Bu çalışma Deutsche Forschungsgemeinschaft (DFG), Fritz-Thyssen-Stiftung ve Charité / MDC hibe tarafından finanse edildi.

1. Hayvan Anestezi ve Organ Toplama <ol><li> Ketamin (200 mg / kg) ve ksilazin (20 mg / kg) intraperitoneal (ip) enjeksiyonu ile fare narkoz. </li><li> Kan pıhtılaşması ve trombus oluşumunu önlemek için 250 IU Heparin ip enjekte antikoagülan için. </li><li> Derin narkoz arefleksi ile karakterize olan, ulaşılana kadar bekleyin. Hafifçe kuyruk çimdikleme kornea veya test uçuşu refleks dokunarak arefleksi, test korneal refleks kontrol etmek için. </li><li> Ameliyat masasının üzerine fare aktarın ve yatar pozisyonda sabitleyin. </li><li> Cilt ve kılıç şeklinde aşağıda karın duvarı kesilirken ve kapaklı torakotomi gerçekleştirin: kosta kemer boyunca her iki taraf için kesme uzatın ve daha sonra medial aksiller hat kaburga kesilmiş, göğüs kafesi yukarı saptırmak. </li><li> , Perikard aç büyük damarları bulmak. Yavaşça iyi aorta görüntülemek için kalp kaudal basın. Forseps kullanarak aort Kelepçe. </li><li> Nin bir çift içbükeylik olarak kalp yerleştirin<html> cissors ve tek kesim ile tüm bağlantı damarları teşrih. Langendorff kanülasyon için asendan aort yeterince büyük bir bölümünü korumak için emin olun. </li><li> Buz gibi soğuk ve pre-Oksijenli çözümü 1 (çözümler için, bkz Tablo 1) ile dolu bir Petri kabı hemen çıkarılarak kalp aktarın. </li></ol> 2. Kalp ve Langendorff Perfüzyon hazırlanması <ol><li> Bir 1.8F çelik kanül ile aorta kanüle. Hava embolisi önlemek için emin olun. </li><li> Çözelti 1 1 ml olan bir cerrahi dikiş ve çalkalama ile koroner aort kanülünün üzerinde sabitleştirmek. </li><li> Langendorff aparatı ile kanül takın. </li><li> Torakotomi gelen Langendorff kanülasyon için zaman emin olun miyokarda Uzamış iskemi / reperfüzyon hasarı önlemek için 120 sn aşmaz. </li><li> Ca 2 10 ml serpmek kalp + serbest çözelti 2 (4 ml / dak.) </li><li> 8 dak (4 mL / dak) kollagenaz için çözelti 3 ile serpmek kalp. </li></ol>Kardiyak Chambers le &quot;&gt; 3. Mikrocerrahi Ayrışma <ol><li> Düşük Ca 2 + çözümü 4 içeren önceden ısıtılmış 100 mm Petri kabı içine Transferi kalp. </li><li> Dikkatlice makas ile aort ve diğer non-kardiyak doku kaldırmak ve atın. </li><li> Ayrı atriyumlar ve ventriküller ve ayrı ayrı her odası ile devam edin. Çözüm 4 dalmış hücreleri tutun. Küçük hacimli (az 5 ml) kullanın. </li></ol> 4. Kardiyomiyositlerin Diğer Ayrışma <ol><li> Atriyal kardiyomiyositlerde: <ol><li> Atriyal kardiyomiyositlerde ayrı önceden ısıtılmış 100-mm kültür çanak içine kulakçıklar aktarmak ve nazikçe ince forseps ile didik didik dokuya disosiye bireyselleştirmeye için. Dokusunun neredeyse tam bir ayrışma olun. </li><li> 5 dakika boyunca çözelti 5, 1 ml hücre askıya almak için büyütülmüş bir ateş cilalı bir plastik pipet ucu ile bir 1 ml pipet kullanın. </li><li> Bir hücre filtresi (200 mikron göz açıklığı) kullanılarak artıklardan hücreleri ayırın. </li> &lt;li&gt; 5 ml hücre süspansiyonu ile çözelti 5 ve oda sıcaklığında 16 x g&#39;de 2 dakika süre ile santrifüje ekleyin. </li><li> Aşağıdaki adımlar, bir hücre kültürü kaputu altında işletilmektedir. 6 çözelti 5 ml süpernatant ve yeniden askıya pelet atın. </li><li> Bir 15 ml tüp içinde 10 dakika boyunca yerçekimi ile sedimantasyon sonra, oda sıcaklığında 16 xg&#39;de 1 dakika süreyle santrifüjlenir. Süpernatantı. Solüsyonu 6 1-5 ml kendi miktar bağlı hücreleri yeniden askıya. </li></ol></li><li> Ventriküler kardiyomiyositlerde: <ol><li> 4. çözelti 5 ml ince forseps ile söz konusu sol ventrikül bölge teşrih. </li><li> Hücrelerin en ayrılır kadar yavaşça pipetle hücreler süspanse edin. 50 ml&#39;lik bir tüpe (200 mikron göz açıklığı) filtrelenmesinden sonra çözelti hücre transferi, 25 ml&#39;lik bir hacme ekleyin. </li><li> Oda sıcaklığında 16 x g&#39;de 2 dakika süre ile santrifüje. </li><li> Aşağıdaki adımlar, bir hücre kültürü kaputu altında işletilmektedir. Süpernatantı, 25 ml pelet yeniden askıya6 ve çözelti 10 dk için hücrelerin sedimantasyon izin verir. </li><li> Hücreleri sayın ve süpernatant kaldırmak, 25 eklemek - 50 ml çözelti 6 hücreleri üzerinde. </li></ol></li></ol> 5. Biyokimyasal Hücresel Elektrofizyoloji, ya Çalışmaları IF kardiyomiyositlerin hazırlanması <ol><li> Elektrofizyolojik çalışmalar için, bir 50 ml tüp içinde çözelti halinde 6 miyositleri tutmak ve sedimantasyon inhibe ederler. </li><li> Için biyokimyasal çalışmalar, örneğin kalsiyum görüntüleme, laminin kaplı hücre kültürü çanak tabak miyositler (son konsantrasyonu 20 mg / PBS ml laminin). </li><li> Immunofloresan boyama cam kapak slipleri ile bir hücre kültürü çanağı plaka hazırlamak ve laminin çözeltisi (nihai konsantrasyon 50 mg / ml PBS içerisinde laminin) ile kaplanmış için. <ol><li> Kaplama miyositlerin önce çözüm çıkarın. Levha hücreleri ve bir mikroskop kullanarak, hücre yoğunluğu kontrol eder. </li><li> % 2 CO2 içinde 37, 1 saat boyunca kayma ° C kapsayacak şekilde miyositleri bağlı olsun, çözelti çıkarmakve standart bir boyama işlemi protokolü ile hemen başlayabilirsiniz. </li><li> Oda sıcaklığında 10 dakika boyunca PBS içinde sabitleştirici, örneğin,% 4 PFA (pH 7.5) ile inkübe edin ve 5 dakika her biri için üç PBS yıkama adımları ile takip. </li><li> Hücreler geçirgenliği ve spesifik olmayan bağlayıcı antikor% 10 serum ve% 0.3 Triton, oda sıcaklığında 30 dakika boyunca PBS içinde% 0.2 BSA ile miyositleri inkübe inhibe etmek. </li><li> 37, 1 saat boyunca birincil antikor ile inkübe ° C ve daha önce anlatıldığı gibi yıkayın. </li><li> Oda sıcaklığında 1 saat süreyle ikincil antikor ile inkübe edin. Çekirdekler counterstain ve α-aktinin kullanımı DAPI ve florokrom konjuge phalloidin (Alexa Fluor 488, Invitrogen) için. </li><li> Yıkadıktan sonra, cam kapak floresans montaj orta silan tedavi mikroskop slaytlar ve embed hücreleri üzerinde dikkatle kayıyor aktarın. </li></ol></li></ol> 6. Hücresel Elektrofizyoloji <ol><li> Fre üzerine Tüm-hücre yama klemp kayıtları yapınshly oda sıcaklığında atriyal ve ventriküler kardiyomiyositlerde izole. </li><li> Ekstrasellüler banyo solüsyonu tanımlı ses ile dolu perfüzyon odasının içine sağlıklı görünmesini kardiyomiyositlerde aktarın. 117 mM NaCl, 4 mM KCI, 1 mM KH 2 PO 4, 4 mM NaHCO 3, 1,7 mM MgCl2, 3 mM CoCl 2, 10 mM HEPES, 10 mM glukoz, ve 0.02 mM Tetrodotoxin (TTX), (pH 7.4) . PH değeri ayarı için NaOH kullanın. </li><li> (Yani IX71 inverted mikroskop, bir Multiclamp 700B Amplifikatör, pClamp10.3 yazılımı (Moleküler Cihazlar) ile Digidata 1440A toplama sistemi ve PC) düzgün yama kelepçe teçhizat kullanın. </li><li> 2. CP Na 130 mM KCl, 2 mM MgCl2, 11 mM HEPES, 11 mM EGTA, 5 mM Na 2 ATP, 0.4 mM Na 2 GTP, 5 mM ihtiva eden hücre içi solüsyonu ile dolu olduğunda MΩ 3-5 arasında dirençleri ile yama pipet kullanarak ve 4.9 mM CaCl2 (pH 7.2). PH değerinin ayarlanması için KOH kullanın. </li><li> Dışa K + akımları du Evokehalka 4.5-sn voltaj adımları -60 ve 20 ms -40 mV prepulse sonra -70 mV tutarak potansiyelinden 10 mV adımlarla +50 mV arasında potansiyelleri test etmek. </li><li> Sızıntı akımları her zaman &lt;100 pA olduğundan emin olun. </li><li> , Heteropodatoxin 2 (HpTx2; Alomone) veya tetraetilamonyum (TİM, Sigma); farklı K diseksiyonu için + akımları gibi 4-aminopiridin (ICN Biomedicals, Irvine, CA, USA 4-AP) gibi spesifik inhibitörleri kullanmak. Stok çözeltileri ekstraselüler banyo solüsyonu olarak hazırlanmış ve &quot;temel&quot; kayıt sonra bir mikro tip yoluyla hücre olası en yakın çevresine doğrudan doğruya uygulanabilir. Dengeleme &quot;ilaç&quot; kayıtları önce 2-3 dakika süreyle izin verilmelidir. </li><li> Analizi için hücre boyutu ayrı ayrı hücrelerde (tam hücre zarındaki kapasitans) akım genlikleri normalleştirmek. PClamp10.3 yazılımı (Moleküler Cihazlar) veya benzer yazılımları kullanarak çevrimdışı verileri analiz. </li></ol>

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Biz ifşa etmek başka bir şey var.

Gen delesyonu ile ilgili kardiyak fonksiyon ve kardiak patoloji incelemek için GDO&#39;lu fare suşları artan gelişimi ile in vitro spesifik gen silinmesi etkilerini incelemek için özel yöntemler giderek artan bir ilgi de var. Laboratuarımızda kardiyak repolarizasyon üzerine Kv kanal yan alt birimden bir aile rollerini incelemek. KCNE genler KCNEs kendi üzerinde herhangi bir potasyum akımları geçemez tek transmembran alanı Kv kanal yan alt birimden vardır. Insan ventriküler ve ...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td></tr><tr><td> Tetrodotoxin </td><td> Alomone </td></tr><tr><td> 4-aminopiridin </td><td> ICN Biomedicals </td></tr><tr><td> 2 Heteropodatoxin </td><td> Alomone </td></tr><tr><td> Tetraetilamonyum </td><td> Sigma Kimyasallar </td></tr><tr><td> Kollajenaz Tip 2 </td><td> Worthington </td></tr></tbody></table>

isolation and kv channel recordings in murine atrial and ventricular cardiomyocytes

KCNE genlerin fonksiyonel özelliklerini değiştirmek için Kv kanalı alfa alt birimden heteromerik kompleksler oluştururlar Kv kanal yan altbirimden küçük bir aile için kodlamak. KCNE genlerdeki mutasyonlar gibi uzun QT sendromu ve / veya atriyal fibrilasyon gibi kardiyak aritmiler olan hastalarda bulunmuştur. Bununla birlikte, bu hastalıklara yol açan belirli moleküler patofizyolojisi belirsiz kalır. Önceki çalışmalarda bu genlerdeki mutasyonların sebep olan hastalığın elektrofizyolojik özellikleri çoğunlukla heterolog ekspresyon sistemleri incelenmiş ve rapor edilmiş etkileri doğrudan yerli kardiyomiyositlerde tercüme edilebilir eğer biz emin olamayız. Laboratuvarımızda bu yüzden farklı bir yaklaşım kullanır. Biz görüntülenmiştir Deneyleri Dergisi bu sayısında tanımlayan benzersiz bir teknik - Biz doğrudan hücresel elektrofizyoloji tarafından nakavt farelerin izole kardiyomiyositlerde KCNE gen delesyonu etkilerini incelemek. Genetica gelen kalplerlly mühendislik KCNE farelerin hızla eksize ve aort kanülasyon tarafından Langendorff aparatı monte edilirler. Miyokardda Serbest Ca 2 + EGTA tarafından bağlanır ve kardiyak miyosit dissosiasyon ardından özel bir düşük Ca 2 + tampon içeren kollajenaz ile koroner arterlerin retrograd perfüzyon elde edilir. Atria, serbest sağ ventriküler duvara ve sol ventrikül sonra, mikrocerrahi teknikler ile ayrılabilmektedir. Kalsiyum sonra yavaş yavaş işlem çamaşır içeren bir çoklu adım izole kardiyomiyositler tekrar ilave edilir. Spontan kontraksiyonları ile sağlıklı görünüm Atriyal ve ventriküler kardiyomiyositlerde sonra hemen izolasyonu takiben ilk 6 saat içinde yama klemp tekniği veya diğer biyokimyasal analizler ile elektrofizyolojik analizlere tabi tutulmaktadır.

In vitro ilgi belirli genlerin işlevini incelemek için genetiği değiştirilmiş farelerin erişkin fare kardiyomiyositlerde izolasyonu daha da kardiyak patofizyoloji anlamak için güçlü bir araç haline gelmiştir. Bu yöntem, şu anda dünya çapında temel bilim laboratuarları sadece bir küçük ama artan sayıda tarafından kullanılır. Ancak, yetişkin ventriküler murin kardiyomiyositlerde izolasyonu zor ve tecrübeli eller tarafından iyice ve tekrarlı yapılması gerekiyor olabilir. Ş...

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 kanal disfonksiyonu kardiyak aritmiler ile ilişkilidir. Biz Kv kanal yan altbirim knockout farelerde atriyal ve ventriküler kardiyomiyositlerde izolasyonu için sistematik bir protokol kullanmak gibi aritmiler neden moleküler mekanizmaları incelemek için. İzole kardiyomiyositlerde hemen sonra hücresel elektrofizyolojik çalışmalar, biyokimyasal ya da immunofloresan (IF) deneyleri için de kullanılabilir.

Mürin Atriyal ve Ventriküler kardiyomiyositlerde izolasyonu ve Kv Kanal Kayıtları

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

isolation-kv-channel-recordings-murine-atrial-ventricular

Research

JoVE Journal

Biology

13.3K Görüntüleme.  Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine (MDC). Kv kanal disfonksiyonu kardiyak aritmiler ile ilişkilidir. Biz Kv kanal yan altbirim knockout farelerde atriyal ve ventriküler kardiyomiyositlerde izolasyonu için sistematik bir protokol kullanmak gibi aritmiler neden moleküler mekanizmaları incelemek için. İzole kardiyomiyositlerde hemen sonra hücresel elektrofizyolojik çalışmalar, biyokimyasal ya da immunofloresan (IF) deneyleri için de kullanılabilir.

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.

Mürin Hematopoetik Kök Hücreler ve Lineage-kararlı progenitörlerin Fenotipik Analizi ve İzolasyon

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

Biyoloji

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.

İzolasyon ve Kültür Yenidoğan Fare kardiyomiyositlerin

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.

Yapısal ve İşlevsel Araştırmalar için Lipid Membranlar bir Kv Kanal Sulandırma

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.

Fare kardiyomiyositlerde İzolasyon ve Fizyolojik Analizi

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.

İzolasyon ve Kültür Yetişkin Fare Hücre Sinyal için kardiyomiyositlerin ve<em&gt; In vitro</em&gt; Kalp hipertrofisi

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.

Tek kanallı bir hücre-bağlı Patch-clamp kayıt

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.

Saf Yabanitip ve Mutant CFTR Protein Fonksiyonel Sulandırma ve Kanal Aktivite Ölçümleri

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.

Kültürlü HL-1 Atriyal miyositlerdeki Monolayer Gerilim ve Kalsiyum Çift Kanal Optik Haritalama

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.

İzolasyon, Kültür ve Yetişkin Fare kardiyomiyositlerde İletimi

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.