Este trabalho foi financiado por doações da Deutsche Forschungsgemeinschaft (DFG), Fritz Thyssen--Stiftung e Charité / MDC.

1. Anestesia animal e extração de órgãos <ol><li> Anestesiar o rato por injeção intraperitoneal (ip) de cetamina (200 mg / kg de peso corporal) e xilazina (20 mg / kg de peso corporal). </li><li> Para anticoagulate injetar 250 ip UI de heparina para evitar a formação de coágulos sangüíneos e trombo. </li><li> Espere até narcose profunda é atingido, o qual é caracterizado por arreflexia. Para verificar arreflexia reflexo teste, córnea por tocar suavemente a córnea ou reflexo voo de teste por beliscar a cauda. </li><li> Transferir o mouse para mesa de operação e corrigi-lo em posição supina. </li><li> Faça uma incisão na pele ea parede abdominal abaixo do xifóide e toracotomia garra: estender o corte para ambos os lados ao longo do arco costal e posteriormente cortado costelas na linha axilar média, desviar para cima da caixa torácica. </li><li> Abra pericárdio, localizar grandes vasos. Pressione suavemente caudal coração, para melhor visualizar a aorta. Grampo da aorta com a pinça. </li><li> Colocar o coração no concavidade de um par de s<html> cissors e dissecar todos os navios de conexão com um único corte. Certifique-se de preservar uma parte suficientemente grande da aorta ascendente para a canulação Langendorff. </li><li> Transferir coração excisado imediatamente para uma placa de Petri cheia de gelo frio e pré-oxigenado solução 1 (para soluções, ver Tabela 1). </li></ol> 2. Preparação de perfusão do coração e Langendorff <ol><li> Canular a aorta, com uma cânula de aço 1.8F. Certifique-se de evitar a embolia aérea. </li><li> Fixar a cânula na aorta, com uma sutura cirúrgica e coronárias, lave com 1 ml de solução 1. </li><li> Ligue cânula com um aparelho de Langendorff. </li><li> Certifique-se de tempo a partir de toracotomia para canulação Langendorff não excede 120 s, para evitar lesão de isquemia / reperfusão prolongada para o miocárdio. </li><li> Coração perfundir com 10 ml de solução de Ca 2 + 2 livre (4 ml / min). </li><li> Coração perfundir com colagenase solução 3 para 8 min (4 ml / min). </li></ol>le &quot;&gt; 3. Microsurgical Dissociação de Cardíaco Chambers <ol><li> Transferir para um centro de pré-aquecido placa de Petri de 100 mm contendo Ca 2 + baixo solução 4. </li><li> Remova cuidadosamente aórtica e outros tecidos não-cardíaca com tesoura e descartá-lo. </li><li> Átrios e ventrículos separados e continuar com cada câmara separadamente. Manter as células imersas em solução 4. Use volumes pequenos (menos de 5 ml). </li></ol> 4. Dissociação adicional de cardiomiócitos <ol><li> Cardiomiócitos atriais: <ol><li> Para individualizar cardiomiócitos atriais transferir os átrios em um prato separado cultura pré-aquecido de 100 mm e dissociar o tecido através puxando-o distante com uma pinça fina. Assegurar uma dissociação quase completa do tecido. </li><li> Usar uma pipeta de 1 ml com uma ponta de pipeta alargada fogo polida plástico para suspender as células em 1 ml de solução a 5 por 5 min. </li><li> Separa-se as células a partir de detritos por meio de um filtro celular (200 malhagem jim). </li> &lt;li&gt; Adicionar 5 ml de solução de 5 a suspensão de células e centrifugação durante 2 min a 16 xg à temperatura ambiente. </li><li> Os passos seguintes são operados sob uma capa de cultura de células. Descartar o sobrenadante e re-suspensão do sedimento em 5 ml de uma solução de 6. </li><li> Após a sedimentação por acção da gravidade, durante 10 minutos em um tubo de 15 ml e centrifugar durante 1 min a 16 xg à temperatura ambiente. Remover o sobrenadante. Re-suspender as células, dependendo da sua quantidade em 1-5 ml de uma solução de 6. </li></ol></li><li> Cardiomiócitos ventriculares: <ol><li> Dissecar a região ventricular esquerda de interesse com uma pinça fina em 5 ml de solução 4. </li><li> Suspender as células por pipetagem suave até que a maioria das células são separadas. Transferir a solução de células após a filtragem (200 malhagem um) para um tubo de 50 ml, adicionar um volume de 25 ml. </li><li> Centrifugar durante 2 minutos a 16 xg à temperatura ambiente. </li><li> Os passos seguintes são operados sob uma capa de cultura de células. Remover o sobrenadante, re-suspender o sedimento em 25 ml desolução a 6 e permitir a sedimentação das células durante 10 min. </li><li> Contar as células e remover o sobrenadante, adicionar 25-50 ml de solução 6 sobre as células. </li></ol></li></ol> 5. Preparação de cardiomiócitos para celular Eletrofisiologia, Bioquímica ou se os estudos <ol><li> Para os estudos de electrofisiologia, manter os miócitos em solução a 6 num tubo de 50 ml e inibir a sedimentação. </li><li> Para estudos bioquímicos, de imagem de cálcio, por exemplo, miócitos placa sobre placas de cultura de células revestidas de laminina (concentração final de 20 ug / ml de laminina em PBS). </li><li> Para coloração por imunofluorescência preparar um prato de cultura de células da placa com lamelas de vidro e revestida com laminina solução (concentração final de 50 ng / ml de laminina em PBS). <ol><li> Remover a solução de revestimento antes de miócitos. Placa das células e controlar a densidade de células, usando um microscópio. </li><li> Deixe os miócitos aderir a tampa deslizante, durante 1 hora a 37 ° C em 2% de CO 2, remover a solução dee começar imediatamente com um protocolo padrão de coloração procedimento. </li><li> Incubar com fixador, por exemplo, PFA 4% em PBS (pH 7,5) durante 10 min à temperatura ambiente e seguir com três passos de lavagem com PBS durante 5 minutos cada. </li><li> Para permeabilizar as células e para inibir a ligação do anticorpo não específico incubar os miócitos com soro a 10%, Triton 0,3%, BSA a 0,2% em PBS durante 30 min à temperatura ambiente. </li><li> Incubar com o anticorpo primário durante 1 hora a 37 ° C e lava-se, tal como descrito antes. </li><li> Incubar com o anticorpo secundário durante 1 h à temperatura ambiente. Para contracoloração os núcleos e α-actinina utilização DAPI e faloidina conjugada fluorocromo (Alexa Fluor 488, Invitrogen). </li><li> Após a lavagem, transferir a tampa de vidro desliza cuidadosamente sobre silano lâminas de microscópio tratados e células embed meio de montagem de fluorescência. </li></ol></li></ol> 6. Eletrofisiologia celular <ol><li> Realize Whole-cell patch-clamp gravações em freshly isolado cardiomiócitos atriais e ventriculares, à temperatura ambiente. </li><li> Transferir saudáveis ​​cardiomiócitos que aparecem na câmara de perfusão preenchido com um determinado volume de solução de banho extracelular. 117 mM de NaCl, 4 mM de KCl, 1 mM de KH 2 PO 4, 4 mM de NaHCO3, 1,7 mM MgCl 2, 3 mM de CoCl2, 10 mM de HEPES, 10 mM de glucose, e 0,02 mM de tetrodotoxina (TTX), (pH 7,4) . Use NaOH para ajuste a valor de pH. </li><li> Use equipamento adequado de fixação adesivo (isto é, microscópio invertido IX71, um amplificador Multiclamp 700B, um sistema de aquisição de Digidata 1440A e PC com pClamp10.3 software (Molecular Devices)). </li><li> Usar pipetas de remendo com resistências de 3-5 MQ quando preenchidos com a solução intracelular contendo 130 mM de KCl, 2 mM de MgCl2, 11 mM de HEPES, 11 mM de EGTA, 5 mM de Na 2 ATP, 0,4 mM de Na 2 GTP, 5 mM de Na 2 CP e 4,9 mM de CaCl 2 (pH 7,2). Use KOH para ajuste a valor de pH. </li><li> Evoke exterior K + correntes dupassos do anel 4,5-sec tensão para testar potenciais entre -60 e +50 mV em incrementos de 10 mV a partir de um potencial de manutenção de -70 mV, depois de um 20-mseg prepulse mV para -40. </li><li> Certifique-se de correntes de fuga são sempre &lt;100 pA. </li><li> Para dissecção de K + diferente correntes utilizar inibidores específicos, tais como a 4-aminopiridina (4-AP; ICN Biomedicals, Irvine, CA, EUA), Heteropodatoxin 2 (HpTx2; Alomone) ou de tetraetilamónio (TEA, Sigma). As soluções-mãe devem ser preparados em uma solução de banho extracelular, e aplicado directamente na vizinhança mais próxima possível da célula através de um microtip depois &quot;de base&quot; de gravações. Equilíbrio deve ser permitida por 2-3 minutos antes de &quot;drogas&quot; gravações. </li><li> Para a análise de normalizar as amplitudes de corrente nas células individuais de tamanho de célula (células inteiras capacitância de membrana). Analisar dados offline usando pClamp10.3 software (Molecular Devices) ou software comparável. </li></ol>

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Com o desenvolvimento crescente de estirpes de ratos geneticamente modificados para estudar a função cardíaca e patologia cardíaca relacionada com deleção do gene também existe um crescente interesse em métodos especializados para estudar os efeitos da supressão do gene específico in vitro. Em nosso laboratório estudamos os papéis de uma família de subunidades Kv canais auxiliares na repolarização cardíaca. Os genes KCNE compreendem uma família de cinco genes (KCNE1-5) que des...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td></tr><tr><td> Tetrodotoxina </td><td> Alomone </td></tr><tr><td> 4-aminopiridina </td><td> ICN Biomedicals </td></tr><tr><td> Heteropodatoxin 2 </td><td> Alomone </td></tr><tr><td> Tetraethylammonium </td><td> Sigma Chemicals </td></tr><tr><td> Colagenase do Tipo 2 </td><td> Worthington </td></tr></tbody></table>

isolation and kv channel recordings in murine atrial and ventricular cardiomyocytes

KCNE genes codificam para uma pequena família de subunidades de canais Kv auxiliares que formam complexos heteroméricos com subunidades alfa dos canais Kv para modificar as suas propriedades funcionais. Mutações em genes KCNE foram encontrados em pacientes com arritmias cardíacas, tais como a síndroma de QT longo e / ou fibrilação atrial. No entanto, a fisiopatologia molecular preciso que conduz a estas doenças permanece elusiva. Em estudos anteriores, as propriedades eletrofisiológicas da doença que causa mutações nestes genes têm sido mais estudada em sistemas de expressão heterólogos e não podemos ter certeza se os efeitos relatados podem ser diretamente traduzidos em cardiomiócitos nativos. Em nosso laboratório, portanto, usar uma abordagem diferente. Nós estudar diretamente os efeitos da KCNE deleção do gene em cardiomiócitos isolados de camundongos knockout por eletrofisiologia celular - uma técnica única que descrevemos nesta edição do Jornal de Experimentos visualizado. Os corações de geneticaratinhos KCNE lly modificadas são rapidamente excisado e montado num aparelho de Langendorff por canulação. Grátis Ca 2 + no miocárdio está vinculado por EGTA, e dissociação de miócitos cardíacos é então obtida por perfusão retrógrada das artérias coronárias com um baixo Ca 2 especializado colagenase tampão contendo +. Átrios, parede livre do ventrículo direito e no ventrículo esquerdo pode, então, ser separados por técnicas microcirúrgicas. O cálcio é então adicionado lentamente de volta para cardiomiócitos isolados em um passo múltiplo compreendendo procedimento de lavagem. Cardiomiócitos atriais e ventriculares de aparência saudável sem contracções espontâneas são então imediatamente sujeitas a análises eletrofisiológicas pela técnica patch clamp ou outras análises bioquímicas dentro das primeiras 6 horas após o isolamento.

Isolamento de cardiomiócitos murinos adultos de camundongos geneticamente modificados para estudar a função de genes específicos de interesse in vitro tornou-se uma ferramenta poderosa para entender a fisiopatologia cardíaca. Este método é usado atualmente por apenas um número pequeno mas crescente de laboratórios de ciências básicas em todo o mundo. No entanto, o isolamento dos cardiomiócitos do ventrículo adultos murinos pode ser complicado e precisa ser feito cuidadosamente e repetidamente por m...

Watch this Scientific Journal Video about Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes at JoVE.com

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

Kv canal disfunção está associada a arritmias cardíacas. Para estudar os mecanismos moleculares que conduzem à ocorrência de arritmias tais que utilizam um protocolo sistemático para o isolamento dos cardiomiócitos atriais e ventriculares do canal Kv auxiliares ratinhos knockout de subunidade. Cardiomiócitos isolados podem então ser imediatamente utilizados para celulares estudos eletrofisiológicos, bioquímicos ou imunofluorescência (IF) ensaios.

Isolamento e gravações de canais Kv em Murino cardiomiócitos atriais e ventriculares

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

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13.3K visualizações.  Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine (MDC). Kv canal disfunção está associada a arritmias cardíacas. Para estudar os mecanismos moleculares que conduzem à ocorrência de arritmias tais que utilizam um protocolo sistemático para o isolamento dos cardiomiócitos atriais e ventriculares do canal Kv auxiliares ratinhos knockout de subunidade. Cardiomiócitos isolados podem então ser imediatamente utilizados para celulares estudos eletrofisiológicos, bioquímicos ou imunofluorescência (IF) ensaios.

Vídeo: Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Análise fenotípica e Isolamento de Células-Tronco Hematopoéticas Murino e Lineage comprometidos Progenitores

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Biologia

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Isolamento e Cultura de Neonatais rato Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

Reconstituição de um canal de Kv para membranas lipídicas de Estudos estruturais e funcionais

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Isolamento e Fisiológica Análise de rato Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Isolamento e Cultura de rato adulto Cardiomyocytes de sinalização celular e<em&gt; In vitro</em&gt; A hipertrofia cardíaca

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

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 ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Reconstituição funcional e atividade do canal Medidas de purificada tipo selvagem e mutante CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Tensão e cálcio Dual Channel Mapping Optical do Cultivadas HL-1 Atrial miócitos Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Isolamento, cultura e Transdução de rato adulto Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.