Buna ek olarak, onların mükemmel teknik destek için insan atriyal doku ve Claudia Liebetrau, Katrin Kupser ve Ramona Nagel sağlanması için Heidelberg Üniversitesi&#39;nde kalp cerrahlar teşekkür ederim. Onun yararlı öneriler ve Ca 2 + geçici ölçümlerin kuruluş aşamasında danışmanlık için de Andy W. Trafford için teşekkür etmek istiyorum. Onlar hücresel elektrofizyoloji ve kardiyomiyosit izolasyon temel teknikleri ve becerileri öğrenmek için bize sunulan fırsatı için Teknoloji Dresden Üniversitesi: Yazarlar Farmakoloji ve Toksikoloji Anabilim Dalı (Ursula Ravens kafa) üyelerine en derin şükranlarımızı ifade etmek istiyoruz. Yazarlar araştırma Alman Araştırma Vakfı (Do769/1-1-3), Atriyal fibrilasyon Yetkinlik Ağı aracılığıyla Eğitim ve Araştırma Alman Federal Bakanlığı (01Gi0204) ve Kardiyovasküler Araştırma için Alman Merkezi aracılığıyla Avrupa Birliği tarafından desteklenmektedir EuAtriyal fibrilasyon içinde Translational Araştırma Sanatlar Lisesi, Avrupa Ağı (EUTRAF, FP7-SAĞLIK-2010, büyük ölçekli entegre proje, Teklif No 261057) ve Avrupa-Kuzey Amerika Atriyal fibrilasyon Araştırma Birliği (ENAFRA) Vakfı Leducq (07CVD03) bir hibe. Video dosyası gösterilen şematik bir genel Servier, tıp tekniğinde kullanılarak üretildi.

Deneysel protokolleri, yerel etik kurul tarafından onaylanmış ve tüm hastalarda yazılı onay vermek gerekir gerekir. Araştırma Tıp Fakültesi Mannheim etik kurul, Heidelberg Üniversitesi (# 2011-216N-MA) tarafından onaylandı ve insan refahı için tüm, kurumsal, ulusal ve uluslararası kurallar uygun olarak yapıldı. Tüm hastalar yazılı onam verdi. 0. İnsan Atriyal Doku Alınması Aşılama kardiyopulmoner bypass için açık kalp ameliyatı geçiren hastalarda rutin kanülasyon prosedürleri sırasında, sağ atriyal apendiks ucunda genellikle kaldırılır ve atriyal kardiyomiyositlerinin izolasyonu için kullanılabilir. BDM, kasılma önleyici, miyosit c önlenmesi; eksizyon sonrası doku örneği 2,3-bütandion monoxime (Tablo I içeren +-ücretsiz ulaşım çözümü steril Ca 2 ile 50 ml Falcon tüp içine hemen aktarılır) ontracture. 30 ve 45 dakika arasında ulaşım zaman ile, izole atriyal kardiyomiyositlerinin sayısı ve kalitesini göre soğutma veya oksijenasyon bir avantaj tanımadı. Laboratuara genel ulaşım mümkün olduğunca hızlı olmalıdır. Daha uzun süre taşıma süresi önlenemez Ancak, 4 de ulaşım ° C oksijenli çözüm avantajlı olabilir. 1. Prearrangements <ol><li> Ceketli beher (Tablo II) sıcaklık kontrol thermocirculator, açın. Kabı içindeki sıcaklık 37 ° C ye eşit olduğundan emin olun Tüm süreç boyunca izolasyon beher içinde sabit bir sıcaklığı korumak için bir cam kapak ile kabı kapsamaktadır. </li><li> Üç cam bardak içine kollajenaz I ve Proteaz XXIV tartılır ve oda sıcaklığında saklayın. Enzimler, kullanımdan hemen önce seyreltildi edilecektir. </li><li> Ca 2 Mağaza 20 ml 4 bir Petri kabındaki + ücretsiz bir çözüm ° C </li><li>Ca 2 deposu 250 ml 37 ° C&#39;de su banyosunda + serbest çözeltisi ° C </li></ol> 2. Doku temizliği <ol><li> Bir Petri kabı (~ 10 cm çapında) içine taşıma çözümü ile birlikte doku örneği aktarın ve yaklaşık makas kullanılarak yağ doku kaldırmak. </li><li> Doku örneği tartılır. 200 ila 600 mg hücre izolasyonu için kullanılır. Kalan doku daha sonra biyokimyasal analiz için likit nitrojende dondurulmuş olabilir. </li><li> 20 ml Ca 2 içeren Petri kabı içine doku örneği aktarmak 4 de + ücretsiz bir çözüm ° C (adım 1.3) ve boyutu yaklaşık 1 mm 3 küçük parçalar halinde doku örneği doğrayın. </li><li> Aşağıdaki adımlar% 100 O 2 ile 37 ° C de ve sürekli gazlama altında yürütülmelidir. Bir pipet güçlü köpüren önlemek ve sürekli hava akışı oluşturmak için oksijen tüpü üzerine yerleştirilebilir. </li><li> Içinde Ca2 + içermeyen bir çözüm ile birlikte doku parçaları transferceketli beher ve bir manyetik karıştırma çubuğu ile yaklaşık 3 dakika boyunca dikkatli bir şekilde karıştırın. </li><li> Karıştırma durdurmak ve doku parçaları birkaç saniye yerleşmek için izin verir. Bir naylon örgü (200 mikron, Tablo II) ile süpernatant dikkatlice süzün. Forseps kullanarak beher içine örgü gelen ölçülü doku parçaları dönün. </li><li> Ca 2 ile doldurun beher + serbest çözeltisi ile yıkama aşaması 2.4 ve 2.5 &#39;da iki kez tekrarlayın. </li></ol> 3. İlk Enzimatik Sindirim <ol><li> Kolajenaz ve proteaz ihtiva eden 20 ml enzim çözeltisi E1 (Tablo I) ile doku parçaları yeniden askıya ve 10 dakika boyunca dikkatli bir şekilde karıştırın. </li><li> 20 uM Ca2 + bir son konsantrasyon elde etmek için 10 mM CaCl2-çözeltisinin 40 ul ekle. </li><li> 35 dakika doku parçaları çoğu beher kalır bir şekilde bir naylon örgü (200 mikron, Tablo II) dikkatle süpernatant süzün sonra. Dinlenme dönörgü gelen behere doku parçaları yağmur yağdı. </li></ol> 4. İkinci Enzimatik Sindirim <ol><li> Sadece 20 mL enzim çözeltisi içeren E2 kolajenaz (Tablo I) ile tekrar doku parçaları yeniden süspanse edin. Hemen 20 uM Ca2 bir son konsantrasyon elde etmek için 10 mM CaCl2-çözeltisi 40 ul +. </li><li> İkinci sindirim kullanımı makas sırasında daha fazla bazen doku pıhtıları kesmek için. </li><li> 5 dakika sonra hücrelerin ayrışma kontrol etmek için bir Pasteur pipeti kullanarak bir örnek almak. Çubuk şeklinde, çizgili kardiyomiyositler görünene kadar bu 2-3 dk her tekrarlayın. </li><li> Karıştırma durdurmak ve doku parçaları yaklaşık 20-30 saniye yerleşmek için izin verir. </li><li> 50 ml&#39;lik bir Falcon tüp (tüp A) içine, naylon elekten (200 mm, Tablo II) ile dikkatli bir şekilde yüzer süzün. Behere örgü gelen ölçülü doku parçaları dönün. </li><li> 20 ml Stora ile beher doku parçaları yeniden askıyaGE çözeltisi (Tablo I) &#39;in 10 ve daha fazla dağıtıcı içeren bir 20 mL serolojik bir pipet kullanarak yumuşak mekanik öğütülerek hücreleri ayırmak. Kabarcıklar hücre sağkalım ve kalite için zararlı olduğu için kabarcık oluşumunu önlemek için deneyin. </li><li> 50 ml Falcon Tüp (Tüp B) bir naylon örgü (200 mikron, Tablo II) dikkatle süpernatant süzün. </li></ol> 5. Final Ca 2 Son Hazırlık ve Ayar + Konsantrasyon <ol><li> 10 dakika 95 xg&#39;de Falcon tüpler A ve B (adım 4.5 ve 4.7) hem de santrifüj. </li><li> Aspirasyon tarafından dikkatli bir şekilde her iki tüpleri süpernatantı. Pelet rahatsız etmemek için emin olun. Süpernatantı atın. </li><li> 1.5 ml saklama solüsyonu (Tablo I) &#39;in her biri (oda sıcaklığı) her iki pelet yeniden askıya. </li><li> İki kez her Falcon için 10 mM CaCl2 çözüm 7.5 ul ekleyin ve dikkatli bir şekilde karıştırın. Her adım, sonra 10 dakika süreyle inkübe edin. </li><li>0.2 mM nihai bir Ca2 + konsantrasyonu elde etmek için 10 mM CaCl2 çözeltisi 15 ul ekle. </li></ol> 6. Floresan Ca 2 +-göstergesi Fluo-3 AM (Şekil 1) ile miyositlerin yükleme <ol><li> 2 ml mikrosantrifüj tüpü (Eppendorf tüp) içine hücre süspansiyonu, 1.5 ml (tüp A ve / ya da tüp B) aktarın. </li><li> Aşağıdaki adımlar floresan Ca 2 + Gösterge Fluo-3 ışık hassasiyeti dikkate alınarak yürütülmelidir. </li><li> Pluronik F-127 (Tablo III) stok çözeltisi (% 20 susuz DMSO içerisinde ağırlık / hacim olarak) elde etmek için 44 ul içerisinde Fluo-3 membran geçirgen asetoksimetil ester türevi (Fluo-3 AM, Tablo III) &#39;ün 50 mg çözülür 1 mM Fluo-3 en az 1 hafta boyunca -20 ° C&#39;de saklanabilir stok solüsyonu, Am. </li><li> 15 ul ekle Fluo-3 hücre süspansiyonu, 1.5 ml (adım 6.1) ve ajitasyon içeren mikrosantrifüj tüpüne stok solüsyonu AMdikkatli. </li><li> Bir optik olarak donuk olan bir kutuda 10 dakika boyunca hücre süspansiyonu inkübe edin. </li><li> Kısaca yaklaşık 6000 rpm santrifüj. </li><li> Süpernatantı atın ve 1.5 ml banyo solüsyonu (Tablo IV) pelet yeniden askıya. </li><li> Deneyleri ile başlamadan önce de-esterifikasyon için yaklaşık 30 dakika boyunca bir hücre süspansiyonu bırakın. </li></ol> 7. Eşzamanlı Patch-kelepçe ve epifluorescent Ca 2 + Ölçümler Yama-kelepçe ölçümleri bu yorumu en önemli konu olmadığı, bu tekniğin derinlemesine bir açıklama daha sağlayan diğer yayınlar için ilgilenen okuyucu bakın. 11-14 bütünlüğü uğruna biz ölçmek için bir protokol kısa bir özetini aksiyon potansiyelleri veya L-tipi Ca 2 + akımları, eş zamanlı Ca 2 +-geçici kayıtları ile birlikte hem. Deneyler sırasında miyositler banyo soluti ile 37 ° C&#39;de superfused edilirhızlı bir perfüzyon sistemi (Octaflow IITM, ALA Scientific Instruments, NY) ile (Tablo IV). Voltaj-kıskaç deneyleri için, K + akımlarının banyo solüsyonuna 4-aminopiridin (5 mg / L) ve BaCl 2 (0.1 mmol / L) eklenerek bloke edilmiştir. Borosilikat cam Mikroelektronlar kullanılır ve pipet çözüm (Tablo V) ile dolu zaman MQ 2-5 dirençleri ucu olmalıdır. Fluo-3 ek olarak (adım 6), miyosit yükleme PM, Flu-3, aynı zamanda pipet çözeltisi (Tablo V) &#39;de yer almaktadır. Floresan 488 nm heyecanlı ve yayılan ışık (&lt;520 nm) [Ca 2 +] i varsayarak dönüştürülür <img alt="Denklem 1" fo:content-width="1.540in" fo:src="/files/ftp_upload/50235/50235eq1highres.jpg" src="/files/ftp_upload/50235/50235eq1.jpg" /> burada K, D Fluo-3 sabiti = çözünme (864 KM), K = Fluo-3 floresan;. Fmax = Ca2 her deney sonunda elde edilen + doymuş floresan 12 elektrik sinyalleri ve Epifluorescent Ca2 + hem de görüntü sinyallerini da kaydedilir. Aksiyon potansiyelleri 1.2x eşik gücü 1 msn akım darbeleri kullanarak akım kelepçe modunda 0,5 Hz uyarılır. L tipi Ca2 + akımları, 100 milisaniye ile hızlı-Na + akımı, ardından etkisiz hale getirmek için -80 mV değerinde bir tutma potansiyeli -40 mV&#39;a bir 100-msn rampa darbe ile voltaj-kelepçe modunda ölçülür 0,5 Hz +10 mV test darbe.

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Pharmacol. 154, 1619-1630 (2008).</li><li>Dobrev, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+downregulation+of+G-protein-coupled+inward+rectifying+K++current+IK,ACh+in+chronic+human+atrial+fibrillation:+decrease+in+GIRK4+mRNA+correlates+with+reduced+IK,ACh+and+muscarinic+receptor-mediated+shortening+of+action+potentials.">Molecular basis of downregulation of G-protein-coupled inward rectifying K+ current IK,ACh in chronic human atrial fibrillation: decrease in GIRK4 mRNA correlates with reduced IK,ACh and muscarinic receptor-mediated shortening of action potentials.</a> Circulation. 104, 2551-2557 (2001).</li><li>Hatem, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+compartments+of+sarcoplasmic+reticulum+participate+in+the+excitation-contraction+coupling+process+in+human+atrial+myocytes.">Different compartments of sarcoplasmic reticulum participate in the excitation-contraction coupling process in human atrial myocytes.</a> Circ. Res. 80, 345-353 (1997).</li><li>Heidbuchel, H., Vereecke, J., Carmeliet, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+different+potassium+channels+in+human+atrium.+Contribution+to+the+basal+potassium+conductance.">Three different potassium channels in human atrium. Contribution to the basal potassium conductance.</a> Circ. Res. 66, 1277-1286 (1990).</li><li>Hove-Madsen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial+fibrillation+is+associated+with+increased+spontaneous+calcium+release+from+the+sarcoplasmic+reticulum+in+human+atrial+myocytes.">Atrial fibrillation is associated with increased spontaneous calcium release from the sarcoplasmic reticulum in human atrial myocytes.</a> Circulation. 110, 1358-1363 (2004).</li><li>Molina, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+adenosine+monophosphate+phosphodiesterase+type+4+protects+against+atrial+arrhythmias.">Cyclic adenosine monophosphate phosphodiesterase type 4 protects against atrial arrhythmias.</a> J. Am. Coll. Cardiol. 59, 2182-2190 (2012).</li></ol>

Worthington Biyokimyasal Corp video dosyasının üretim maliyetleri kapsayan bu yayın destekledi. Fon bir çalışma tasarımında rol, veri toplama ve analizi, yayınlamak karar, ya da yazının hazırlanması vardı.

Burada açık kalp cerrahisi uygulanan hastalarda elde edilen sağ atriyal uzantıları insan atriyal miyositlerin izolasyonu için bir yöntem açıklanmaktadır. Sitosolik Ca2 ölçümleri için bu miyositler kullanmak için + Eğer saklama çözeltisinden EGTA ihmal edilmesi ile daha önce tarif edilen yöntem 4-11 uyarlanmıştır. Zaten 1970 yılında bu miyositler + sindirim sırasında Ca 2 varlığında ayırmak rağmen, hepsi nedenle kontraktürü ve cansız. 16,17 içinde olduğu gözlenmiştir, hücre izolasyonu Ca 2 +-ücretsiz bir çözüm yapılır. Ancak, Ca 2 fizyolojik konsantrasyonlarının yeniden giriş + hızlı Ca 2 + akını ve hücre ölümüne neden oldu. Bu aslında Zimmerman ve Hulsman ile perfüze gözlenmiştir Ca2 + paradoks bir olgu olarak tarif edilmiştir. 7.0 pH azaltılması da dahil olmak üzere yalıtım ortamı 18 Değişiklikler &lt;taurin 20 veya Ca 2 + (adım 3.2 ve 4.1 &#39;e bakınız), 21 yanı sıra, depolama çözelti 22 EGTA ihtiva eden izole miyosit depolama küçük miktarlarda&gt; 19, ek destek Ca2 + paradoksu. 17 engellemek için öne sürülmüştür Bununla birlikte, iyi bir Ca2 + EGTA ile tampon L-tipi Ca 2 + akımı genliği ile indüklenen Ca2 + geçici genlik ve Ca2 + geçici bifazik bir çürüme ile sonuçlanır azalttığı bilinmektedir. 23. Bu nedenle, EGTA ihmal Tipik özellikleri ve tek fazlı bozunur olan Ca2 + geçici elde etmek için, tüm süreç boyunca izolasyon. Ca 2 + hücreleri korumak için paradoks biz 0.2 mM kadar kademeli bir şekilde depolama çözeltinin nihai Ca2 + konsantrasyonu artar. Kollajenaz seçimi muhtemelen başarılı miyosit izolasyon için en önemli adımdır. Geleneksel collagenases Clostridium histolyticum elde edilen ham preparatlar olup diğer proteinazlar polysaccharidases ve lipazlar arasında bir dizi ek kolajenaz içerir. Kendi genel kompozisyon kolajenazlar göre farklı bölünmüştür. 24 Worthington kollajenaz Türleri I ve II başarıyla anda açıklanan protokolde insan atriyal miyositlerin izolasyonu. 4-10,15,25-30 için kullanılmıştır biz kollajenaz kullanılmasını tavsiye biz de kollajenaz Tip II kullanarak canlı hücreler kabul edilebilir miktarda elde etmek mümkün olmasına rağmen, ben yazın. Ancak, hatta tek bir kollajenaz tip içinde enzim faaliyetleri ile ilgili önemli bir toplu-seri farklılıklar vardır. Bu varyasyonlar dikkatli toplu seçimi ve izolasyon prosedürü optimize etmek için çeşitli gruplar test gerektirir. Worthington Biyokimyasal Corp (den online olarak mevcuttur toplu-seçim aracı <a href="http://www.worthington-biochem.com/cls/match.php" target="_blank">http://www.worthington-bio</a>chem.com / CLS / match.php), insan atrial miyosit izolasyonu için uygun olduğu gösterilmiştir bir bileşim ile uygun gruplar bulmak için de kullanılabilir. Şu anda kollajenaz 250 U / mg kollajenaz aktivite, 345 U / mg caseinase aktivite, 2.16 U / mg clostripain aktivite ve 0.48 U / mg triptik aktivite (lot # 49H11338) ile tip I kullanın. Bu makalede tarif edilen prosedür kullanılarak elde edilen hücreler patch-clamp çalışmaları, Ca2 + geçici ölçümleri ve ek olarak her iki. 15 bir kombinasyonu için 8 saat içinde kullanılabilir, bu hücrelerin elektrik alanı uyarılmasına tepki olarak hücre daralma ölçümü sağlar ya da elektrik stimülasyonu yama-kelepçe pipet (yayınlanmamış gözlemler) kullanarak.

Yama klemp tekniği ve + taşıma anormallikleri izole kardiyomiyositlerin gerektiren hücresel Ca 2 arama ile kardiyak iyon kanallarının elektrofizyolojik özellikleri incelenmesi. Bunlar genellikle sindirim enzimleri kardiyak doku örneklerinin in vitro maruz kalma (kolajenaz, hiyaluronidaz, peptidaz vb) ardından elde edilir. 1.955 2&#39;de canlı kalp miyositlerin izolasyonu ilk rapordan bu yana protokoller büyük miktarda fare, sıçan, tavşan, köpek, kobay ve insan dahil olmak üzere farklı türlerden tek atriyal ve ventriküler kardiyomiyositlerde hasat için geliştirilmiştir. Bu derlemede insan atriyal miyositlerin izolasyonu odaklanın. Biz Worthington Biyokimyasal Corp, ABD (tarafından sağlanan &quot;Worthington Doku Ayrılma kılavuzu&quot; bölümüne bakın diğer türlerden miyositlerin izolasyonu için prosedürleri ile ilgili <a href="http://www.tissuedissociation.com" target="_blank">www.tissuedissociation.com</a> ). <p class= &quot;Jove_content&quot;&gt; İnsan atriyal miyosit izolasyon protokolleri genellikle Burada amacıyla önceden yayınlanmış bir yöntem uyarlanan bir teknik bir adım-adım bir açıklaması, sağlamak Bustamante, ve diğ. 3 açıklanan yöntem kullanılarak elde edilmiştir Patch-clamp deneyler için değil, aynı zamanda eş zamanlı olarak hücre içi Ca2 + ölçümü için sadece uygun atrial miyositlerinde elde edilir. 4-11

<table border="1">
 <tbody>
 <tr>
 <td> &nbsp;</td>
 <td> &nbsp;</td>
 <td> &nbsp;</td>
 <td>Transport solution</td>
 </tr>
 <tr>
 <td>Albumin</td>
 <td>Sigma-Aldrich</td>
 <td>A3059</td>
 <td>Storage solution: 1%</td>
 </tr>
 <tr>
 <td>BDM</td>
 <td>Sigma-Aldrich</td>
 <td>31550</td>
 <td>Transport solution: 30</td>
 </tr>
 <tr>
 <td>DL-b-Hydroxy-butyric acid</td>
 <td>Sigma-Aldrich</td>
 <td>H6501</td>
 <td>Storage solution: 10</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G8270</td>
 <td>Transport solution: 20 
 Ca2+-free solution: 20 
 Enzyme solution E1 and E2: 20 
 Storage solution: 10</td>
 </tr>
 <tr>
 <td>L-Glutamic acid</td>
 <td>Sigma-Aldrich</td>
 <td>G1251</td>
 <td>Storage solution: 70</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Merck</td>
 <td>1049360250</td>
 <td>Transport solution: 10 
 Ca2+-free solution: 10 
 Enzyme solution E1 and E2: 10 
 Storage solution: 20</td>
 </tr>
 <tr>
 <td>KH2PO4</td>
 <td>Sigma-Aldrich</td>
 <td>P5655</td>
 <td>Transport solution: 1.2 
 Ca2+-free solution: 1.2 
 Enzyme solution E1 and E2: 1.2 
 Storage solution: 10</td>
 </tr>
 <tr>
 <td>MgSO4</td>
 <td>Sigma-Aldrich</td>
 <td>M9397</td>
 <td>Transport solution: 5 
 Ca2+-free solution: 5 
 Enzyme solution E1 and E2: 5</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>Sigma-Aldrich</td>
 <td>M1254</td>
 <td>Transport solution: 5 
 Ca2+-free solution: 5 
 Enzyme solution E1 and E2: 5</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma-Aldrich</td>
 <td>S3014</td>
 <td>Transport solution: 100 
 Ca2+-free solution: 100 
 Enzyme solution E1 and E2: 100</td>
 </tr>
 <tr>
 <td>Taurin</td>
 <td>Sigma-Aldrich</td>
 <td>86330</td>
 <td>Transport solution: 50 
 Ca2+-free solution: 50 
 Enzyme solution E1 and E2: 50 
 Storage solution: 10</td>
 </tr>
 <tr>
 <td>Collagenase I</td>
 <td>Worthington</td>
 <td>4196</td>
 <td>Enzyme solution E1 and E2: 286 U/ml</td>
 </tr>
 <tr>
 <td>Protease XXIV</td>
 <td>Sigma-Aldrich</td>
 <td>P8038</td>
 <td>Enzyme solution E1 and E2: 5 U/ml*</td>
 </tr>
 <tr>
 <td>pH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Transport solution: 7.00 
 Ca2+-free solution: 7.00 
 Enzyme solution E1 and E2: 7.00 
 Storage solution: 7.40</td>
 </tr>
 <tr>
 <td>adjusted with</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Transport solution: 1 M NaOH 
 Ca2+-free solution: 1 M NaOH 
 Enzyme solution E1 and E2: 1 M NaOH 
 Storage solution: 1 M KOH</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Concentrations in mM unless otherwise stated. BDM, 2,3-Butanedione monoxime. *Protease XXIV is included in Enzyme solution E1 only.</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table I. Solutions.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nylon mesh (200 &mu;m)</td>
 <td>VWR-Germany</td>
 <td>510-9527</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Jacketed reaction beaker</td>
 <td>VWR</td>
 <td>KT317000-0050</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table II. Specific equipment.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dimethyl-sulphoxide</td>
 <td>Sigma-Aldrich</td>
 <td>D2650</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fluo-3 AM (special packaging)</td>
 <td>Invitrogen</td>
 <td>F-1242</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pluronic F-127</td>
 <td>Invitrogen</td>
 <td>P6867</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table III. Substances for loading of myocytes with Fluo-3 AM.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Bath solution</td>
 </tr>
 <tr>
 <td>4-aminopyridine*</td>
 <td>Sigma-Aldrich</td>
 <td>A78403</td>
 <td>Bath solution: 5</td>
 </tr>
 <tr>
 <td>BaCl2*</td>
 <td>Sigma-Aldrich</td>
 <td>342920</td>
 <td>Bath solution: 0.1</td>
 </tr>
 <tr>
 <td>CaCl2 &times; 2H2O</td>
 <td>Sigma-Aldrich</td>
 <td>C5080</td>
 <td>Bath solution: 2</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G8270</td>
 <td>Bath solution: 10</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H9136</td>
 <td>Bath solution: 10</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Merck</td>
 <td>1049360250</td>
 <td>Bath solution: 4</td>
 </tr>
 <tr>
 <td>MgCl &times; 6H2O</td>
 <td>Sigma-Aldrich</td>
 <td>M0250</td>
 <td>Bath solution: 1</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma-Aldrich</td>
 <td>S3014</td>
 <td>Bath solution: 140</td>
 </tr>
 <tr>
 <td>Probenecid</td>
 <td>Sigma-Aldrich</td>
 <td>P8761</td>
 <td>Bath solution: 2</td>
 </tr>
 <tr>
 <td>pH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Bath solution: 7.35</td>
 </tr>
 <tr>
 <td>adjusted with</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Bath solution: 1 M HCl</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table IV. Bath solution for patch-clamp. *4-aminopyridine and BaCl were included for voltage-clamp experiments only.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Pipette solution</td>
 </tr>
 <tr>
 <td>DL-aspartat K+-salt</td>
 <td>Sigma-Aldrich</td>
 <td>A2025</td>
 <td>Pipette solution: 92</td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td>Sigma-Aldrich</td>
 <td>E4378</td>
 <td>Pipette solution: 0.02</td>
 </tr>
 <tr>
 <td>GTP-Tris</td>
 <td>Sigma-Aldrich</td>
 <td>G9002</td>
 <td>Pipette solution: 0.1</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H9136</td>
 <td>Pipette solution: 10</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Merck</td>
 <td>1049360250</td>
 <td>Pipette solution: 48</td>
 </tr>
 <tr>
 <td>MgATP</td>
 <td>Sigma-Aldrich</td>
 <td>A9187</td>
 <td>Pipette solution: 1</td>
 </tr>
 <tr>
 <td>Na2ATP</td>
 <td>Sigma-Aldrich</td>
 <td>A2383</td>
 <td>Pipette solution: 4</td>
 </tr>
 <tr>
 <td>Fluo-3**</td>
 <td>Invitrogen</td>
 <td>F3715</td>
 <td>Pipette solution: 0.1</td>
 </tr>
 <tr>
 <td>pH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>7.20</td>
 </tr>
 <tr>
 <td>adjusted with</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>1 M KOH</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Table V. Pipette solution for patch-clamp*. *On experimental days pipette solution is stored on ice until use. **Fluo-3 is added from a 1 mM stock solution on experimental days.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation of human atrial myocytes for simultaneous measurements of ca2+ transients and membrane currents

Yama klemp tekniği ve kalp hücresel Ca 2 arama ile kardiyak iyon kanallarının elektrofizyolojik özelliklerinin çalışma + taşıma anomaliler izole kardiyomiyositlerin gerektirir. Buna ek olarak, bu yöntemler kullanılarak hastaların miyositleri araştırmak için olasılık, bu tür atriyal fibrilasyon (AF) ve kalp hastalıklarının moleküler temeli aydınlatmak için çok değerli bir gerekliliktir. 1 Burada her iki için uygun olan insan atrial miyositlerinde bölgesinin izolasyonu için bir yöntem tarif Patch-clamp çalışmaları ve hücre içi Ca2 + konsantrasyonu aynı anda ölçümleri. İlk olarak, açık kalp cerrahisi uygulanan hastalarda elde edilen sağ atriyal uzantıları küçük doku parçaları (&quot;yığın yöntemi&quot;) içine doğranmış ve +-ücretsiz bir çözüm Ca 2 yıkanır. Daha sonra doku parçaları 20 mcM Ca 2 + ile çözümler içeren kollajenaz ve proteaz sindirilir. Bundan sonra, izole edilmiş olan miyosit Harvedoku süspansiyonu filtrasyon ve santrifüj ile sted. Son olarak, hücre depolama çözelti içinde Ca2 + konsantrasyonu 0.2 mM kademeli bir şekilde ayarlanır. Biz kısaca + ve Ca 2 + izolasyon işlemi sırasında tampon ve aksiyon potansiyelleri ve membran akımlarının temsilcisi kayıtları sağlamak, her ikisi birlikte aynı anda Ca 2 + geçici ölçümler, bu izole miyositler yapılan ile Ca 2 anlamını tartışmak.

Şekil 2A izole insan sağ atriyal miyositlerden üç temsilci örnekler gösterir. Bir CellFinder mikroskop lamı (üzerine hücre süspansiyonu (adım 5.5) 10 ul pipet hücre verimi ölçmek için <a href="http://www.antenna.nl/microlab/index-uk.html" target="_blank">http://www.antenna.nl/microlab/index-uk.html</a> ). 16.5 ± 3.1 cells/10 ul (n = 29) vs 5.1 ± 2.3 cells/10 ul: Şekil 2B&#39;de Ortalama hücre verim açıkça kronik AF (CAF) daha düşük hücre verim eğilimi hasta örnekleri (tüp bir olduğunu gösterir (n = 10) sırasıyla SR ve Caf, p &lt;0.05; tüp B: 17.9 ± 3.9 cells/10 ul (n = 29) vs 5.9 ± SR ve Caf 2.0 cells/10 ul (n = 9), , p = 0.107). Aksiyon potansiyel ölçümleri ve sitosolik Ca2 +, aynı anda geçici kayıtları için temsili örnekler, Şekil 3&#39;te verilmiştir. Incelenmiştir hücreler, T arasında yaklaşık% 90o aksiyon potansiyeli-tetiklenen Ca 2 + sürümü açık ve normal bir cep kasılmalar neden olur. Önceden bildirildiği gibi, hücre bütünlüğü için kabul edilen bir göstergesidir istirahat membran potansiyeli, -73.9 hakkında ortalama ± 2.7 mV (n = 23/10 miyositler / hasta) ve -77,7 ± 1,8 mV (n = 19/8 miyositler / hasta ) sırasıyla SR ve Caf (p&gt; 0,05). 15 Şekil 4 voltaj bağımlı L-tipi Ca 2 + akımları ve sitozolik Ca 2 + geçici temsilcisi eş zamanlı kayıtları gösterir. Seçici olmayan β-adrenoseptör agonisti izoprenalin (1 uM) Uygulama sağlam β-adrenerjik sinyal transdüksiyon kaskadının düşündüren bir Ca, L ve sitosolik Ca2 + hem de geçici amplitüdlerinin artırır. <img alt="Şekil 1" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig1highres.jpg" src="/files/ftp_upload/50235/50235fig1.jpg" /> FŞEKIL 1. miyositler Fluo-3 akış şeması (adım 6.1-6.5 bakınız) protokolü yükleme AM. m / v, kütle / hacim. <img alt="Şekil 2," fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig2highres.jpg" src="/files/ftp_upload/50235/50235fig2.jpg" /> Şekil 2. Depolama çözümü bir saat sonra bir, izole insan sağ atriyal miyositler. B, depolama çözümü hücrelerin 10 ul sayılır hücre verim ± SEM (adım 5.5) ortalama. n, her bir grup içinde preparatların sayısını ifade eder. * P &lt;0.05. <img alt="Şekil 3," fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig3highres.jpg" src="/files/ftp_upload/50235/50235fig3.jpg" /> Aksiyon potansiyeli-tetiklenen Ca 2 sinüs ritmi bir atriyal miyosit içinde +-geçici (Cat) ve chr Şekil 3. Temsilcisi kayıtlarıonic atriyal fibrilasyon hasta. Top: Enjekte stimülasyon (0.5 Hz) için kullanılan akım membran (I M). Aşağıda: membran potansiyeli (V M) ve tetiklenen Cat (alt) aynı anda kayıt. (Voigt ve ark izniyle Replotted. 2012) 15 <img alt="Şekil 4," fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235fig4highres.jpg" src="/files/ftp_upload/50235/50235fig4.jpg" /> L-tipi Ca 2 izoprenalin (1 mcM) etkisi Şekil 4. Temsilcisi kayıtları + akım tetiklenen Ca 2 sinüs ritmi ve kronik atriyal fibrilasyon hasta bir atriyal miyosit içinde +-geçici (Cat). Top: Gerilim-kelepçe protokolü (0.5 Hz). Aşağıda: Mevcut toplam net membran Eş zamanlı kayıt (I M), ağırlıklı olarak L-tipi Ca 2 + akım (orta) ve tetiklenen Cat (alt) yansıtan. (Voigt izni ile Replotted,ve ark. 2012) 15 <img src="/files/ftp_upload/50235/50235table1.jpg" alt="Tablo 1" fo:content-width="4in" fo:src="/files/ftp_upload/50235/50235table1highres.jpg" /> Tablo I. Çözümler. <img src="/files/ftp_upload/50235/50235table2.jpg" alt="Tablo 2" fo:content-width="2in" fo:src="/files/ftp_upload/50235/50235table2highres.jpg"/> Tablo II. Özel ekipman. <img src="/files/ftp_upload/50235/50235table3.jpg" alt="Tablo 3" fo:content-width="2in" fo:src="/files/ftp_upload/50235/50235table3highres.jpg"/> Tablo III. Fluo-3 ile miyositlerin yükleme için maddeler var. <img src="/files/ftp_upload/50235/50235table4.jpg" alt="Tablo 4" fo:content-width = &quot;2,5 inç&quot; fo: src = &quot;/ files/ftp_upload/50235/50235table4highres.jpg&quot; /&gt; Tablo IV. Yama-kelepçe için banyo çözüm. <img src="/files/ftp_upload/50235/50235table5.jpg" alt="Tablo 5" fo:content-width="2.5in" fo:src="/files/ftp_upload/50235/50235table5highres.jpg"/> Yama-kelepçe * için Tablo V. Pipet çözüm.

Watch this Scientific Journal Video about Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents at JoVE.com

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

Bu hücre içi Ca için kullanılabilecek insan atriyal miyosit izolasyonunu anlatır<sup&gt; 2 +</supElektrofizyolojik patch-clamp çalışmaları ile birlikte&gt; ölçümleri.

Ca aynı anda Ölçümler İnsan Atriyal miyositlerin izolasyonu<sup&gt; 2 +</sup&gt; Geçici ve Membran Akımlar

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

isolation-human-atrial-myocytes-for-simultaneous-measurements-ca2

Research

JoVE Journal

Biology

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

20.3K Görüntüleme.  University of Duisburg-Essen. Bu hücre içi Ca için kullanılabilecek insan atriyal miyosit izolasyonunu anlatır 2 + ölçümleri.

Video: Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

Konfokal Görüntüleme İzolasyon ve Yetişkin kardiyak miyositler Genetik Manipülasyon

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Biyoloji

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Görüntüleme Ca Teknikleri 2 + İnsan Sperm sinyal

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Lipidik Mesophases Membran Proteinleri Yüksek throughput Kristalizasyon için bir robot kullanımı

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

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 of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Yüksek Verimli Mikroplate Solunum Ölçümler için Fare İskelet Kası Minimal Miktarları gelen Mitokondri İzolasyonu

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Gelişim ve karakterizasyonu<em&gt; İn Vitro</em&gt; Mikrodamar Ağ ve Endotel kantitatif ölçümleri [Ca<sup&gt; 2+</sup&gt;]<sub&gt; I</sub&gt; Ve Nitrik Oksit Üretimi

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Yetişkin fareler ile Sinoatriyal Düğüm miyositlerin İzolasyonu, Kültür ve Fonksiyonel Karakterizasyonu Yöntemleri

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Floresan Mikroskopi Canlı hücreler Potansiyel Mitokondriyal Kalsiyum ve Mitokondriyal Membran Eşzamanlı Ölçümü

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5&#180;-OH ends or 5&#180;-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5&#180;-end processing, DNA damage, and DNA repair.

Here we describe a method amenable to simultaneously quantitate and genome-wide map ribonucleotides in highly intact DNA at single-nucleotide resolution, combining enzymatic cleavage of genomic DNA with its alkaline hydrolysis and subsequent 5&#180;-end sequencing.

Eş zamanlı haritalama ve Nefelometri Ribonucleotides insan mitokondriyal DNA'ın

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Hücre içi kalsiyum ve taze izole ve olduğu gibi fare serebral endotel membran potansiyeli eşzamanlı ölçümler

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Ca aynı anda Ölçümler İnsan Atriyal miyositlerin izolasyonu

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