이 작품은 독일 Forschungsgemeinschaft (DFG), 프리츠 - 티센 (Thyssen) - Stiftung 및 Charité / MDC에서 교부금의 지원을받는되었다.

1. 동물 마취 및 장기 수확 <ol><li> 케타민 (200 밀리그램 / kg BW)과 Xylazine (20 MG / kg BW)의 intraperitoneal (IP) 주입하여 마우스를 마취시키다. </li><li> 혈액 응고와 혈전 형성을 방지하기 위해 250 IU 헤파린의 IP를 삽입 anticoagulate 할 수 있습니다. </li><li> 깊은 마취법은 areflexia 특징으로하는,에 도달 할 때까지 기다립니다. 부드럽게 꼬리 곤란에 의한 각막이나 시험 비행 반사을 터치하여 areflexia, 테스트 각막 반사를 확인합니다. </li><li> 운영 테이블에 마우스를 전송하고 나태한 위치에서 문제를 해결. </li><li> 피부와 검상 돌기 아래의 복부 벽을 절개하고 탄두 thoracotomy를 수행 늑골 아치를 따라 양쪽으로 컷을 연장하고 이후 중간 겨드랑 선에서 갈비를 잘라, 갈비 위쪽으로 편향. </li><li> , 심낭을 엽니 다 큰 혈관을 찾습니다. 부드럽게 잘 대동맥을 표시 할 심장 꼬리를 누르십시오. 집게를 사용하여 대동맥을 고정. </li><li> s의 쌍 오목에 마음을 놓으십시오<html> cissors 한 싱글 컷 모든 연결 혈관을 해부. Langendorff의 cannulation에 대한 오름차순 대동맥의 큰 부족 부분을 보존해야합니다. </li><li> 얼음 추위와 사전 oxygenized 솔루션 1 (솔루션, 표 1 참조)로 가득 찬 페트리 접시에 즉시 excised 마음을 전송합니다. </li></ol> 2. 마음을 Langendorff 살포의 작성 <ol><li> 1.8F 강철 캐뉼라과 대동맥을 Cannulate. 공기 색전증을 방지해야합니다. </li><li> 솔루션 1 개 중 1 ML과 수술 봉합 및 수세식 coronaries와 캐뉼라에 대동맥을 흥분시키는. </li><li> Langendorff 장치와 캐뉼라를 연결합니다. </li><li> thoracotomy에서 Langendorff의 cannulation 할 시간이 있는지 확인 심근에 확장 국소 빈혈 / reperfusion 부상을 방지하기 위해 120 초를 초과하지 않습니다. </li><li> 칼슘이 10 ML과 Perfuse 마음 + 무료 솔루션이 (4 ML ​​/ 분). </li><li> 8 분 (4 ML / 분)에 대한 collagenase 솔루션 3 Perfuse 마음. </li></ol>심장 챔버 르 &quot;&gt; 3. Microsurgical 분리 <ol><li> 낮은 칼슘 2 + 솔루션 4를 포함하는 미리 예열 100 mm 페트리 접시에 전송 심장. </li><li> 조심스럽게 가위로 대동맥 및 기타 비 심장 조직을 제거하고 폐기합니다. </li><li> 별도의 심방과 심실하여 따로 챔버를 계속합니다. 해결 방법 4에 익숙해 세포세요. 작은 볼륨을 (이하 5 ML)을 사용합니다. </li></ol> 4. Cardiomyocytes의 추가 분리 <ol><li> 심방 cardiomyocytes : <ol><li> 심방 cardiomyocytes은 별도의 사전 예열 100-mm 배양 접시에 심방를 전송하고 부드럽게 고급 집게로 떨어져 당기는를 통해 조직을 떼어 놓다 개별화합니다. 조직의 거의 완전한 분리를 확인하십시오. </li><li> 5 분을위한 솔루션을 5 단계 중 1 ML에있는 세포를 일시 중지 할 수 확대 화재 광택 플라스틱 피펫 팁으로 1 ML의 피펫을 사용합니다. </li><li> 셀 필터 (200 μm의 메쉬 크기)를 사용하여 쓰레기들로부터 세포를 분리합니다. </li> &lt;리&gt; 5 ML의 세포 현탁액에 대한 솔루션 5 실내 온도 16 XG에서 2 분을위한 원심 분리기를 추가합니다. </li><li> 다음 단계는 세포 배양 후드 아래 운영하고 있습니다. 솔루션 6 5 ML의 표면에 뜨는하고 다시 일시 중지 펠릿을 폐기하십시오. </li><li> 15 ML 튜브에 10 분에 중력에 의해 침강 후, 상온 16 XG에서 1 분 동안 원심 분리기. 표면에 뜨는을 제거합니다. 솔루션 6 1-5 ML에서 자신의 양에 따라 세포를 다시 일시 중지합니다. </li></ol></li><li> 심실 cardiomyocytes : <ol><li> 해결 방법 4 5 ML에서 고급 집게와 관심의 왼쪽 심실 지역을 해부. </li><li> 세포의 대부분이 분리 될 때까지 부드럽게 pipetting하여 세포를 일시 중지합니다. 50 ML 튜브에 (200 μm의 메쉬 크기) 필터링 한 후 셀 솔루션을 전송 25 ML의 볼륨을 추가 할 수 있습니다. </li><li> 실내 온도 16 XG에서 2 분 동안 원심 분리기. </li><li> 다음 단계는 세포 배양 후드 아래 운영하고 있습니다. 표면에 뜨는 제거의 25 ML에있는 펠릿을 다시 일시 중지솔루션 6 10 분의 세포의 침강 할 수 있습니다. </li><li> 세포를 계산하고 표면에 뜨는을 제거, 25를 추가 - 50 ML 솔루션 6 셀에. </li></ol></li></ol> 5. 생물 세포 전기 생리학, 또는 연구 IF Cardiomyocytes의 작성 <ol><li> 전기 생리학 연구를 들어, 50 ML 튜브에 솔루션 6 근세포를 유지하고 침전을 억제. </li><li> 에 대한 생화학 연구, 예를 들어 칼슘 이미징, laminin 코팅 세포 배양 접시에 판 근세포 (최종 농도 20 μg / PBS에 ML laminin). </li><li> immunofluorescence 얼룩은 유리 커버 용지로 세포 배양 접시 판을 준비하고 laminin 솔루션 (최종 농도 50 μg / PBS에 ML laminin)으로 코팅하십시오. <ol><li> 도금 근세포 전에 솔루션을 제거합니다. 플레이트 세포를과 현미경을 사용하여 세포 밀도를 제어합니다. </li><li> 2퍼센트 CO 2에 37 번 1 시간에 슬립 ° C를 충당하기 위해 근세포가 준수 가자, 솔루션을 제거및 표준 착색 절차 프로토콜 즉시 시작합니다. </li><li> 실온에서 10 분 동안 PBS의 정착액, 예를 4퍼센트 PFA (산도 7.5)으로 배양하고 5 분마다 세 PBS 세척 단계를 따르십시오. </li><li> 세포를 permeabilize하고 바인딩 불특정 항체가 10 % 혈청, 0.3 % 트리톤, 실온에서 30 분을위한 PBS에 0.​​2 % BSA와 근세포을 길러 억제 할 수 있습니다. </li><li> 37 1 시간에 대한 기본 항체와 함께 배양 ° C와 같은 이전에 설명 씻는다. </li><li> 상온에서 1 시간을위한 차 항체를 품다. 핵을 counterstain 및 α-actinin 사용 DAPI와 fluorochrome 복합 phalloidin (알렉사 형석 488, Invitrogen)합니다. </li><li> 세척 후, 유리 커버 형광 장착 매체에 실란 처리 현미경 슬라이드와 소스 세포에 조심스럽게 미끄러 전송할 수 있습니다. </li></ol></li></ol> 6. 세포 전기 생리학 <ol><li> fre에 전체 세포 패치 - 클램프 녹음을 수행shly 실온에서 심방과 심실 cardiomyocytes을 분리. </li><li> 세포 목욕 솔루션의 정의 볼륨 가득 재관류 챔버에 건강 나타나지 cardiomyocytes을 전송합니다. 117 MM NaCl, 4 MM KCl, 1 ㎜ KH 2 PO 4, 4 MM NaHCO 3, 1.7 MM MgCl 2, 3 MM CoCl 2, 10 밀리미터 HEPES, 10 MM 포도당과 0.02 밀리미터 테트로도톡신 (TTX), (산도 7.4) . 산도 값 조정 NaOH를 사용하십시오. </li><li> (예 : IX71 역 현미경, Multiclamp 700B 앰프, pClamp10.3 소프트웨어 (분자 ​​장치)와 Digidata 1440A 수집 시스템 및 PC) 적절한 패치 클램프 장비를 사용합니다. </li><li> 이 CP 없음 130 MM KCl, 2 MM MgCl 2, 11 MM HEPES, 11 MM EGTA, 5 MM 그렇단이 ATP, 0.4 MM 그렇단이 GTP, 5 MM를 포함하는 세포 솔루션으로 가득 할 때 MΩ 3-5의 저항과 패치 피펫을 사용하여 및 4.9 MM CaCl 2 (산도 7.2). 산도 값 조정 코를 사용하십시오. </li><li> 외부 K + 전류 뒤를 그대로 보여주고 있습니다링 4.5 초 전압 단계는 -60와 20 밀리 초는 -40 뮤직 비디오에 prepulse 후 -70 mV의 개최 가능성에서 10-MV 단위 50 MV 사이의 잠재력을 테스트합니다. </li><li> 누설 전류는 항상 &lt;100 PA되어 있는지 확인합니다. </li><li> , Heteropodatoxin 2 (HpTx2, Alomone) 또는 Tetraethylammonium (차, 시그마), 다른 K의 해부를 들어 + 전류는 4 aminopyridine (ICN의 Biomedicals, 어바인, CA, USA 4 AP) 등의 특정 억제제를 사용합니다. 증권 솔루션은 세포 목욕 용액에 준비하고, &quot;기준&quot;녹음 후 microtip을 통해 세포의 가장 가까운 가능한 주변에 직접 적용해야합니다. 평균은 &quot;약물&quot;녹음하기 전에 2-3 분 동안 허용해야합니다. </li><li> 분석을 위해 셀 크기에 개별 셀 (전체 세포 막 커패시턴스)의 현재 진폭을 정상화. pClamp10.3 소프트웨어 (분자 장치) 또는 유사한 소프트웨어를 사용하여 오프라인 데이터를 분석합니다. </li></ol>

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	<li>Abbott, G. W., Goldstein, S. A., Sesti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+all+voltage-gated+potassium+channels+use+MiRPs?.">Do all voltage-gated potassium channels use MiRPs?.</a> Circ. Res. 88, 981-983 (2001).</li><li>Abbott, G. W., Sesti, F., Splawski, I., Buck, M. E., Lehmann, M. H., Timothy, K. W., Keating, M. T., Goldstein, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiRP1+forms+IKr+potassium+channels+with+HERG+and+is+associated+with+cardiac+arrhythmia.">MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia.</a> Cell. 97, 175-187 (1999).</li><li>Barry, D. M., Trimmer, J. S., Merlie, J. P., Nerbonne, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+voltage-gated+K++channel+subunits+in+adult+rat+heart.+Relation+to+functional+K++channels?.">Differential expression of voltage-gated K+ channel subunits in adult rat heart. Relation to functional K+ channels?.</a> Circ. Res. 77, 361-369 (1995).</li><li>Benjamin, E. J., Wolf, P. A., D'Agostino, R. B., Silbershatz, H., Kannel, W. B., Levy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+atrial+fibrillation+on+the+risk+of+death:+the+Framingham+Heart+Study.">Impact of atrial fibrillation on the risk of death: the Framingham Heart Study.</a> Circulation. 98, 946-952 (1998).</li><li>Bou-Abboud, E., Nerbonne, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+correlates+of+the+calcium-independent,+depolarization-activated+K++currents+in+rat+atrial+myocytes.">Molecular correlates of the calcium-independent, depolarization-activated K+ currents in rat atrial myocytes.</a> J. Physiol. 517 (Pt 2), 407-420 (1999).</li><li>Gaborit, N., Steenman, M., Lamirault, G., Le, M. N., Le, B. S., Lande, G., Leger, J., Charpentier, F., Christ, T., Dobrev, D., Escande, D., Nattel, S., Demolombe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+atrial+ion+channel+and+transporter+subunit+gene-expression+remodeling+associated+with+valvular+heart+disease+and+atrial+fibrillation.">Human atrial ion channel and transporter subunit gene-expression remodeling associated with valvular heart disease and atrial fibrillation.</a> Circulation. 112, 471-481 (2005).</li><li>Guo, W., Xu, H., London, B., Nerbonne, J. M. <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+transient+outward+K++current+diversity+in+mouse+ventricular+myocytes.">Molecular basis of transient outward K+ current diversity in mouse ventricular myocytes.</a> J. Physiol. 521 (Pt 3), 587-599 (1999).</li><li>Lundby, A., Ravn, L. S., Svendsen, J. H., Hauns, S., Olesen, S. P., Schmitt, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCNE3+mutation+V17M+identified+in+a+patient+with+lone+atrial+fibrillation.">KCNE3 mutation V17M identified in a patient with lone atrial fibrillation.</a> Cell Physiol. Biochem. 21, 47-54 (2008).</li><li>Ma, K. J., Li, N., Teng, S. Y., Zhang, Y. H., Sun, Q., Gu, D. F., Pu, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+KCNQ1+current+by+atrial+fibrillation-associated+KCNE4+(145E/D)+gene+polymorphism.">Modulation of KCNQ1 current by atrial fibrillation-associated KCNE4 (145E/D) gene polymorphism.</a> Chin Med. J. (Engl.). 120, 150-154 (2007).</li><li>McCrossan, Z. A., Abbott, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MinK-related+peptides.">The MinK-related peptides.</a> Neuropharmacology. 47, 787-821 (2004).</li><li>Pongs, O., Schwarz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ancillary+subunits+associated+with+voltage-dependent+K++channels.">Ancillary subunits associated with voltage-dependent K+ channels.</a> Physiol. Rev. 90, 755-796 (2010).</li><li>Ravn, L. S., Aizawa, Y., Pollevick, G. D., Hofman-Bang, J., Cordeiro, J. M., Dixen, U., Jensen, G., Wu, Y., Burashnikov, E., Haunso, S., Guerchicoff, A., Hu, D., Svendsen, J. H., Christiansen, M., Antzelevitch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain+of+function+in+IKs+secondary+to+a+mutation+in+KCNE5+associated+with+atrial+fibrillation.">Gain of function in IKs secondary to a mutation in KCNE5 associated with atrial fibrillation.</a> Heart Rhythm. 5, 427-435 (2008).</li><li>Ravn, L. S., Hofman-Bang, J., Dixen, U., Larsen, S. O., Jensen, G., Haunso, S., Svendsen, J. H., Christiansen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+of+97T+polymorphism+in+KCNE5+to+risk+of+atrial+fibrillation.">Relation of 97T polymorphism in KCNE5 to risk of atrial fibrillation.</a> Am. J. Cardiol. 96, 405-407 (2005).</li><li>Roepke, T. K., Abbott, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacogenetics+and+cardiac+ion+channels.">Pharmacogenetics and cardiac ion channels.</a> Vascul. Pharmacol. 44, 90-106 (2006).</li><li>Roepke, T. K., Kontogeorgis, A., Ovanez, C., Xu, X., Young, J. B., Purtell, K., Goldstein, P. A., Christini, D. J., Peters, N. S., Akar, F. G., Gutstein, D. E., Lerner, D. J., Abbott, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+deletion+of+kcne2+impairs+ventricular+repolarization+via+disruption+of+I(K,slow1)+and+I(to,f).">Targeted deletion of kcne2 impairs ventricular repolarization via disruption of I(K,slow1) and I(to,f).</a> FASEB J. 22, 3648-3660 (2008).</li><li>Sesti, F., Abbott, G. W., Wei, J., Murray, K. T., Saksena, S., Schwartz, P. J., Priori, S. G., Roden, D. M., George, A. L., Goldstein, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+polymorphism+associated+with+antibiotic-induced+cardiac+arrhythmia.">A common polymorphism associated with antibiotic-induced cardiac arrhythmia.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 10613-10618 (2000).</li><li>Temple, J., Frias, P., Rottman, J., Yang, T., Wu, Y., Verheijck, E. E., Zhang, W., Siprachanh, C., Kanki, H., Atkinson, J. B., King, P., Anderson, M. E., Kupershmidt, S., Roden, D. M. <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+in+KCNE1-null+mice.">Atrial fibrillation in KCNE1-null mice.</a> Circ. Res. 97, 62-69 (2005).</li><li>Yang, Y., Xia, M., Jin, Q., Bendahhou, S., Shi, J., Chen, Y., Liang, B., Lin, J., Liu, Y., Liu, B., 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=Identification+of+a+KCNE2+gain-of-function+mutation+in+patients+with+familial+atrial+fibrillation.">Identification of a KCNE2 gain-of-function mutation in patients with familial atrial fibrillation.</a> Am. J. Hum. Genet. 75, 899-905 (2004).</li><li>Zeng, Z. Y., Pu, J. L., Tan, C., Teng, S. Y., Chen, J. H., Su, S. Y., Zhou, X. Y., Zhang, S., Li, Y. S., Wang, F. Z., 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=[The+association+of+single+nucleotide+polymorphism+of+slow+delayed+rectifier+K++channel+genes+with+atrial+fibrillation+in+Han+nationality+Chinese].">[The association of single nucleotide polymorphism of slow delayed rectifier K+ channel genes with atrial fibrillation in Han nationality Chinese].</a> Zhonghua Xin. Xue. Guan. Bing. Za Zhi. 33, 987-991 (2005).</li><li>Zhang, M., Jiang, M., Tseng, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=minK-related+peptide+1+associates+with+Kv4.2+and+modulates+its+gating+function:+potential+role+as+beta+subunit+of+cardiac+transient+outward+channel?+Circ.">minK-related peptide 1 associates with Kv4.2 and modulates its gating function: potential role as beta subunit of cardiac transient outward channel? Circ.</a> Circ. Res. 88, 1012-1019 (2001).</li></ol>

우리는 공개 할 아무 것도 없습니다.

유전자 삭제와 관련된 심장 기능 및 심장 병리학을 연구하는 유전자 변형 마우스 변종의 증가 개발 체외에서 특정 유전자 삭제 효과를 공부하는 전문 방법에 증가 관심도 있습니다. 저희 연구실에서는 심장 repolarization에 KV 채널 보조의 subunits의 가족의 역할을 공부합니다. KCNE 유전자는 KCNEs 자신에 대한 칼륨 전류를 전달할 수 없습니다 하나의 transmembrane 도메인 KV 채널 부수적...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td></tr><tr><td> 테트로도톡신 </td><td> Alomone </td></tr><tr><td> 4 aminopyridine </td><td> ICN의 Biomedicals </td></tr><tr><td> Heteropodatoxin 2 </td><td> Alomone </td></tr><tr><td> Tetraethylammonium </td><td> 시그마 화학 물질 </td></tr><tr><td> Collagenase의 제 2 형 </td><td> 워싱턴 </td></tr></tbody></table>

isolation and kv channel recordings in murine atrial and ventricular cardiomyocytes

KCNE 유전자는 기능적 속성을 수정할 수 KV 채널 알파 subunits로 heteromeric 단지를 형성 KV 채널 보조의 subunits의 작은 가족 인코딩합니다. KCNE 유전자의 변이는 같은 긴 QT 증후군 및 / 또는 심방 세동 등의 심장 arrhythmias 환자에서 발견되었습니다. 그러나, 이러한 질병에 이르게 정확한 분자 pathophysiology은 어려운 남아 있습니다. 이전 연구에서이 유전자에 변이를 일으키는 질병의 electrophysiological 속성은 대부분 heterologous 표현 시스템에서 공부하고 있으며보고 된 효과가 직접 네이티브 cardiomyocytes로 번역 할 수 있다면 우리는 확신 할 수 없습니다. 저희 연구실에서는 따라서 다른 방법을 사용합니다. 우리가 시각화 실험의 저널이 문제에 대해 설명하는 고유 한 기술 - 우리가 직접 세포 전기 생리학의 녹아웃 마우스에서 분리 된 cardiomyocytes에 KCNE 유전자 삭제의 효과를 공부합니다. genetica에서 마음베드로 설계 KCNE 마우스는 빠르게 excised과 대동맥 cannulation로 Langendorff 장치에 장착되어 있습니다. 심근에서의 자유 칼슘 2 +는 EGTA에 의해 구속되고, 심장 근세포의 분리는 다음 전문 낮은 칼슘 2 + 버퍼를 포함하는 collagenase와 관상 동맥의 역행 재관류에 의해 달성된다. 심방, 무료 오른쪽 심실 벽과 왼쪽 심실 그런 다음 microsurgical 기법으로 구분 될 수있다. 칼슘은 천천히 세척 절차를 포함하는 여러 단계에서 분리 된 cardiomyocytes으로 추가됩니다. 더 자연 수축과 건강한 모양의 심방과 심실 cardiomyocytes는 다음 즉시 분리 후 첫 6 시간 내에 패치 클램프 기술 또는 다른 생화학 분석하여 electrophysiological 분석의 대상이됩니다.

체외에 대한 관심의 특정 유전자의 기능을 연구하기 위해 유전자 변형 생쥐에서 성인용 손쥐 cardiomyocytes의 절연은 또한 심장 pathophysiology을 이해 할 수있는 강력한 도구가되었습니다. 이 방법은 현재 전 세계적으로 기초 과학 연구소 만 작지만 증가에 의해 사용됩니다. 그러나, 성인 심실 손쥐 cardiomyocytes의 격리하지 않냐는 경험이 풍부한 손으로 철저하고 반복적으로 수행해야 할 수 있습?...

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 채널 장애는 심장 arrhythmias과 연결되어 있습니다. 우리가 KV 채널 보조의 subunit의 녹아웃 마우스에서 심방과 심실 cardiomyocytes의 분리에 대한 체계적인 프로토콜을 활용 이러한 arrhythmias로 이어질 분자 메커니즘을 연구하기 위해. 절연 cardiomyocytes는 다음 즉시 세포 electrophysiological 연구, 생화학 또는 immunofluorescence (IF) assays에 사용하실 수 있습니다.

손쥐 심방과 심실 Cardiomyocytes의 절연 및 KV 채널 레코딩

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 조회수.  Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine (MDC). KV 채널 장애는 심장 arrhythmias과 연결되어 있습니다. 우리가 KV 채널 보조의 subunit의 녹아웃 마우스에서 심방과 심실 cardiomyocytes의 분리에 대한 체계적인 프로토콜을 활용 이러한 arrhythmias로 이어질 분자 메커니즘을 연구하기 위해. 절연 cardiomyocytes는 다음 즉시 세포 electrophysiological 연구, 생화학 또는 immunofluorescence (IF) assays에 사용하실 수 있습니다.

비디오: 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.

Murine 조혈 줄기 세포와 리니지 - 헌신 Progenitors의 Phenotypic 분석 및 절연

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

연구

생물학

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.

분리 및 문화 신생아 마우스 심근

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.

구조 및 기능 연구를위한 지질 막에 Kv 값 채널의 재구성

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.

마우스 심근의 분리 및 생리 학적 분석

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.

분리 및 문화 성인 마우스 세포 신호 전달 용의 Cardiomyocytes과의<em&gt; 시험관</em&gt; 심장 비대

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.

1 채널의 세포 부착 된 패치 클램프 녹음

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.

정제 된 야생형과 돌연변이 체 CFTR 단백질의 기능의 재구성 및 채널 활동 측정

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.

배양 된 HL-1 심방 근육 세포 단층의 전압 및 칼슘 듀얼 채널 광학 매핑

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.

격리, 문화 및 성인 마우스 심근 세포의 형질 도입

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.