Questo lavoro è stato finanziato da sovvenzioni da Deutsche Forschungsgemeinschaft (DFG), Fritz-Thyssen-Stiftung e Charité / MDC.

1. Animal Anestesia e prelievo di organi <ol><li> Anestetizzare il mouse intraperitoneale (ip) iniezione di ketamina (200 mg / kg di peso corporeo) e TMA (20 mg / kg di peso corporeo). </li><li> Per anticoagulate iniettare 250 IP UI di eparina per evitare la coagulazione del sangue e la formazione di trombi. </li><li> Attendere fino narcosi viene raggiunto, che è caratterizzato da areflessia. Per verificare la presenza di areflessia, riflesso corneale prova toccando delicatamente la cornea o del volo reflex test pizzicamento coda. </li><li> Trasferire il mouse sul tavolo operatorio e fissarlo in posizione supina. </li><li> Incidere la pelle e la parete addominale al di sotto del xifoide ed eseguire toracotomia clamshell: estendere il taglio su entrambi i lati lungo l&#39;arco costale e successivamente tagliare le costole nella linea ascellare mediale, deviare verso l&#39;alto gabbia toracica. </li><li> Aprire pericardio, individuare grossi vasi. Premere delicatamente caudale cuore per visualizzare meglio l&#39;aorta. Bloccare l&#39;aorta con pinza. </li><li> Posizionare il cuore in concavità di una coppia di s<html> cissors e sezionare tutte le navi di collegamento con un unico taglio. Assicurarsi di conservare una parte sufficientemente grande della aorta ascendente per incannulazione Langendorff. </li><li> Trasferire cuore staccato immediatamente ad una piastra Petri riempito con ghiaccio freddo e pre-ossigenato soluzione di 1 (per le soluzioni, vedi Tabella 1). </li></ol> 2. Preparazione di perfusione Cuore e Langendorff <ol><li> Incannulare l&#39;aorta con una cannula di acciaio 1.8F. Assicurati di evitare embolia gassosa. </li><li> Aorta fissarsi sulla cannula con una sutura chirurgica e coronarie filo con 1 ml di soluzione 1. </li><li> Collegare cannula con un apparato Langendorff. </li><li> Assicurarsi che il tempo di toracotomia per incannulazione Langendorff non supera i 120 secondi per evitare di estesa ischemia / riperfusione al miocardio. </li><li> Cuore profumato con 10 ml di soluzione di Ca 2 + libero 2 (4 ml / min). </li><li> Cuore profumato con una soluzione di collagenasi 3 per 8 min (4 ml / min). </li></ol>le &quot;&gt; 3. microchirurgica dissociazione del Cardiac Chambers <ol><li> Cuore trasferimento in un pre-riscaldato a 100 mm piastra Petri contenente partire Ca 2 + soluzione 4. </li><li> Rimuovere con attenzione aortica e altri non-tessuto cardiaco con le forbici e scartarla. </li><li> Atri e ventricoli separati e continuare con ciascuna camera separatamente. Mantenere le cellule immersi in soluzione al 4. Utilizzare piccoli volumi (meno di 5 ml). </li></ol> 4. Dissociazione ulteriore Cardiomyocytes <ol><li> Cardiomiociti atriali: <ol><li> Per individuare cardiomiociti atriali trasferire gli atri in un apposito pre-riscaldata da 100 mm piastra di coltura e dissociare il tessuto attraverso delicatamente spinge via con una pinza sottile. Assicurare un dissociazione quasi completa del tessuto. </li><li> Usare una pipetta da 1 ml con allargata fuoco lucidato punta di plastica pipetta per sospendere le cellule in 1 ml di soluzione di 5 per 5 min. </li><li> Separare le cellule dai detriti utilizzando un filtro cella (200 maglia um). </li> &lt;li&gt; Aggiungere 5 ml di soluzione al 5 sospensione cellulare e centrifugare per 2 minuti a 16 xg a temperatura ambiente. </li><li> Le seguenti operazioni sono gestite sotto una cappa di coltura cellulare. Eliminare il supernatante e risospeso il pellet in 5 ml di soluzione 6. </li><li> Dopo sedimentazione per gravità per 10 min in una provetta da 15 ml, centrifugare per 1 min a 16 xg a temperatura ambiente. Rimuovere il surnatante. Risospendere le cellule a seconda della loro quantità in 1-5 ml di soluzione 6. </li></ol></li><li> Cardiomiociti ventricolari: <ol><li> Sezionare la regione di interesse ventricolare sinistra con pinza sottile in 5 ml di soluzione 4. </li><li> Sospendere cellule pipettando gentilmente finché la maggior parte delle cellule sono separate. Trasferire la soluzione cellulare dopo il filtraggio (200 maglia micron) in un tubo da 50 ml, si aggiunge un volume di 25 ml. </li><li> Centrifugare per 2 minuti a 16 xg a temperatura ambiente. </li><li> Le seguenti operazioni sono gestite sotto una cappa di coltura cellulare. Rimuovere il surnatante, risospendere il pellet in 25 ml disoluzione 6 e consentire la sedimentazione delle cellule per 10 min. </li><li> Contare le cellule e rimuovere il surnatante, aggiungere 25-50 ml di soluzione 6 sulle cellule. </li></ol></li></ol> 5. Preparazione di Cardiomyocytes per elettrofisiologia cellulare, biochimica o se gli studi <ol><li> Per gli studi di elettrofisiologia, tenere i miociti in soluzione 6 in una provetta da 50 ml e inibire la sedimentazione. </li><li> Per studi biochimici, calcium imaging esempio, miociti piastra sul piatto laminina rivestite coltura cellulare (concentrazione finale di 20 pg / ml di laminina in PBS). </li><li> Per colorazione per immunofluorescenza preparare una coltura di piastra di cella piatto con vetrini coprioggetto rivestiti di vetro e con soluzione laminina (concentrazione finale di 50 pg / ml di laminina in PBS). <ol><li> Rimuovere la soluzione prima di miociti placcatura. Piastra le cellule e controllare la densità cellulare utilizzando un microscopio. </li><li> Lasciare i miociti aderire fodera per 1 ora a 37 ° C in 2% di CO 2, rimuovere la soluzionee iniziare subito con un protocollo standard procedura di colorazione. </li><li> Incubare con fissativo, ad esempio PFA 4% in PBS (pH 7,5) per 10 min a temperatura ambiente e seguire con tre passaggi di lavaggio PBS per 5 minuti ciascuna. </li><li> Per permeabilizzare le cellule e di inibire l&#39;anticorpo non specifico legame incubare i miociti con 10% di siero, 0,3% Triton, 0,2% BSA in PBS per 30 minuti a temperatura ambiente. </li><li> Incubare con l&#39;anticorpo primario per 1 ora a 37 ° C e lavare come descritto prima. </li><li> Incubare con l&#39;anticorpo secondario per 1 ora a temperatura ambiente. Per i nuclei di contrasto e di α-actinina uso e fluorocromo DAPI falloidina coniugato (Alexa Fluor 488, Invitrogen). </li><li> Dopo il lavaggio, trasferire il coperchio di vetro scivola attentamente silano vetrini trattati e cellule embed in fluorescenza mezzo di montaggio. </li></ol></li></ol> 6. Cellular Elettrofisiologia <ol><li> Eseguire Whole-cell patch-clamp su FREshly isolati cardiomiociti atriali e ventricolari a temperatura ambiente. </li><li> Trasferire sani cardiomiociti compaiono nella camera di perfusione riempita con un volume definito della soluzione del bagno extracellulare. 117 mM NaCl, 4 mM KCl, 1 mM KH 2 PO 4, 4 mM NaHCO 3, 1,7 mM MgCl 2, 3 mM CoCl 2, HEPES 10 mM, glucosio 10 mM, e 0,02 mM Tetrodotoxina (TTX), (pH 7,4) . Utilizzare NaOH per la regolazione del valore di pH. </li><li> Usare un equipaggiamento patch clamp (cioè IX71 microscopio invertito, un amplificatore Multiclamp 700B, una acquisizione Digidata 1440A sistema e PC con pClamp10.3 software (Molecular Devices)). </li><li> Utilizzare pipette patch con resistenze di 3-5 MW quando riempito con soluzione intracellulare contenente 130 mM KCl, 2 mM MgCl2, 11 mM di HEPES, 11 mM EGTA, 5 mM Na 2 ATP, 0,4 mM Na 2 GTP, 5 mM Na 2 CP e 4,9 mM CaCl 2 (pH 7,2). Utilizzare KOH per la regolazione del valore di pH. </li><li> Evoke verso l&#39;esterno K + correnti duanello di 4,5 sec gradini di tensione per testare potenziali tra -60 e +50 mV a 10 mV, con incrementi da un potenziale di mantenimento di -70 mV dopo 20 msec prepulse a mV -40. </li><li> Assicurarsi di correnti di fuga sono sempre &lt;100 pA. </li><li> Per la dissezione di K + diverse correnti di usare inibitori specifici come 4-aminopiridina (4-AP; Biomedicals ICN, Irvine, CA, USA), Heteropodatoxin 2 (HpTx2; Alomone) o Tetraethylammonium (TEA, Sigma). Le soluzioni madre dovrebbero essere preparati in soluzione del bagno extracellulare, e applicato direttamente sulla zona più vicina possibile della cellula tramite una micropunta dopo &quot;baseline&quot; registrazioni. Equilibramento dovrebbe essere consentito per 2-3 minuti prima di &quot;droga&quot; registrazioni. </li><li> Per l&#39;analisi normalizzare ampiezze attuali singole celle a dimensione della cella (a cellula intera capacità di membrana). Analizzare i dati non in linea utilizzando pClamp10.3 software (Molecular Devices) o software simile. </li></ol>

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Non abbiamo nulla da rivelare.

Con lo sviluppo crescente di ceppi di topi geneticamente per studiare la funzione cardiaca e patologia cardiaca relativa a gene delezione c&#39;è anche un crescente interesse per metodi specializzati per studiare gli effetti della delezione genica specifica in vitro. Nel nostro laboratorio studiamo i ruoli di una famiglia di Kv canale subunità accessorie sulla ripolarizzazione cardiaca. I geni KCNE comprendono una famiglia di 5 geni (KCNE1-5), che svolgono un ruolo importante in ventricolare...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td></tr><tr><td> Tetrodotoxina </td><td> Alomone </td></tr><tr><td> 4-amminopiridina </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> Collagenasi di tipo 2 </td><td> Worthington </td></tr></tbody></table>

isolation and kv channel recordings in murine atrial and ventricular cardiomyocytes

KCNE geni codificano per una piccola famiglia di Kv canale subunità accessorie che formano complessi con eteromerici Kv canale subunità alfa di modificare le loro proprietà funzionali. Mutazioni in geni KCNE sono stati trovati in pazienti con aritmia cardiaca, come la sindrome del QT lungo e / o fibrillazione atriale. Tuttavia, precisa la fisiopatologia molecolare che porta a queste malattie resta sfuggente. In studi precedenti le proprietà elettrofisiologiche della malattia che provoca mutazioni in questi geni sono in gran parte state studiate in sistemi di espressione eterologa e non possiamo essere sicuri se gli effetti segnalati possono essere direttamente tradotti in cardiomiociti nativi. Nel nostro laboratorio abbiamo quindi utilizzare un approccio diverso. Siamo direttamente studiare gli effetti della delezione del gene KCNE in cardiomiociti isolati da topi knockout per elettrofisiologia cellulare - una tecnica unica che descriviamo in questo numero del Journal of esperimenti visualizzati. I cuori di geneticatopi ingegnerizzati KCNE lly stanno rapidamente asportato e montato su un apparato Langendorff da incannulazione aortica. Gratuito Ca 2 + nel miocardio è vincolato da EGTA, e la dissociazione dei miociti cardiaci viene poi raggiunto da perfusione retrograda delle arterie coronarie con una specializzata a basso Ca 2 + tampone contenente collagenasi. Atria, parete libera del ventricolo destro e il ventricolo sinistro possono essere separati da tecniche microchirurgiche. Il calcio è poi lentamente aggiunti ai cardiomiociti isolati in un passo multiplo comprendente procedura di lavaggio. Cardiomiociti atriali e ventricolari di aspetto sano senza contrazioni spontanee vengono immediatamente sottoposti ad analisi elettrofisiologiche dalla tecnica di patch clamp o altre analisi biochimiche nelle prime 6 ore dopo l&#39;isolamento.

Isolamento degli adulti cardiomiociti murini di topi geneticamente modificati per studiare la funzione dei geni di interesse specifico in vitro è diventato un potente strumento per capire meglio fisiopatologia cardiaca. Questo metodo è attualmente utilizzato da un numero piccolo ma crescente di base laboratori scientifici di tutto il mondo. Tuttavia, l&#39;isolamento dei cardiomiociti ventricolari murini adulti può essere difficile e deve essere fatto accuratamente e ripetutamente da mani esperte. Fi...

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 canale disfunzione è associata ad aritmie cardiache. Al fine di studiare i meccanismi molecolari che portano a tali aritmie che utilizziamo un protocollo sistematico per l&#39;isolamento di cardiomiociti atriali e ventricolari da Kv topi knockout subunità dei canali ausiliari. Cardiomiociti isolati possono essere utilizzate immediatamente per cellulari studi elettrofisiologici, biochimici o di immunofluorescenza (IF) saggi.

Isolamento e registrazioni dei canali Kv in cardiomiociti murini atriale e ventricolare

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 Views.  Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine (MDC). Kv canale disfunzione è associata ad aritmie cardiache. Al fine di studiare i meccanismi molecolari che portano a tali aritmie che utilizziamo un protocollo sistematico per l'isolamento di cardiomiociti atriali e ventricolari da Kv topi knockout subunità dei canali ausiliari. Cardiomiociti isolati possono essere utilizzate immediatamente per cellulari studi elettrofisiologici, biochimici o di immunofluorescenza (IF) saggi.

Video: 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.

L&#39;analisi fenotipica e l&#39;isolamento di murini cellule staminali ematopoietiche e Lineage-committed progenitori

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

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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 della neonatali mouse 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.

La ricostituzione di un canale Kv in membrane lipidiche per gli studi strutturali e funzionali

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 fisiologica Analisi di mouse 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 del topo adulto Cardiomyocytes per Cell Signaling e<em&gt; In vitro</em&gt; Cardiaca Ipertrofia

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.

Un canale Cell-allegato patch-clamp registrazione

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.

Ricostituzione funzionale e attività Canale Misure di purificata di tipo selvatico e mutante CFTR proteine

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.

Tensione e Calcio mappatura ottica a doppio canale di Colta HL-1 atriale miociti monostrato

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 trasduzione di topo adulto cardiomiociti

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.

Registrazioni di potenziale di azione ottica sottotipo-specifici in essere umano indotto Cardiomyocytes ventricolari derivati da cellule staminali di Pluripotent