chroma, Inc., Rockingham, VT</td>
<td>HQ530/50</td>
<td>&nbsp;</td>
<tr>
<td>Dichroic mirror</td>
<td>&nbsp;</td>
<td>Chroma, Inc., Rockingham, VT</td>
<td>565 nm (50% transmission)</td>
<td>&nbsp;</td>
<tr>
<td>Long-pass filter</td>
<td>&nbsp;</td>
<td>Chroma, Inc., Rockingham, VT</td>
<td>HQ572LP</td>
<td>&nbsp;</td>
<tr>
<td>Acoustic curtains</td>
<td>&nbsp;</td>
<td>Acoustical Surfaces, Inc.</td>
<td>BSC-25</td>
<td>&nbsp;</td>
<tr>
<td>Neuroplex</td>
<td>&nbsp;</td>
<td>RedShirtImaging, Inc., Decatur, GA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>IDL</td>
<td>&nbsp;</td>
<td>ITT Visual Information Solutions, Boulder, CO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>High-resolution CCD camera, with its own camera controller</td>
<td>&nbsp;</td>
<td>Hamamatsu Photonics, KK, Hamamatsu City, Japan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Frame-grabber for high-resolution camera</td>
<td>&nbsp;</td>
<td>Data Translation, Marlboro, MA</td>
<td>DT3120K-1</td>
<td>&nbsp;</td>
<tr>
<td>Imaging software for high-resolution camera</td>
<td>&nbsp;</td>
<td>Data Translation, Marlboro, MA</td>
<td>Global Lab Image/2</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

optical recording of electrical activity in guinea-pig enteric networks using voltage-sensitive dyes

O sistema nervoso entérico (ENS) é uma rede independente com funções identificadas, capaz de executar comportamentos complexos em isolamento. Seus neurônios (10 a 25 m de diâmetro) são arranjados em plexos que se limitam a planos distintos da parede do intestino<sup&gt; 1</sup&gt;; Do plexo mioentérico podem ser encontrados entre as camadas musculares longitudinal e circular, eo plexo submucoso entre a camada muscular circular e da mucosa. Uma vez que os sistemas efetores para estes plexos (transporte de epitélio, as células endócrinas, elementos imunológico, vasos sanguíneos e músculo liso) também estão contidos dentro da parede do intestino, semi-intactas preparações podem ser dissecados que preservam os componentes individuais de diferentes vias reflexas. O comportamento dos sistemas efetores é controlado pelo plexos submucoso e mioentérico atuando em conjunto. Portanto, o conhecimento detalhado das interações sinápticas dentro e entre os gânglios, e de comunicação entre os plexos, é essencial para a compreensão da função gastrointestinal normal. A ENS, como um sistema nervoso intacto, é um modelo único experimental em que se pode correlacionar eventos moleculares e celulares com o comportamento elétrico da rede neuronal e suas saídas fisiológicas. Por causa da organização quase-bidimensional de sua plexos, a ENS é particularmente adequado para o estudo de redes neurais usando vários sites técnicas de gravação óptica que empregam corantes sensíveis à voltagem<sup&gt; 2,7,8,9</sup&gt;. Vamos ilustrar aqui o uso de um relativamente novo naphthylstyryl-piridínio corante (di-4-ANEPPDHQ)<sup&gt; 3</sup&gt; Que oferece múltiplas vantagens sobre seus antecessores, incluindo fototoxicidade muito baixa, baixa taxa de internalização, e estabilidade química notável. Quando usado em conjunto com uma câmera que permite sub-milissegundo tempo de resolução, este corante nos permite monitorar a atividade elétrica de todos os neurônios no campo de visão com uma resolução máxima espacial de ~ 2,5 mM-aumento de 100X. Com menor ampliação (10X ou 20X), o sacrifício de uma única célula de resolução é compensada por um ganho em perspectiva, revelando a complexidade do circuito inter-ganglionares.

Watch this Scientific Journal Video about Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes at JoVE.com

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

Este protocolo ilustra como sensíveis à voltagem corantes permitir a gravação ótica da atividade elétrica de redes neurais intactos, como os plexos do cobaia sistema nervoso entérico, com uma resolução ajustável que varia de single-células ganglionares para multi-circuitos.

A gravação óptica de atividade elétrica na Guiné-porco Networks Enteric usando Tensão sensível Corantes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons ...

optical-recording-electrical-activity-guinea-pig-enteric-networks

Research

JoVE Journal

Biology

9.9K visualizações.  University of Pennsylvania-School of Medicine. Este protocolo ilustra como sensíveis à voltagem corantes permitir a gravação ótica da atividade elétrica de redes neurais intactos, como os plexos do cobaia sistema nervoso entérico, com uma resolução ajustável que varia de single-células ganglionares para multi-circuitos.

Vídeo: Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Rotulagem células-tronco com corantes fluorescentes para detecção não invasiva com imagens ópticas

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Biologia

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Cuidados e limpeza do microscópio

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Contraste de fase e de interferência diferencial Contrast Microscopy (DIC)

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye  DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan  DIGE technology and analysis of protein expression changes using DeCyder  2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan&#x2122; DIGE technology.

Rotulagem seletiva de proteínas de superfície celular usando Dyes CyDye DIGE Fluor Minimal

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Mapeamento bacterianas redes funcionais e vias<em&gt; Escherichia Coli</em&gt; Usando sintéticos matrizes genéticas

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Um canal de gravação de patch-clamp anexado-Cell

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

Strand DNA Deslocamento Portas para redes de reação de Execução químicos derivados do plasmídeo

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

Síntese de DNA sondas de hibridação de mudança de comprimento de onda usando fotoestável Cyanine Corantes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

Drosophila Preparação e imagem longitudinal do coração Função In Vivo Usando Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

Divergência de Microbiota Raiz em Diferentes Habitats baseados em Redes de Correlação Ponderada

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...