Os canais microfluídicos são fabricados em uma poliolefina cíclico (Zeonex ® 690R, Zeon Chemical Inc., Louisville, KY). Zeonex tem uma temperatura de transição vítrea (T g) de 136 ° C e é UV transparente, o que é essencial para a química de luz dirigido usado na formação porosa monólito. Os microcanais são fabricados por micro-hot-relevo com um eletroformada Nico mestre-mofo, que tem a negativa (inversa) os recursos da plataforma microfluídica. A estampagem a quente é feito da seguinte forma: <ol><li> O molde eo substrato de polímero são colocados entre duas Placas metálicas paralelas. </li><li> Os pratos são aquecidos até a temperatura embossing (166 ° C), que é de 30 º C acima da T g de Zeonex. </li><li> Os pratos são então pressionados juntos por cerca de 5 min ea pressão é mantida em 250 psi. </li><li> O molde eo substrato são então removidos da platens aquecida, deixou esfriar por 1-2 min, e depois manualmente separados uns dos outros. </li><li> Poços de 1,5 mm de diâmetro são perfurados nas extremidades dos canais para a introdução da amostra e coleta. </li><li> O substrato plástico é, então, em relevo termicamente ligado com outro pedaço de Zeonex fechado para formar canais microfluídicos. O substrato em relevo e a peça de tampa pode ser de ozônio UV ou plasma tratados antes da colagem térmica para melhorar a eficiência de vedação e garantir a fidelidade da estrutura de canal fechado (Bhattacharyya e Klapperich, 2007). </li></ol> Fabricação de extração de fase sólida coluna (SPE) dentro dos canais microfluídicos de plástico Fabricação da fase sólida é um processo de duas etapas. Na primeira, os microcanais fabricados são superfície modificada, a fim de melhorar a aderência da coluna SPE para o dispositivo de plástico. Enxertia via fotopolimerização de superfície é usado para funcionalizar as paredes dos microcanais poliméricos. O procedimento de enxerto é a seguinte: <ol><li> Os microcanais são preenchidos com uma mistura 1:1 de diacrilato de etileno (EDA) e metacrilato de metila (MMA) com benzofenona 3%, que é um fotoiniciador hidrogênio abstrair. A EDA, MMA e benzofenona forem comprados de Sigma-Aldrich, St. Louis, MO. </li><li> O chip é então UV-irradiado por 10 min a 254 nm de comprimento de onda UV e 200 mJ / cm 2 de energia em um instrumento exposição aos raios ultravioleta (CL-1000 Agente de crosslinking UV, UPV Inc., Upland, CA). A enxertia ocorre via abstração H-polimerização e resulta em correntes de superfície amarrados polímero, que são incorporadas dentro da mistura de polimerização utilizado para a preparação do monolito na segunda etapa. </li><li> O monômero em excesso é removido os canais de lavagem com 15 mL de metanol por meio de aspiração a vácuo. </li></ol> Após o processo de photografting, um polímero microporoso monólito é formada dentro da microcanais que prender as partículas de sílica para extração de DNA. O monólito poroso é preparado in situ da seguinte forma: <ol><li> Os canais de superfície modificada são preenchidos com uma mistura de precursor monólito consistindo de Buma (15% em peso), EDMA (10% em peso), 1-dodecanol (52,5% em peso), ciclohexanol (22,5% em peso), DMPAP (wt 1% com relação de monômeros) e 0,7 mM partículas de sílica (15% em relação ao volume total da mistura pré-polímero). A sílica microesferas foram adquiridos da Polysciences, Inc. (Warrington, PA) e os monômeros e solventes porogenic forem comprados de Sigma-Aldrich, St. Louis, MO. </li><li> O chip é irradiada com UV no reticulador UV a 200 mJ / cm 2 para 1,1 min para iniciar a polimerização e do monômero em excesso é removido os canais de lavagem com 15 mL de metanol por meio de aspiração a vácuo. </li><li> As conexões fluídicas (TM NanoPorts conexões, Upchurch Científico, Oak Harbor, WA) são fixadas sobre a introdução e poços de captação dos canais microfluídicos. </li><li> O monólito poroso é lavada novamente com metanol por pelo menos 30 min usando a bomba de seringa a uma vazão de 100mL/hour. </li></ol> Fabricação de CNT lise monólito dentro dos canais microfluídicos de plástico Fabricação do (nanotubos de carbono) lise CNT monólito é um processo de duas etapas. Primeiro, os microcanais fabricados são superfície modificada (enxertada) da mesma maneira como para as colunas SPE no dispositivo de plástico. Após o processo de photografting, um polímero microporoso monólito é formada dentro da microcanais que está impregnada com nanotubos de carbono de paredes múltiplas. O monólito poroso é preparado in situ da seguinte forma: <ol><li> Multi-walled nanotubos são ressuspenso em ciclohexanol em uma concentração de 2.27M. Esta é a ressuspensão ultrasonicated durante 30 minutos a amplitude de 50% e 50% ciclo de trabalho com uma célula sonifier disruptor (Sonifier S-250D, fabricado pela Branson Ultrasonic, Danbury, CT). A sonicação desde uma suspensão eficaz e estável 0.5M de nanotubos de carbono. Os nanotubos de carbono foram comprados da Nanolabs (Brighton, MA). </li><li> Os canais de superfície modificada são preenchidos com uma mistura semelhante precursor monólito exceto que agora a solução de ciclohexanol + nanotubos de carbono é utilizado. A mistura monólito consiste em Buma (15% em peso), EDMA (10% em peso), 1-dodecanol (52,5% em peso), e + ciclohexanol CNTs (22,5% em peso). Para esta solução a DMPAP fotoiniciador é adicionado (1,13% em peso com respeito ao monômeros). Os monômeros, solventes e porogenic DMPAPA forem comprados de Sigma-Aldrich, St. Louis, MO. </li><li> O chip é irradiada com UV no reticulador UV a 120 mJ / cm 2 para 0,9 min para iniciar a polimerização. O dispositivo é girado 180 graus no crossliker e irradiado para uma outra min 0,9 a 120 mJ / cm 2. O monômero em excesso é removido os canais de lavagem com 50 mL de metanol por meio de aspiração a vácuo. </li><li> As conexões fluídicas (TM NanoPorts conexões, Upchurch Científico, Oak Harbor, WA) são fixadas sobre a introdução e poços de captação dos canais microfluídicos. </li></ol> Preparação de amostras de bactérias para uso com CNT lise monólito A preparação da amostra para uma amostra bacteriana envolve tomar uma medida de densidade óptica a 600nm para garantir que a amostra não é extremamente concentrado, de modo a evitar o entupimento do canal. <ol><li> Tome uma leitura de OD 600nm com as bactérias na mídia, isso é feito com um biophotometer (Eppendorf BioPhotometer, Eppendorf Scientific, Inc., Westbury, NY). Diluir a amostra até que as leituras OD estão dentro do intervalo 0,18-0,25 A. </li><li> Centrifugar a amostra a 6.500 rpm por 5 minutos e decantar o sobrenadante. </li></ol> Lise <ol><li> Ressuspender as bactérias em GuSCN * + 0,01% SDS + 4% Proteinase K (0.8mg/ml). Brevemente Vortex (1ml de bactérias em meios de comunicação deve ser ressuspenso em 1ml GuSCN). Proteinase K foi adicionada à solução para ajudar com desnaturação de proteínas que estão ligadas ao DNA bacteriano e inibir DNAases e RNAases. Um benefício adicional do K proteinase é que ele também ajuda com a quebra das proteínas da parede celular que é desejável uma vez que tanto bactérias gram-positivas e gram-negativas paredes celulares de bactérias contêm grandes quantidades de proteína (embora bactérias gram-negativas geralmente têm mais proteínas ). O buffer caotrópicos adicionalmente auxilia o K proteinase com desnaturação de proteínas, enquanto o detergente rompe a parede celular. Proteinase K e tiocianato guanidinium (GuSCN) foram adquiridos da Qiagen Inc. (Valencia, CA). SDS (dodecil sulfato de sódio) foi comprado de Pierce (Rockford, IL). </li><li> Imediatamente carga da amostra em uma seringa conectada ao tubo de PEEK. Aspirar a seringa de modo que nenhum ar é no sistema. Conecte o tubo à nanoport e canal. </li></ol> A bomba de seringa KDS100 (fabricado pela KD Scientific, Holliston, MA) foi utilizado para os experimentos ea vazão utilizada é de 450 mL / hora para todas as etapas. Fluxo do canal lise da amostra em 450ul/hr. <ol><li> Coletar a amostra e proceder à extracção de DNA através de SPE (ver protocolo de extração de DNA). Observação: Este exemplo bacteriana não precisará seguir passo de carga 2, desde a suspensão é GuSCN *. </li></ol> Extração de DNA Protocolo O procedimento de extração consiste em 3 passos: <ol><li> Carga. </li><li> Washington </li><li> Eluir. </li></ol> KDS100 bomba de seringa (fabricado pela KD Scientific, Holliston, MA) foi utilizado para os experimentos ea vazão utilizada foi de 300 mL / hora para todas as etapas. Carga <ol><li> Condição de canal com tampão caotrópicos (GuSCN *, tiocianato guanidinium) por 1-2 min antes de iniciar o experimento. </li><li> Misturar a amostra com tampão caotrópicos na relação de 1:1. </li><li> Fluxo de mistura da amostra através do canal durante 3 min. </li></ol> Lavar <ol><li> Lavar o canal com etanol 70% por 2 min. </li><li> Lavar o canal com etanol 100% por 2 min. </li></ol> Eluir <ol><li> Eluir o DNA extraído em água millipore por 3 min. Colete 15 mL de purificado, pronto PCR-DNA. </li></ol> * O GuSCN do kit Qiagen (buffer RLT) foi utilizado.

<ol>
	<li>Bhattacharyya, A., Klapperich, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+chemical+analysis+of+plasma+and+ultraviolet-ozone+surface+treatments+for+thermal+bonding+of+polymeric+microfluidic+devices.">Mechanical and chemical analysis of plasma and ultraviolet-ozone surface treatments for thermal bonding of polymeric microfluidic devices.</a> Lab Chip. 7, 876-882 (2007).</li></ol>

The authors have nothing to disclose.

fabrication of the thermoplastic microfluidic channels

Em nosso laboratório, conseguimos isolados ácidos nucléicos diretamente microlitro e submicroliter volumes de sangue humano, urina e fezes utilizando polímero / composto de nanopartículas de lise micro e colunas de extração em fase sólida. As amostras recuperadas estão concentradas, as amostras de pequeno volume que são PCRable, sem qualquer limpeza adicional. Aqui, demonstramos como fabricar chips microfluídicos termoplástico usando estampagem a quente e selagem a quente. Então, demonstrar como usar a luz na superfície situ dirigido enxertia e polimerização através do chip lacrado para formar o composto colunas fase sólida. Demonstramos enxertia e de polimerização de um nanotubo de carbono coluna / polímero para a lise celular bacteriana. Em seguida, mostrar o processo de lise seguido por extração em fase sólida de ácidos nucléicos a partir da amostra no chip usando um sílica / polímero coluna composta. Os protocolos anexos contêm instruções detalhadas sobre como fazer tanto lise e colunas de extração em fase sólida.

Watch this Scientific Journal Video about Fabrication of the Thermoplastic Microfluidic Channels at JoVE.com

Fabrication of the Thermoplastic Microfluidic Channels

Aqui demonstramos como fabricar chips microfluídicos termoplástico usando estampagem a quente e selagem a quente. Então demonstrar como usar a luz na superfície situ dirigido enxertia e polimerização através do chip lacrado para formar o composto colunas fase sólida.

Fabricação dos canais microfluídicos Termoplásticos

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

fabrication-of-the-thermoplastic-microfluidic-channels

Research

JoVE Journal

Biology

13.3K visualizações.  Boston University. Aqui demonstramos como fabricar chips microfluídicos termoplástico usando estampagem a quente e selagem a quente. Então demonstrar como usar a luz na superfície situ dirigido enxertia e polimerização através do chip lacrado para formar o composto colunas fase sólida.

Vídeo: Fabrication of the Thermoplastic Microfluidic Channels

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Fabricação de um dispositivo micro para a compartimentação de Neuron Soma e axônios

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Biologia

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

Não-plasma Colagem de PDMS para Fabricação barato e de dispositivos microfluídicos

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

Microfluidic Applications for Disposable Diagnostics

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

Aplicações microfluídicos para diagnóstico descartáveis

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Gravação de patch-clamp de canais iônicos Expresso em Oócitos Xenopus

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

Fabricação de eletrodos amperométrica

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

Fabricação de Miogênica Constrói engenharia de tecidos

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Proteômica para identificar proteínas Interagindo com P2X2 Canais Ligand-Gated Cation

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

O Probe microfluídicos: Operação e Uso de Processamento de Superfície localizada

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagênese e Análise Funcional de canais iônicos Heterologously Expresso em células de mamíferos

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Grampo patch e Técnicas de Perfusão para estudar canais iônicos Expresso em Xenopus Oócitos

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...